Characterisation of the CspA paralogues of Salmonella Typhimurium

In cold temperatures, the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) requires the action of cold shock protein A (CspA) paralogues. These are thought to melt misfolded ribonucleic acids, facilitating their translation at low temperatures. However, through phenotypic analysis of our SL1344 csp null mutant (lacking all CspA paralogues), it has been shown that CspA paralogues function during other environmental stresses, outwith temperature reduction, and play an essential role in colony formation of an SL1344 rpoS mutant at 37°C. The general stress σ subunit, RpoS, plays an important role in adapting cells to a number of stresses including oxidative stress, temperature changes, low pH and stationary phase. Under such conditions, RpoS acts as an ‘emergency co-ordinator’, subsequently inducing the transcription of necessary stress response genes. In Escherichia coli, RpoS is regulated posttranscriptionally by at least three small RNAs (sRNAs): OxyS, DsrA and RprA; that require interactions with the Sm-like RNA chaperone, Hfq. In S. Typhimurium, the stability of the RpoS protein itself is regulated by ClpXP, an ATP-dependent protease responsible for RpoS degradation, and a specific recognition factor that targets RpoS to this protease, MviA. The present study has shown that the CspA paralogues of S. Typhimurium are involved in the expression of RpoS and aims to elucidate the role of these proteins in RpoS production. Comparative phenotypic tests were carried out in strains carrying mutations in rpoS, hfq and the csp genes to gain insight into the interactions of Hfq and CspA paralogues, with respect to RpoS expression. Both significant phenotypic overlaps, such as peroxide sensitivity, and phenotypes unique to certain mutant strains, such as cold acclimation in the csp null strain, were observed. CspA paralogues and Hfq are functionally distinct, not only in their involvement in RpoS expression, but also in RpoS-independent processes, such as cold acclimation, motility and to some extent, growth at 37°C. The roles of Hfq and the CspA paralogues, in RpoS expression, were also assessed at the molecular level. A combination of qRT-PCR analysis, transcriptional fusions and immunoblotting (with anti-σ antibodies) has shown that DsrA and RprA are not essential for RpoS expression in S. Typhimurium, during stationary phase or exponential cold shock, and do not require Hfq under these conditions. Contrary to reports in E. coli, DsrA is not induced upon cold shock in SL1344. Northern blots have shown that neither Hfq nor the CspA paralogues are involved in regulating rpoS transcription during either stationary phase at 37°C or cold shock in exponential phase. Immunoblotting and translational fusions have identified different pathways for the regulation of RpoS during stationary phase at 37°C and cold shock in exponential phase. Hfq is involved during the former condition only, whilst CspA paralogues are involved in both. Protein stability experiments have shown that the CspA paralogues do not play a major role in stabilising RpoS protein against degradation. Together, these results have pointed to a role for both the CspA paralogues and Hfq in facilitating the efficient translation of rpoS mRNA. An SL1344 csp null rpoS mutant is unable to form colonies on LB agar at 37°C, a phenomenon found when introducing combinations of mutations to SL1344 for phenotypic assessment. A conditional rpoS mutant revealed that the SL1344 csp null rpoS strain is viable but non-culturable. From the csp gene family, only cspA and cspB were able to restore colony forming ability to the rpoS mutant. Further complementation experiments pointed to faulty cell division, due to abnormal RNase E activity, as the cause

Sleep restriction and serving accuracy in performance tennis players, and effects of caffeine

