Towards a Structural Understanding of Spore Germination in Clostridium Difficile

Clostridium difficile is a Gram-positive bacterium that causes a toxin-mediated disease, typically in individuals whose normal intestinal flora has been compromised by antibiotic therapy. C. difficile is naturally resistant to many antibiotics and produces spores that can withstand harsh environmental conditions and many disinfectants, making the infection difficult to clear and easy to spread. The infection begins when spores from the environment are ingested and germinate upon exposure to taurocholate and glycine in the digestive tract. This germination process is required to initiate infection and thus represents a good target for the development of novel therapeutics. Although spore germination is necessary for disease transmission, the molecular mechanisms regulating this process are poorly understood. Germination relies on sensing a germinant and triggering degradation of the cortex layer of the spore, which is important for spore resistance. Once the cortex is degraded, the spore can undergo outgrowth to a vegetative cell and secrete toxins to cause disease symptoms. There are several discrete steps to the proteolytic cascade that ultimately lead to cortex hydrolysis. First, the pseudoprotease CspC acts as a germinant receptor for the bile salt taurocholate; CspC then relays this signal to the subtilisin-like serine protease, CspB. CspB is required for efficient cleavage and activation of the cortex hydrolase. SleC. Upon proteolytic activation of SleC, cortex hydrolysis can proceed, which allows subsequent outgrowth. To better understand the mechanistic basis of the germination process, we solved the 1.6 Å structure of the required germination protease, CspB, from C. perfringens (a related pathogen). This structure revealed that CspB is comprised of three domains: an associated prodomain, a subtilase domain, and a jellyroll domain. Our work significantly advanced our understanding of the proteolytic cascade that leads to germination; in particular the structure and function of the CspB protease, and the role of its three domains. We have described the four domains of the cortex hydrolase, SleC, and how they contribute to the activity of SleC. We have recently obtained diffraction-quality crystals of the pseudoprotease, CspC, from an organism more closely related to C. difficile, C. bifermentans. Our latest work, focusing on the germination receptor, CspC, has brought us closer to a three-dimensional structure of this protein, which will likely reveal how it binds ligands and functions in germination

Identifying Adaptor Proteins for Selective mRNA Export in S. cerevisiae

The export of mature mRNA from the nucleus of the cell to the cytoplasm is crucial for a cell’s survival, as this process is necessary to produce proteins encoded by these transcripts. In S. cerevisiae (yeast) cells, a protein known as Mex67 must bind to the mRNA while it’s in the nucleus so it can cross through the nuclear pore complex (NPC). Mex67 cannot bind to the mRNA directly, instead it needs to be accompanied by adaptor proteins to aid it in getting across the NPC. Stress conditions such as heat shock interferes with the function of these adaptor proteins, causing Mex67 to not be able to cross the NPC. However, during heat shock cells induce the production and export of heat shock transcripts that encode proteins that allow for recovery from the stress. One of these transcripts is SSA4, which encodes a protein chaperone that helps cellular proteins refold after denaturation during heat shock. It is unknown what molecular mechanism enables the selective export of SSA4. We hypothesized that there is an unknown adaptor protein that binds to SSA4 so it can recruit Mex67 for export under stress conditions. To test this hypothesis, we first tested various growth conditions in which wild-type yeast cells could grow but mRNA export mutants could not. Following this, an overexpression plasmid library was prepared and will be transformed into this mutant to search for proteins which could rescue this growth defect. We anticipate that this project will aid in identification of adaptors that permit selective mRNA export during cellular stress

Testing SSA4::ADE3 Reporters for MCS Screening

Gene expression is essential to life and occurs through the processes of transcription of mRNA in the nucleus, export of transcripts to the cytoplasm through the nuclear pore complex (NPC), and translation of the mRNA into protein in the cytosol. The budding yeast S. cerevisiae is a eukaryotic model system used to explore the regulation of mRNA export. Transcripts are able to exit the nucleus through interaction with Mex67 which binds mRNA via adaptor proteins and allows crossing through NPCs. However, during heat shock (42°C) known adaptor proteins are rendered dysfunctional thus halting general mRNA export. Under these conditions, specific transcripts are able to exit the nucleus to enable the cell to response to stress. For example, SSA4 encodes a chaperone, which helps denatured proteins refold following heat shock is able to selectively export in response to stress. However, the mechanism for this selective export is not understood. I hypothesize that an unknown adaptor protein recruits Mex67 to allow for its selective export from the nucleus. In order to test this hypothesis, I have cloned a pair of vectors to be used as phenotypic reporters for SSA4 export. These reporters include the ADE3 ORF and SSA4 regulatory sequence (promoter, 5’ and 3’ UTRs). We anticipated that these reporters would yield red yeast cells if selectively exported during heat shock conditions. Analysis of the first series of reporter plasmids indicated that the plasmids weren’t fully cloned as expected, and subsequent experiments have been aimed at generating the proper clones. Future analysis will test whether these reporters successfully recapitulate selective SSA expression and export and use of these reporters for genetic screening to identify proteins involved in selective mRNA export

