Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy.

This is the final version of the article. Available from BioMed Central via the DOI in this record.BACKGROUND: Clonal microbial populations often harbor rare phenotypic variants that are typically hidden within the majority of the remaining cells, but are crucial for the population's resilience to external perturbations. Persister and viable but non-culturable (VBNC) cells are two important clonal bacterial subpopulations that can survive antibiotic treatment. Both persister and VBNC cells pose a serious threat to human health. However, unlike persister cells, which quickly resume growth following drug removal, VBNC cells can remain non-growing for prolonged periods of time, thus eluding detection via traditional microbiological assays. Therefore, understanding the molecular mechanisms underlying the formation of VBNC cells requires the characterization of the clonal population with single-cell resolution. A combination of microfluidics, time-lapse microscopy, and fluorescent reporter strains offers the perfect platform for investigating individual cells while manipulating their environment. METHODS: Here, we report a novel single-cell approach to investigate VBNC cells. We perform drug treatment, bacterial culturing, and live/dead staining in series by using transcriptional reporter strains and novel adaptations to the mother machine technology. Since we track each cell throughout the experiment, we are able to quantify the size, morphology and fluorescence that each VBNC cell displayed before, during and after drug treatment. RESULTS: We show that VBNC cells are not dead or dying cells but share similar phenotypic features with persister cells, suggesting a link between these two subpopulations, at least in the Escherichia coli strain under investigation. We strengthen this link by demonstrating that, before drug treatment, both persister and VBNC cells can be distinguished from the remainder of the population by their lower fluorescence when using a reporter strain for tnaC, encoding the leader peptide of the tnaCAB operon responsible for tryptophan metabolism. CONCLUSION: Our data demonstrates the suitability of our approach for studying the physiology of non-growing cells in response to external perturbations. Our approach will allow the identification of novel biomarkers for the isolation of VBNC and persister cells and will open new opportunities to map the detailed biochemical makeup of these clonal subpopulations.This work was supported by a Royal Society Research Grant (RG140203), a Welcome Trust ISSF (WT097835/Z/11/Z) and a Start up Grant from the University of Exeter awarded to SP. AS acknowledges support from the BBSRC through a SWBio-DTP studentship (BB/M009122/1). JM was generously supported by a Wellcome Trust Institutional Strategic Support Award (WT097835MF). This work was partly supported by BBSRC award BB/1024631/1 to RWT

The Culture Environment Influences Both Gene Regulation and Phenotypic Heterogeneity in Escherichia coli

This is the final version of the article. Available from Frontiers Media via the DOI in this record.Microorganisms shape the composition of the medium they are growing in, which in turn has profound consequences on the reprogramming of the population gene-expression profile. In this paper, we investigate the progressive changes in pH and sugar availability in the medium of a growing Escherichia coli (E. coli) culture. We show how these changes have an effect on both the cellular heterogeneity within the microbial community and the gene-expression profile of the microbial population. We measure the changes in gene-expression as E. coli moves from lag, to exponential, and finally into stationary phase. We found that pathways linked to the changes in the medium composition such as ribosomal, tricarboxylic acid cycle (TCA), transport, and metabolism pathways are strongly regulated during the different growth phases. In order to quantify the corresponding temporal changes in the population heterogeneity, we measure the fraction of E. coli persisters surviving different antibiotic treatments during the various phases of growth. We show that the composition of the medium in which β-lactams or quinolones, but not aminoglycosides, are dissolved strongly affects the measured phenotypic heterogeneity within the culture. Our findings contribute to a better understanding on how the composition of the culture medium influences both the reprogramming in the population gene-expression and the emergence of phenotypic variants.This work was supported by a Royal Society Research Grant (RG140203), a Wellcome Trust Strategic Seed Corn Fund (WT097835/Z/11/Z), and a start up Grant from the University of Exeter awarded to SP. AS acknowledges support from the BBSRC through a SWBio-DTP studentship (BB/M009122/1). KP, KM, and PO would like to acknowledge support from the following awards: Wellcome Trust Institutional Strategic Support Fund (WT097835MF), Wellcome Trust Multi User Equipment Award (WT101650MA), and Medical Research Council Clinical Infrastructure Funding (MR/M008924/1). This work was partly supported by BBSRC award BB/1024631/1 to RT

Repetitive arm functional tasks after stroke (RAFTAS): a pilot randomised controlled trial