Athletes often lose sleep on the night before a competition. Whilst it is unlikely that sleep loss will impair sports mostly relying on strength and endurance, little is known about potential effects on sports involving psychomotor performance requiring high level cognitive skills necessitating judgement and accuracy, as in tennis, and where caffeine is ‘permitted’’. Two studies were undertaken on the effects of 5h sleep (33%) restriction versus normal sleep, on an objective measure of serving accuracy in semi-professional tennis players. Testing (14:00h-16:00h) for both studies comprised 40 serves into a (1.8m x 1.1m) ‘service box’ diagonally, over the net. Study 2 was identical to that of Study 1, except that there was an extra sleep restriction condition. All conditions involved a sugar free drink given 30 min before testing, but with the drink for one sleep restriction containing 80mg caffeine (double blind). Study 1 comprised 16 men and women, in a within Ss counterbalanced design (normal versus sleep restriction). Study 2 involved 12 different men and women undergoing three conditions in a latin square design. Conditions were as for Study 1, with a replicated sleep restriction. All conditions incorporated a sugar-free drink given 30 min before testing, with one restriction including 80mg caffeine (double blind). Both studies showed significant impairments to serving accuracy following sleep restriction, particularly with women. At this dose, caffeine had no beneficial effect. These results reflect laboratory based, non-sports related findings pointing to detrimental effects of sleep loss on executive function

Fatigue in ferry crews: a pilot study

Since the 1950s, the shipping industry has undergone profound changes, comparable in scale to those resulting from the advent of steam in the last century. This transformation has been shaped by many historical events and facilitated by the introduction of new technology and new commercial, operational and managerial practices, and it has clear implications for current research into the causality of marine accidents; technological developments, for example, have led to the so-called ‘radar-assisted collisions’. It is often said in the industry that 70-80% of maritime accidents are attributable to human error. What is less well known, however, is that in the majority of these cases, the factor which has consistently been identified as a major contributory link in the chain of events leading to an accident, is fatigue. Because of this, it is important to continue research into the causes and incidence of fatigue in seafarers. The objectives of this pilot study were: to investigate the quality and quantity of sleep among crew employed on the short-sea ferry sector; to evaluate the extent to which poor quality sleep occurs; and to identify the factors contributing to poor sleep quality. The subjects of the study were 12 crew members of different rank and with different work patterns from two UK-flagged short-sea pax/ro ferries. Background information on the participants was collected via questionnaires. Data on the duration and quality of sleep were collected by the use of wrist-worn actimeters and by self-report sleep logs, while self reports of alertness were collected at two-hourly intervals during each wakeful period. All the above data were collected during a complete tour of duty comprising one week at sea followed by one week of leave. The week-on / week-off work schedule allowed comparisons to be made between the sleep patterns of a working week at sea and those of a rest week at home. The findings indicate that differences in both sleep quality and duration of sleep between the work and non-work weeks were greater for those crew members required to work split shifts. Furthermore, they experienced greater sleep disturbance and generally shorter sleep periods than crew members who worked a single shift every 24 hours. The statistical analyses presented in the current study show a significant difference in the total number of hours sleep between the home and work schedules, and reveal a similar difference in the incidence of sleep disturbance. While further and more substantial examination into different shift patterns is required, there are already clear indications of a need to reappraise traditional watch regimes on board. This pilot study is the first phase of an extensive investigation into sleep and fatigue in seafarers’ work patterns, which is being carried out under the name of SEAFATIGUE. It is to include personal and environmental factors and is to be conducted within different shipping sectors of deep-sea and coastal trade. The final objective of the SEAFATIGUE project is to provide the maritime industry with a resource of detailed technical data on fatigue among seafarers, in order to facilitate the formulation and implementation of sound, proactive policies in areas of employment practices, manning levels, shipboard ergonomics, shiftwork patterns and the training of seafarers in fatigue management. This pilot study is the result of a joint collaborative project between the Sleep Research Laboratory at Loughborough University and the Seafarers International Research Centre at the University of Wales, Cardiff

Mild hypohydration increases the frequency of driver errors during a prolonged, monotonous driving task