Home-prepared food, dietary quality and socio-demographic factors: a cross-sectional analysis of the UK National Diet and nutrition survey 2008–16

Abstract: Background: Evidence suggests eating home-prepared food (HPF) is associated with increased dietary quality, while dietary quality varies across socio-demographic factors. Although it has been hypothesised that variation in HPF consumption between population sub-groups may contribute to variation in dietary quality, evidence is inconclusive. This study takes a novel approach to quantifying home-prepared food (HPF) consumption, and describes HPF consumption in a population-representative sample, determining variation between socio-demographic groups. It tests the association between HPF consumption and dietary quality, determining whether socio-demographic characteristics moderate this association. Methods: Cross-sectional analysis of UK survey data (N = 6364, aged≥19; collected 2008–16, analysed 2018). High dietary quality was defined as ‘DASH accordance’: the quintile most accordant with the Dietary Approaches to Stopping Hypertension (DASH) diet. HPF consumption was estimated from 4-day food diaries. Linear regressions were used to determine the association between HPF consumption and socio-demographic variables (household income, education, occupation, age, gender, ethnicity and children in the household). Logistic regression was used to determine the association between HPF consumption and DASH accordance. Interaction terms were introduced, testing for moderation of the association between HPF consumption and DASH accordance by socio-demographic variables. Results: HPF consumption was relatively low across the sample (Mean (SD) % of energy consumption = 26.5%(12.1%)), and lower among white participants (25.9% v 37.8 and 34.4% for black and Asian participants respectively, p < 0.01). It did not vary substantially by age, gender, education, income or occupation. Higher consumption of HPF was associated with greater odds of being in the most DASH accordant quintile (OR = 1.2 per 10% increase in % energy from HPF, 95% CI 1.1–1.3). Ethnicity was the only significant moderator of the association between HPF consumption and DASH accordance, but this should be interpreted with caution due to high proportion of white participants. Conclusions: While an association exists between HPF consumption and higher dietary quality, consumption of HPF or HPF’s association with dietary quality does not vary substantially between socio-demographic groups. While HPF may be a part of the puzzle, it appears other factors drive socio-demographic variation in dietary quality

How Does Time Use Differ between Individuals Who Do More versus Less Foodwork? A Compositional Data Analysis of Time Use in the United Kingdom Time Use Survey 2014–2015

Background: Increased time spent on home food preparation is associated with higher diet quality, but a lack of time is often reported as a barrier to this practice. We compared time use in individuals who do more versus less foodwork (tasks required to feed ourselves and our households, including home food preparation). Methods: Cross-sectional analysis of the UK Time Use Survey 2014−15, participants aged 16+ (N = 6143). Time use over 24 h was attributed to seven compositional parts: personal care; sleep; eating; physical activity; leisure screen time; work (paid and unpaid); and socialising and hobbies. Participants were categorised as doing no, ‘some’ (70 min), or ‘more’ foodwork (≥70 min). We used compositional data analysis to test whether time-use composition varied between these participant groups, determine which of the parts varied between groups, and test for differences across population subgroups. Results: Participants who spent more time on foodwork spent less time on sleep, eating, and personal care and more time on work. Women who did more foodwork spent less time on personal care, socialising, and hobbies, which was not the case for men. Conclusion: Those who seek to encourage home food preparation should be aware of the associations between foodwork and other activities and design their interventions to guard against unintended consequences

The Immunoglobulin Superfamily Members syg-2 and syg-1 Regulate Neurite Development in C. elegans

