The regulation of peptidoglycan hydrolysis in Escherichia coli

PhD ThesisThe bacterial cell envelope heteropolymer, peptidoglycan (PG), is essential for maintaining the osmotic stability and shape of most bacteria. PG biosynthesis is the target of our most successful antibiotics, the β-lactams and glycopeptides. However, the spread of antibiotic resistant strains highlights the need for novel antibiotic targets. Gram-negative bacteria possess a mainly single layered PG, which is enlarged in growing and dividing bacteria by the coordinated action of PG synthases and hydrolases. PG synthesis in Gram-negative bacteria is regulated from the cytoplasmic membrane (CM), by prokaryotic cytoskeletal elements, and from the outer membrane (OM) by the lipoproteins, LpoA and LpoB. LpoA/B interact with, and are essential for the in vivo activity of, the major PG synthases PBP1A and PBP1B, respectively. While the regulation of PG synthesis has been well studied in recent years, the mechanisms of PG hydrolysis regulation in E. coli remain poorly understood. E. coli possesses ~30 PG hydrolases with relatively few known regulators. In this work, we have structurally characterised LpoA from E. coli using nuclear magnetic resonance (NMR) spectroscopy of the N-terminal domain and use this to further the understanding of the in vitro and in vivo interaction of LpoA/PBP1A. We also studied PBP1A and LpoA in Haemophilus influenzae; in this species LpoA is essential. In a search for novel LpoA interaction partners we discovered the in vitro and in vivo interaction with the PG hydrolase, PBP4 and show that PBP4 also interacts with PBP1A. Subsequently, we optimised a process for the rapid identification of in vitro interactions and identified >20 interactions between PG synthases, PG hydrolases and other cell envelope proteins. We therefore present a putative PG hydrolysing complex with direct associations to the PG synthesis machinery. Through direct functional interactions with at least five PG hydrolases, we present the characterisation of the OM-anchored lipoprotein NlpI, of currently unknown cellular function, as a regulator of hydrolase activity. We show the in vitro regulation of activity by NlpI and the in vivo relevance of these interactions using a β-lactamase induction assay. This work significantly enhances our understanding of how PG synthesis and hydrolysis are coordinated as multi-enzyme complexes and presents the characterisation of a novel regulator of hydrolase activity, NlpI

Effects of Hurricane-Felled Tree Trunks on Soil Carbon, Nitrogen, Microbial Biomass, and Root Length in a Wet Tropical Forest

Decaying coarse woody debris can affect the underlying soil either by augmenting nutrients that can be exploited by tree roots, or by diminishing nutrient availability through stimulation of microbial nutrient immobilization. We analyzed C, N, microbial biomass C and root length in closely paired soil samples taken under versus 20–50 cm away from large trunks of two species felled by Hugo (1989) and Georges (1998) three times during wet and dry seasons over the two years following the study conducted by Georges. Soil microbial biomass, % C and % N were significantly higher under than away from logs felled by both hurricanes (i.e., 1989 and 1998), at all sampling times and at both depths (0–10 and 10–20 cm). Frass from wood boring beetles may contribute to early effects. Root length was greater away from logs during the dry season, and under logs in the wet season. Root length was correlated with microbial biomass C, soil N and soil moisture (R = 0.36, 0.18, and 0.27, respectively; all p values < 0.05). Microbial biomass C varied significantly among seasons but differences between positions (under vs. away) were only suggestive. Microbial C was correlated with soil N (R = 0.35). Surface soil on the upslope side of the logs had significantly more N and microbial biomass, likely from accumulation of leaf litter above the logs on steep slopes. We conclude that decaying wood can provide ephemeral resources that are exploited by tree roots during some seasons

Predicting Invasion Risk Using Measures of Introduction Effort and Environmental Niche Models