The aim of the present study was to examine the effect of mild hypohydration on performance during a prolonged, monotonous driving task. Methods: Eleven healthy males (age 22 ± 4 y) were instructed to consume a volume of fluid in line with published guidelines (HYD trial) or 25% of this intake (FR trial) in a crossover manner. Participants came to the laboratory the following morning after an overnight fast. One hour following a standard breakfast, a 120 min driving simulation task began. Driver errors, including instances of lane drifting or late breaking, EEG and heart rate were recorded throughout the driving task. Results: Pre-trial bodymass (P=0.692), urine osmolality (P=0.838) and serumosmolality (P=0.574)were the same on both trials. FR resulted in a 1.1±0.7% reduction in bodymass, compared to−0.1±0.6% in the HYD trial (P = 0.002). Urine and serum osmolality were both increased following FR (P b 0.05). There was a progressive increase in the total number of driver errors observed during both the HYD and FR trials, but significantly more incidents were recorded throughout the FR trial (HYD 47 ± 44, FR 101 ± 84; ES = 0.81; P = 0.006). Conclusions: The results of the present study suggest that mild hypohydration, produced a significant increase in minor driving errors during a prolonged, monotonous drive, compared to that observed while performing the same task in a hydrated condition. The magnitude of decrement reported,was similar to that observed following the ingestion of an alcoholic beverage resulting in a blood alcohol content of approximately 0.08% (the current UK legal driving limit), or while sleep deprived

Characterisation of the CspA paralogues of Salmonella Typhimurium

In cold temperatures, the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) requires the action of cold shock protein A (CspA) paralogues. These are thought to melt misfolded ribonucleic acids, facilitating their translation at low temperatures. However, through phenotypic analysis of our SL1344 csp null mutant (lacking all CspA paralogues), it has been shown that CspA paralogues function during other environmental stresses, outwith temperature reduction, and play an essential role in colony formation of an SL1344 rpoS mutant at 37°C. The general stress σ subunit, RpoS, plays an important role in adapting cells to a number of stresses including oxidative stress, temperature changes, low pH and stationary phase. Under such conditions, RpoS acts as an ‘emergency co-ordinator’, subsequently inducing the transcription of necessary stress response genes. In Escherichia coli, RpoS is regulated posttranscriptionally by at least three small RNAs (sRNAs): OxyS, DsrA and RprA; that require interactions with the Sm-like RNA chaperone, Hfq. In S. Typhimurium, the stability of the RpoS protein itself is regulated by ClpXP, an ATP-dependent protease responsible for RpoS degradation, and a specific recognition factor that targets RpoS to this protease, MviA. The present study has shown that the CspA paralogues of S. Typhimurium are involved in the expression of RpoS and aims to elucidate the role of these proteins in RpoS production. Comparative phenotypic tests were carried out in strains carrying mutations in rpoS, hfq and the csp genes to gain insight into the interactions of Hfq and CspA paralogues, with respect to RpoS expression. Both significant phenotypic overlaps, such as peroxide sensitivity, and phenotypes unique to certain mutant strains, such as cold acclimation in the csp null strain, were observed. CspA paralogues and Hfq are functionally distinct, not only in their involvement in RpoS expression, but also in RpoS-independent processes, such as cold acclimation, motility and to some extent, growth at 37°C. The roles of Hfq and the CspA paralogues, in RpoS expression, were also assessed at the molecular level. A combination of qRT-PCR analysis, transcriptional fusions and immunoblotting (with anti-σ antibodies) has shown that DsrA and RprA are not essential for RpoS expression in S. Typhimurium, during stationary phase or exponential cold shock, and do not require Hfq under these conditions. Contrary to reports in E. coli, DsrA is not induced upon cold shock in SL1344. Northern blots have shown that neither Hfq nor the CspA paralogues are involved in regulating rpoS transcription during either stationary phase at 37°C or cold shock in exponential phase. Immunoblotting and translational fusions have identified different pathways for the regulation of RpoS during stationary phase at 37°C and cold shock in exponential phase. Hfq is involved during the former condition only, whilst CspA paralogues are involved in both. Protein stability experiments have shown that the CspA paralogues do not play a major role in stabilising RpoS protein against degradation. Together, these results have pointed to a role for both the CspA paralogues and Hfq in facilitating the efficient translation of rpoS mRNA. An SL1344 csp null rpoS mutant is unable to form colonies on LB agar at 37°C, a phenomenon found when introducing combinations of mutations to SL1344 for phenotypic assessment. A conditional rpoS mutant revealed that the SL1344 csp null rpoS strain is viable but non-culturable. From the csp gene family, only cspA and cspB were able to restore colony forming ability to the rpoS mutant. Further complementation experiments pointed to faulty cell division, due to abnormal RNase E activity, as the cause.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Moderate sleep restriction in treated older male OSA participants: Greater impairment during monotonous driving compared with controls

