Structural and biochemical studies of an NB-ARC domain from a plant NLR immune receptor.

Plant NLRs are modular immune receptors that trigger rapid cell death in response to attempted infection by pathogens. A highly conserved nucleotide-binding domain shared with APAF-1, various R-proteins and CED-4 (NB-ARC domain) is proposed to act as a molecular switch, cycling between ADP (repressed) and ATP (active) bound forms. Studies of plant NLR NB-ARC domains have revealed functional similarities to mammalian homologues, and provided insight into potential mechanisms of regulation. However, further advances have been limited by difficulties in obtaining sufficient yields of protein suitable for structural and biochemical techniques. From protein expression screens in Escherichia coli and Sf9 insect cells, we defined suitable conditions to produce the NB-ARC domain from the tomato NLR NRC1. Biophysical analyses of this domain showed it is a folded, soluble protein. Structural studies revealed the NRC1 NB-ARC domain had co-purified with ADP, and confirmed predicted structural similarities between plant NLR NB-ARC domains and their mammalian homologues

Measuring, monitoring, and improving sleep variables: its application to professional football players

After several papers reported that Whole Body Cryotherapy (WBC) can improve objective and subjective markers of sleep, supported by anecdotal reports of post-exposure sleepiness from players at Southampton FC (SFC; PhD sponsor), the original aim of this thesis was to elucidate the effect of WBC on sleep in professional football players. However, after the UK COVID-19 lockdowns, WBC was not considered covid safe and, therefore, sleep became the central theme. Sleep plays an important role in the maintenance of both physiological and psychological homeostasis. During sleep, the release of human growth hormone and other anabolic hormones peak, inflammatory processes are modulated, and memories and skills are consolidated. Therefore, sleep is considered integral to athletic recovery and player well-being. Despite this, professional football players regularly present with sub-optimal sleep duration and/or quality. However, the factors associated with sleep variability are not fully understood, and there is no consensus on what the optimal level of sleep for athletes is. Therefore, this thesis conceptualised the following research questions: (1) What is known about the quality and duration of sleep amongst professional footballers? (2) What factors affect sleep in professional football players, specifically at SFC? (3) What are suitable and effective ways of improving sleep in professional football players? These questions were addressed across 2 systematic reviews (Chapters 2 & 4), an interventional study (Chapter 3), an observational cohort study (Chapter 5), a method agreement study (Chapter 6), and finally a case study (Chapter 7). Chapter 3 presents a study that aimed to (1) investigate the effect of a WBC applied across an in-season microcycle on the objective and subjective sleep quality in under-18 (U18) professional footballers, and (2) determine the effect of WBC on game-day inflammation, testosterone, and cortisol. Unfortunately, this study was curtailed by the COVID lockdowns. Nevertheless, novel findings were reported. Specifically, whilst objective sleep data were not significantly different between groups, players who received WBC during the microcycle preceding a competitive fixture, reported a greater sense of alertness following wake, as determined by the Leeds Sleep Quality Index. Whilst these results are subjective, they could also be indicative of improved sleep architecture following WBC. However, considering objective sleep was determined from wrist-worn activity monitors without the capability to detect sleep stages, this cannot be known with certainty. In Chapter 4, a scoping review of observational studies was performed that suggested that professional football players’ mean sleep duration, sleep latency, and wake after sleep onset (WASO), were all within recommended guidelines (these same reference limits were also used for Chapter 4). This conclusion was made on the basis that over 63% of the included studies reported means that were above the lower reference boundary for sleep duration. Despite this, several papers reported error bars that exceeded the reference limits, suggesting that suboptimal sleep remains common among individual players. In Chapter 5, an observational study was performed on under-18 professional SFC players, and the results matched what was observed from the scoping review in Chapter 4. Specifically, whilst sleep duration on matchday+1 (the day proceeding matchday) presented with a beta estimate (derived from linear mixed models) of 400mins, the remaining day types presented with sleep durations of above 420mins, the lower end of the reference limits. Nevertheless, in this study, confidence intervals breached the reference limits, therefore, further suggesting that suboptimal sleep occurs in this population. In tandem, results from Chapter 4 and Chapter 5 potentially indicate that group-level interventions are unnecessary. Rather, practitioners may find it more efficient to target support to players who report sleep disturbances. The scoping review presented in Chapter 4 also suggested that professional football players' sleep was also more variable compared to age-matched controls and several factors (e.g. scheduling variables) were associated with disrupted sleep. Chapter 5 builds on these findings by demonstrating for the first time that scheduled start time (the time players were scheduled to arrive at training or for a fixture) was associated with