Neurons form elaborate networks by guiding axons and dendrites to appropriate destinations. Neurites require information about the relative body axes during the initial projection from the cell body, and failure to receive or interpret those cues correctly can result in outgrowth errors. We identified a mutation in the Ig superfamily member syg-2 in a screen for animals with anterior/posterior (A/P) axon guidance defects. We found that syg-2 and its cognate Ig family member syg-1 appear to function in a linear genetic pathway to control the outgrowth of GABAergic axons. We determined that this pathway works in parallel to Wnt signaling. Specifically, mutations in syg-2 or syg-1 selectively affected the embryonically derived Dorsal D-type (DD) GABAergic neurons. We found no evidence that these mutations affected the Ventral D-type neurons (VD) that form later, during the first larval stage. In addition, mutations in syg-1 or syg-2 could result in the DD neurons forming multiple processes, becoming bipolar, rather than the expected pseudounipolar morphology. Given SYG-2′s essential function in synaptogenesis of the hermaphrodite-specific neurons (HSNs), we also examined DD neuron synapses in syg-2 mutants. We found syg-2 mutants had a decreased number of synapses formed, but synaptic morphology was largely normal. These results provide further evidence that the GABAergic motorneurons use multiple guidance pathways during development

Concert recording 2022-04-26

[Track 1]. The four seasons. La primavera, op. 8, no. 1. L\u27Estate, Op. 8, No. 2 ; L\u27Autunno, Op. 8, No. 3 ; L\u27Inverno, Op. 8, No. 4 / Antonio Vivaldi -- [Track 2]. Serenade for string orchestra, op. 48. I. Pezzo in forma di sonatina ; III. Elegie ; IV. Finale / Pyotr Ilych Tchaikovsky

Effectiveness of mental health warnings on tobacco packaging in people with and without common mental health conditions:an online randomised experiment

BACKGROUND: Health warning labels on tobacco packaging are a cost-effective means of health risk communication. However, while an extensive range of physical health risks are well-portrayed via current tobacco health warnings in the UK, there are none that currently portray the negative impact of smoking on mental health. AIMS: (i) develop novel mental health warning labels for tobacco packaging and (ii) test perceptions of these warnings in smokers and non-smokers, with and without mental health problems. METHODS: Six mental health warning labels were developed with a consultancy focus group. These warning labels were tested in an online randomised experiment, where respondents (N = 687) rated six Mental Health Warning Labels (MHWLs) and six Physical Health Warning Labels (PHWLs) on measures of perceived effectiveness, believability, arousal, valence, acceptability, reactance and novelty of information. RESULTS: MHWLs were perceived as low to moderately effective (mean = 4.02, SD = 2.40), but less effective than PHWLs (mean = 5.78, SD = 2.55, p < 0.001, η(p)(2) = 0.63). MHWLs were perceived as less believable, arousing, unpleasant, and acceptable than PHWLs. MHWLs evoked more reactance and were rated as more novel. Perceptions of MHWLs did not differ in people with and without mental health problems except for reactance and acceptability, but consistent with the PHWL literature, perceptions of MHWLs differed between non-smokers and smokers. CONCLUSION: MHWLs could be an effective means to communicate novel information about the effects of smoking on mental health. MHWLs are perceived as less effective, believable, arousing, unpleasant, and acceptable than PHWLs, but MHWLs evoke more reactance and are rated as more novel

Concert recording 2022-04-26

[Track 1]. The four seasons. La primavera, op. 8, no. 1. L\u27Estate, Op. 8, No. 2 ; L\u27Autunno, Op. 8, No. 3 ; L\u27Inverno, Op. 8, No. 4 / Antonio Vivaldi -- [Track 2]. Serenade for string orchestra, op. 48. I. Pezzo in forma di sonatina ; III. Elegie ; IV. Finale / Pyotr Ilych Tchaikovsky

Single-photon stored-light Ramsey interferometry using Rydberg polaritons

We demonstrate a single-photon stored-light interferometer, where a photon is stored in a laser-cooled atomic ensemble in the form of a Rydberg polariton with a spatial extent of 10×1×1µm3. The photon is subject to a Ramsey sequence, i.e., “split” into a superposition of two paths. After a delay of up to 450 ns, the two paths are recombined to give an output dependent on their relative phase. The superposition time of 450 ns is equivalent to a free-space propagation distance of 135 m. We show that the interferometer fringes are sensitive to external fields and suggest that stored-light interferometry could be useful for localized sensing applications