Objectives\ud To examine the effects on monotonous driving of normal sleep versus one night of sleep restriction in continuous positive airway pressure (CPAP) treated obstructive sleep apnoea (OSA) patients compared with age matched healthy controls.\ud \ud Methods\ud Nineteen CPAP treated compliant male OSA patients (OSA-treated patients (OPs)), aged 50–75 years, and 20 healthy age-matched controls underwent both a normal night’s sleep and sleep restriction to 5 h (OPs remained on CPAP) in a counterbalanced design. All participants completed a 2 h afternoon monotonous drive in a realistic car simulator. Driving was monitored for sleepiness-related minor and major lane deviations, with ‘safe’ driving time being total time driven prior to first major lane deviation. EEGs were recorded continuously, and subjective sleepiness ratings were taken at regular intervals throughout the drive.\ud \ud Results\ud After a normal night’s sleep, OPs and controls did not differ in terms of driving performance or in their ability to assess the levels of their own sleepiness, with both groups driving ‘safely’ for approximately 90 min. However, after sleep restriction, OPs had a significantly shorter (65 min) safe driving time and had to apply more compensatory effort to maintain their alertness compared with controls. They also underestimated the enhanced sleepiness. Nevertheless, apart from this caveat, there were generally close associations between subjective sleepiness, likelihood of a major lane deviation and EEG changes indicative of sleepiness.\ud \ud Conclusions\ud With a normal night’s sleep, effectively treated older men with OSA drive as safely as healthy men of the same age. However, after restricted sleep, driving impairment is worse than that of controls. This suggests that, although successful CPAP treatment can alleviate potential detrimental effects of OSA on monotonous driving following normal sleep, these patients remain more vulnerable to sleep restriction

Beginning teachers' conceptual understandings of effective history teaching: examining the change from 'subject knowers' to 'subject teachers'

This article reports the investigation of change in preservice teachers' conceptions of effective history teaching across a secondary History methods course in a postgraduate diploma of education program. Using concept mapping to plot shifts in their expressed reflections, data were obtained that indicate personal constructs of effective history teaching based around participants' map structures, curriculum understandings, knowledge of subject matter, knowledge of learners, knowledge of pedagogies, and pedagogical content knowledge. The purpose of this article is twofold: to present the findings of research exploring the growth in conceptual understandings of early career history teachers promoted through the use of; and to discuss the participants' response to tangible opportunities to reflect on their own conceptual understandings of history teaching

One night's CPAP withdrawal in otherwise compliant OSA patients: marked driving impairment but good awareness of increased sleepiness

Purpose Obstructive sleep apnoea (OSA) patients effectively treated by and compliant with continuous positive air pressure (CPAP) occasionally miss a night’s treatment. The purpose of this study was to use a real car interactive driving simulator to assess the effects of such an occurrence on the next day’s driving, including the extent to which these drivers are aware of increased sleepiness. Methods Eleven long-term compliant CPAP-treated 50–75-year-old male OSA participants completed a 2-h afternoon, simulated, realistic monotonous drive in an instrumented car, twice, following one night: (1) normal sleep with CPAP and (2) nil CPAP. Drifting out of road lane (‘incidents’), subjective sleepiness every 200 s and continuous electroencephalogram (EEG) activities indicative of sleepiness and compensatory effort were monitored. Results Withdrawal of CPAP markedly increased sleep disturbance and led to significantly more incidents, a shorter ‘safe’ driving duration, increased alpha and theta EEG power and greater subjective sleepiness. However, increased EEG beta activity indicated that more compensatory effort was being applied. Importantly, under both conditions, there was a highly significant correlation between subjective and EEG measures of sleepiness, to the extent that participants were well aware of the effects of nil CPAP. Conclusions Patients should be aware that compliance with treatment every night is crucial for safe driving