the amount of sleep that U18 players attained. Specifically, for every hour increase in start time, player sleep duration increased by an estimated 19.1mins (CI:9.4–28.79; p<0.001). This occurred in tandem with an 18mins (CI:9.3–26.6; p<0.001) later wake time, per hour increase in scheduled start time. It is not clear to what magnitude start time would have to be extended to generate increases in player performance, secondary to increased sleep duration. However, considering the player's age from this study (age: 17.3 ± 0.7yrs), a later start time may befit their intrinsic chronotype and, therefore, support the players by reinforcing their natural sleep habits. Whilst data from Chapter 5 support the notion that scheduling variables are associated with sleep in U18 professional footballers, they also suggest that sleep is not meaningfully associated with external workload. Global positioning and accelerometry data were collected and collated across 1-day, 7-day, and 28-day periods. For every 100m increase in high-speed running (>5.5 m·s−1), sleep onset and wake time were extended by 4.68min (CI:2.78—6.58mins) and 3.38mins (CI: 1.27—5.5mins), respectively. However, considering that workload had no significant effect on total sleep duration, the changes to wake time and sleep onset time should not concern practitioners. In Chapters 3, 5, and 7, objective sleep monitoring was completed using ReadiBand wrist-worn activity monitors. Though, it was acknowledged that these devices cannot readily link objective sleep quality and performance, and players' data could be missing due to poor band adherence. Therefore, another approach was trialled where the effect that inadequate sleep has on cognitive variables that are sensitive to sleep loss was determined, rather than measuring sleep directly. Consequently, this thesis also assessed the use of a novel virtual reality eye-tracking device that could rapidly administer an oculomotor task which was reported to be sensitive to total sleep deprivation. However, to be efficacious in a footballing environment, the device would have to demonstrate sensitivity to the daily fluctuation of sleep. Target radial variation (a measure of spatial accuracy) was found to be significantly correlated with perceived daytime sleepiness (r=0.33, p=0.005), however, no further relationships were observed between oculomotor function, psychometric vigilance, daytime sleepiness, and sleep metrics. In a retrospective analysis on a second data set from military personnel (that was included to augment the original analysis), only psychomotor vigilance, and not oculomotor function, were associated with the total amount of sleep achieved. This suggested that this device would not be efficacious in a footballing environment as a replacement for sleep monitoring. Following the research presented in Chapters 4 and 5, it was surmised that a bespoke approach to sleep intervention would be more efficacious than team-based interventions. To this end, a framework was conceptualised in collaboration with a multidisciplinary team from SFC (Chapter 7). Next, a player was referred to the scheme after reporting excessive night time awakenings. After consultation, the player completed several subjective questionnaires to assess sleep quality (Pittsburgh Sleep Quality Index), insomnia severity (Insomnia Severity Index), and daytime sleepiness (Epworth Sleepiness Scale) followed by a period of objective sleep monitoring. The sleep monitoring confirmed excessive nighttime awakenings and based on the responses from the initial consultation, a sleep hygiene intervention was applied tailored to the players' responses during the initial consultation. Results revealed improved subjective sleep quality, insomnia severity, and nighttime awakenings. Whilst a case study cannot establish causality, it does provide a potential framework for practitioners looking to provide targeted sleep interventions. Conclusions: In general, professional football players' sleep quantity, latency, and WASO is within available population-based reference limits. Scheduling variables, and not workload variables, are associated with activity monitor-derived objective sleep metrics in professional football players. Scheduled start time is associated with the amount of sleep that professional U18 football players receive. An oculomotor task does not have the requisite sensitivity to detect acute sleep loss in professional football players. A bespoke sleep intervention strategy can be efficacious in an applied footballing environment for players reporting sleep disruption

A specific case in the classification of woods by FTIR and chemometric: discrimination of Fagales from Malpighiales

Fourier transform infrared (FTIR) spectroscopic data was used to classify wood samples from nine species within the Fagales and Malpighiales using a range of multivariate statistical methods. Taxonomic classification of the family Fagaceae and Betulaceae from Angiosperm Phylogenetic System Classification (APG II System) was successfully performed using supervised pattern recognition techniques. A methodology for wood sample discrimination was developed using both sapwood and heartwood samples. Ten and eight biomarkers emerged from the dataset to discriminate order and family, respectively. In the species studied FTIR in combination with multivariate analysis highlighted significant chemical differences in hemicelluloses, cellulose and guaiacyl (lignin) and shows promise as a suitable approach for wood sample classification

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages. © 2013 Rogers et al

Hand-assisted retroperitoneoscopic versus standard laparoscopic donor nephrectomy: HARP-trial