The Chinese mitten crab (Eriocheir sinensis) is native to east Asia, is established throughout Europe, and is introduced but geographically restricted in North America. We developed and compared two separate environmental niche models using genetic algorithm for rule set prediction (GARP) and mitten crab occurrences in Asia and Europe to predict the species\u27 potential distribution in North America. Since mitten crabs must reproduce in water with ≥15‰ salinity, we limited the potential North American range to freshwater habitats within the highest documented dispersal distance (1260 km) and a more restricted dispersal limit (354 km) from the sea. Applying the higher dispersal distance, both models predicted the lower Great Lakes, most of the eastern seaboard, the Gulf of Mexico and southern extent of the Mississippi River watershed, and the Pacific northwest as suitable environment for mitten crabs, but environmental match for southern states (below 35° N) was much lower for the European model. Use of the lower range with both models reduced the expected range, especially in the Great Lakes, Mississippi drainage, and inland areas of the Pacific Northwest. To estimate the risk of introduction of mitten crabs, the amount of reported ballast water discharge into major United States ports from regions in Asia and Europe with established mitten crab populations was used as an index of introduction effort. Relative risk of invasion was estimated based on a combination of environmental match and volume of unexchanged ballast water received (July 1999–December 2003) for major ports. The ports of Norfolk and Baltimore were most vulnerable to invasion and establishment, making Chesapeake Bay the most likely location to be invaded by mitten crabs in the United States. The next highest risk was predicted for Portland, Oregon. Interestingly, the port of Los Angeles/Long Beach, which has a large shipping volume, had a low risk of invasion. Ports such as Jacksonville, Florida, had a medium risk owing to small shipping volume but high environmental match. This study illustrates that the combination of environmental niche- and vector-based models can provide managers with more precise estimates of invasion risk than can either of these approaches alone

A guide to behavioural experiments in bipolar disorder

Behavioural experiments are an important component of cognitive-behavioural therapy. However, there exists little up-to-date guidance on how to conduct these in people with a diagnosis of bipolar disorder. This paper provides recommendations on how to conduct behavioural experiments in this population. The aim is to upskill and empower clinicians to conduct behavioural experiments. The paper combines the expertise of senior clinicians working in the United Kingdom. The article starts by providing general advice on conducting behavioural experiments in people with bipolar disorder. It then offers specific examples of behavioural experiments targeting cognitions around the uncontrollability and danger of affective states, and related behavioural strategies, which have been implicated in the maintenance of bipolar mood swings. The article finishes by providing examples of behavioural experiments for non-mood related difficulties that commonly occur with bipolar experiences including perfectionistic thinking, need for approval, and intrusive memories. Behavioural experiments offer a useful therapeutic technique for instigating cognitive and behavioural change in bipolar disorder. Conducted sensitively and collaboratively, in line with people's recovery-focused goals, behavioural experiments can be used to overcome mood- and non-mood related difficulties

Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20–30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication

Aortic haemodynamics: the effects of habitual endurance exercise, age and muscle sympathetic vasomotor outflow in healthy men

PURPOSE: We determined the effect of habitual endurance exercise and age on aortic pulse wave velocity (aPWV), augmentation pressure (AP) and systolic blood pressure (aSBP), with statistical adjustments of aPWV and AP for heart rate and aortic mean arterial pressure, when appropriate. Furthermore, we assessed whether muscle sympathetic nerve activity (MSNA) correlates with AP in young and middle-aged men. METHODS: Aortic PWV, AP, aortic blood pressure (applanation tonometry; SphygmoCor) and MSNA (peroneal microneurography) were recorded in 46 normotensive men who were either young or middle-aged and endurance-trained runners or recreationally active nonrunners (10 nonrunners and 13 runners within each age-group). Between-group differences and relationships between variables were assessed via ANOVA/ANCOVA and Pearson product-moment correlation coefficients, respectively. RESULTS: Adjusted aPWV and adjusted AP were similar between runners and nonrunners in both age groups (all, P > 0.05), but higher with age (all, P < 0.001), with a greater effect size for the age-related difference in AP in runners (Hedges’ g, 3.6 vs 2.6). aSBP was lower in young (P = 0.009; g = 2.6), but not middle-aged (P = 0.341; g = 1.1), runners compared to nonrunners. MSNA burst frequency did not correlate with AP in either age group (young: r = 0.00, P = 0.994; middle-aged: r = − 0.11, P = 0.604). CONCLUSION: There is an age-dependent effect of habitual exercise on aortic haemodynamics, with lower aSBP in young runners compared to nonrunners only. Statistical adjustment of aPWV and AP markedly influenced the outcomes of this study, highlighting the importance of performing these analyses. Further, peripheral sympathetic vasomotor outflow and AP were not correlated in young or middle-aged normotensive men

Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20–30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication

Eurasian watermilfoil fitness loss and invasion potential following desiccation during simulated overland transport