Contains fulltext : 88436.pdf (publisher's version ) (Open Access)BACKGROUND: Transplantation is the only treatment offering long-term benefit to patients with chronic kidney failure. Live donor nephrectomy is performed on healthy individuals who do not receive direct therapeutic benefit of the procedure themselves. In order to guarantee the donor's safety, it is important to optimise the surgical approach. Recently we demonstrated the benefit of laparoscopic nephrectomy experienced by the donor. However, this method is characterised by higher in hospital costs, longer operating times and it requires a well-trained surgeon. The hand-assisted retroperitoneoscopic technique may be an alternative to a complete laparoscopic, transperitoneal approach. The peritoneum remains intact and the risk of visceral injuries is reduced. Hand-assistance results in a faster procedure and a significantly reduced operating time. The feasibility of this method has been demonstrated recently, but as to date there are no data available advocating the use of one technique above the other. METHODS/DESIGN: The HARP-trial is a multi-centre randomised controlled, single-blind trial. The study compares the hand-assisted retroperitoneoscopic approach with standard laparoscopic donor nephrectomy. The objective is to determine the best approach for live donor nephrectomy to optimise donor's safety and comfort while reducing donation related costs. DISCUSSION: This study will contribute to the evidence on any benefits of hand-assisted retroperitoneoscopic versus standard laparoscopic donor nephrectomy. TRIAL REGISTRATION: Dutch Trial Register NTR1433

Association of Fidaxomicin with C. difficile spores: Effects of Persistence on Subsequent Spore Recovery, Outgrowth and Toxin Production.

Background: We have previously shown that fidaxomicin instillation prevents spore recovery in an in-vitro gut model, whereas vancomycin does not. The reasons for this are unclear. Here, we have investigated persistence of fidaxomicin and vancomycin on C. difficile spores, and examined post-antibiotic exposure spore recovery, outgrowth and toxin production. Methods: Prevalent UK C. difficile ribotypes (n=10) were incubated with 200mg/L fidaxomicin, vancomycin or a non-antimicrobial containing control for 1 h in faecal filtrate or Phosphate Buffered Saline. Spores were washed three times with faecal filtrate or phosphate buffered saline, and residual spore-associated antimicrobial activity was determined by bioassay. For three ribotypes (027, 078, 015), antimicrobial-exposed, faecal filtrate-washed spores and controls were inoculated into broth. Viable vegetative and spore counts were enumerated on CCEYL agar. Percentage phase bright spores, phase dark spores and vegetative cells were enumerated by phase contrast microscopy at 0, 3, 6, 24 and 48 h post-inoculation. Toxin levels (24 and 48h) were determined by cell cytotoxicity assay. Results: Fidaxomicin, but not vancomycin persisted on spores of all ribotypes following washing in saline (mean=10.1mg/L; range= 4.0-14mg/L) and faecal filtrate (mean =17.4mg/L; 8.4-22.1mg/L). Outgrowth and proliferation rates of vancomycin-exposed spores were similar to controls, whereas fidaxomicin-exposed spores showed no vegetative cell growth after 24 and 48 h. At 48h, toxin levels averaged 3.7 and 3.3 relative units (RU) in control and vancomycin-exposed samples, respectively, but were undetectable in fidaxomicin-exposed samples. Conclusion: Fidaxomicin persists on C. difficile spores, whereas vancomycin does not. This persistence prevents subsequent growth and toxin production in vitro. This may have implications on spore viability, thereby impacting CDI recurrence and transmission rates

Left main bronchus resection and reconstruction. A single institution experience

<p>Abstract</p> <p>Background</p> <p>Left main bronchus resection and reconstruction (LMBRR) is a complex surgical procedure indicated for management of inflammatory, benign and low grade malignant lesions. Its application provides maximal parenchymal sparing.</p> <p>Methods</p> <p>Out of 98 bronchoplastic procedures performed at the Authors' Institution in the 1995-2011 period, 4 were LMBRR. Indications were bronchial carcinoid in 2 cases, inflammatory pseudotumor in 1 case, TBC stricture in 1 case. All patients underwent preoperatively a rigid bronchoscopy to restore the airway lumen patency. At surgery a negative resection margin was confirmed by frozen section in the neoplastic patients. In all patients an end-to-end bronchial anastomosis was constructed according to Grillo.</p> <p>Results</p> <p>There were neither mortality nor major complications. Airway lumen was optimal in 3 patients, good in 1.</p> <p>Conclusion</p> <p>LMBRR is a valuable option for the thoracic surgeon. It maximizes the parenchyma-sparing philosophy, broadening the spectrum of potential candidates for cure. It remains a technically demanding procedure, to be carried out by an experienced surgical team. Correct surgical planning affords excellent results, both in the short and long term.</p