Abstract Vegetative reproduction promotes human-mediated dispersal of aquatic invasive plants as fragments &quot;hitchhike&quot; between water bodies on boats and trailers. However, desiccation of plant fragments may also reduce fitness, decreasing the likelihood of fragment survival as transport distances increase. Current inter-lake invasive species spread models do not directly consider fitness loss due to desiccation and mechanical damage of the transport pathway. Here, we estimate survival as a function of desiccation exposure for Eurasian watermilfoil (Myriophyllum spicatum). Following desiccation treatments, we monitored survival and root formation of individual fragments and assessed the differences between treatments. Highest survival rates occurred for short (&lt; one hour) air exposures and coiled fragments with root production for the coiled treatment occurring in less than two weeks, irrespective of fragment length. In contrast, fragments that experienced desiccation for more than 24 hours had little risk of surviving. Our results emphasize the threat posed by same-day overland movements of boats from invaded to uninvaded waterways, and provide managers with a surveillance radius to inform delimitation surveys arising from the discovery of a new invasion

The influence of habitual endurance exercise on carotid artery strain and strain-rate in young and middle-aged men

Central arterial stiffness is an independent predictor of cardiovascular risk that can be modified by exercise training. However, conventional local measures of carotid artery stiffness display conflicting responses to habitual endurance exercise in young and older adults. 2D-Strain imaging of the common carotid artery (CCA) quantifies circumferential deformation (strain) of the arterial wall across the cardiac cycle, which is more sensitive at detecting age-related alterations in CCA stiffness than conventional methods. Therefore, the study was designed to examine the relationship between habitual endurance exercise (running) and CCA 2D-Strain parameters in young and middle-aged men. Short-axis ultrasound images of the CCA were obtained from 13 young nonrunners (23 years [95% CI: 21-26]), 19 young runners (24 [22-26]), 13 middle-aged nonrunners (54 [52-56]) and 19 middle-aged runners (56 [54-58]). Images were analysed for peak circumferential strain (PCS; magnitude of deformation) as well as systolic and diastolic strain-rate (S-SR and D-SR; deformation velocity) and group differences were examined via two-way ANOVA. PCS, S-SR and D-SR were attenuated in middle-aged males when compared to young men (all P ≤ 0.001). PCS and S-SR were elevated in young and middle-aged runners when compared to nonrunners (P = 0.002 and P =0.009 respectively), but no age*training status interaction was observed. In contrast, there was no influence of habitual running on D-SR. Habitual exercise is associated with comparable improvements in CCA 2D-Strain parameters in young and middle-aged men, but the age-related decline in PCS and S-SR may be more amenable to habitual endurance exercise than D-SR

The influence of barosensory vessel mechanics on the vascular sympathetic baroreflex: Insights into ageing and blood pressure homeostasis

Changes in the arterial baroreflex arc contribute to elevated sympathetic outflow and altered reflex control of blood pressure with human ageing. Utilizing ultrasound and sympathetic microneurography (muscle sympathetic nerve activity; MSNA) we investigated the relationships between aortic and carotid artery wall tension (indices of baroreceptor activation) and the vascular sympathetic baroreflex operating point (OP; MSNA burst incidence) in healthy, normotensive young (n = 27, 23 ± 3 years) and middle-aged men (n = 22, 55 ± 4 years). In young men, the OP was positively related to the magnitude and rate of unloading and time spent unloaded in the aortic artery (r = 0.56, 0.65 and 0.51, P = 0.02, 0.003 and 0.03), but not related to the magnitude or rate of unloading or time spent unloaded in the carotid artery (r = -0.32, -0.07 and 0.06, P = 0.25, 0.81 and 0.85). In contrast, in middle-aged men, the OP was not related to either the magnitude or rate of unloading or time spent unloaded in the aortic (r = 0.22, 0.21 and 0.27, P = 0.41, 0.43 and 031) or carotid artery (r = 0.48, 0.28 and -0.01, P = 0.06, 0.25 and 0.98). In conclusion, in young men, aortic unloading mechanics may play a role in determining the vascular sympathetic baroreflex OP. In contrast, in middle-aged men, barosensory vessel unloading mechanics do not appear to determine the vascular sympathetic baroreflex OP, and therefore do not contribute to age-related arterial baroreflex resetting and increased resting MSNA. KEYWORDS: muscle sympathetic nerve activity; barosensory vessel unloading mechanics; healthy ageing; sympathetic nervous system; baroreflex Page Break NEW AND NOTEWORTHY We assessed the influence of barosensory vessel mechanics (magnitude and rate of unloading and time spent unloaded) as a surrogate for baroreceptor unloading. In young men, aortic unloading mechanics are important in regulating the operating point of the vascular sympathetic baroreflex, whereas in middle-aged men, these arterial mechanics do not influence this operating point. The age-related increase in resting muscle sympathetic nerve activity does not appear to be driven by altered baroreceptor input from stiffer barosensory vessels