Background Repetitive functional task practise (RFTP) is a promising treatment to improve upper limb recovery following stroke. We report the findings of a study to determine the feasibility of a multi-centre randomised controlled trial to evaluate this intervention. Methods A pilot randomised controlled trial was conducted. Patients with new reduced upper limb function were recruited within 14 days of acute stroke from three stroke units in North East England. Participants were randomised to receive a four week upper limb RFTP therapy programme consisting of goal setting, independent activity practise, and twice weekly therapy reviews in addition to usual post stroke rehabilitation, or usual post stroke rehabilitation. The recruitment rate; adherence to the RFTP therapy programme; usual post stroke rehabilitation received; attrition rate; data quality; success of outcome assessor blinding; adverse events; and the views of study participants and therapists about the intervention were recorded. Results Fifty five eligible patients were identified, 4-6% of patients screened at each site. Twenty four patients participated in the pilot study. Two of the three study sites met the recruitment target of 1-2 participants per month. The median number of face to face therapy sessions received was 6 [IQR 3-8]. The median number of daily repetitions of activities recorded was 80 [IQR 39-80]. Data about usual post stroke rehabilitation were available for 18/24 (75%). Outcome data were available for 22/24 (92%) at one month and 20/24 (83%) at three months. Outcome assessors were unblinded to participant group allocation for 11/22 (50%) at one month and 6/20 (30%) at three months. Four adverse events were considered serious as they resulted in hospitalisation. None were related to study treatment. Feedback from patients and local NHS therapists about the RFTP programme was mainly positive. Conclusions A multi-centre randomised controlled trial to evaluate an upper limb RFTP therapy programme provided early after stroke is feasible and acceptable to patients and therapists, but there are issues which needed to be addressed when designing a Phase III study. A Phase III study will need to monitor and report not only recruitment and attrition but also adherence to the intervention, usual post stroke rehabilitation received, and outcome assessor blinding

Business experience and start-up size: buying more lottery tickets next time around?

This paper explores the determinants of start-up size by focusing on a cohort of 6247 businesses that started trading in 2004, using a unique dataset on customer records at Barclays Bank. Quantile regressions show that prior business experience is significantly related with start-up size, as are a number of other variables such as age, education and bank account activity. Quantile treatment effects (QTE) estimates show similar results, with the effect of business experience on (log) start-up size being roughly constant across the quantiles. Prior personal business experience leads to an increase in expected start-up size of about 50%. Instrumental variable QTE estimates are even higher, although there are concerns about the validity of the instrument

Medication Persistence Rates and Factors Associated with Persistence in Patients Following Stroke: A Cohort Study

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Study design and methods of the BoTULS trial: a randomised controlled trial to evaluate the clinical effect and cost effectiveness of treating upper limb spasticity due to stroke with botulinum toxin type A

Background Following a stroke, 55–75% of patients experience upper limb problems in the longer term. Upper limb spasticity may cause pain, deformity and reduced function, affecting mood and independence. Botulinum toxin is used increasingly to treat focal spasticity, but its impact on upper limb function after stroke is unclear. The aim of this study is to evaluate the clinical and cost effectiveness of botulinum toxin type A plus an upper limb therapy programme in the treatment of post stroke upper limb spasticity. Methods Trial design : A multi-centre open label parallel group randomised controlled trial and economic evaluation. Participants : Adults with upper limb spasticity at the shoulder, elbow, wrist or hand and reduced upper limb function due to stroke more than 1 month previously. Interventions : Botulinum toxin type A plus upper limb therapy (intervention group) or upper limb therapy alone (control group). Outcomes : Outcome assessments are undertaken at 1, 3 and 12 months. The primary outcome is upper limb function one month after study entry measured by the Action Research Arm Test (ARAT). Secondary outcomes include: spasticity (Modified Ashworth Scale); grip strength; dexterity (Nine Hole Peg Test); disability (Barthel Activities of Daily Living Index); quality of life (Stroke Impact Scale, Euroqol EQ-5D) and attainment of patient-selected goals (Canadian Occupational Performance Measure). Health and social services resource use, adverse events, use of other antispasticity treatments and patient views on the treatment will be compared. Participants are clinically reassessed at 3, 6 and 9 months to determine the need for repeat botulinum toxin type A and/or therapy. Randomisation : A web based central independent randomisation service. Blinding : Outcome assessments are undertaken by an assessor who is blinded to the randomisation group. Sample size : 332 participants provide 80% power to detect a 15% difference in treatment successes between intervention and control groups. Treatment success is defined as improvement of 3 points for those with a baseline ARAT of 0–3 and 6 points for those with ARAT of 4–56

Electron acceleration by wave turbulence in a magnetized plasma

Astrophysical shocks are commonly revealed by the non-thermal emission of energetic electrons accelerated in situ 1-3 . Strong shocks are expected to accelerate particles to very high energies 4-6 ; however, they require a source of particles with velocities fast enough to permit multiple shock crossings. While the resulting diffusive shock acceleration 4 process can account for observations, the kinetic physics regulating the continuous injection of non-thermal particles is not well understood. Indeed, this injection problem is particularly acute for electrons, which rely on high-frequency plasma fluctuations to raise them above the thermal pool 7,8 . Here we show, using laboratory laser-produced shock experiments, that, in the presence of a strong magnetic field, significant electron pre-heating is achieved. We demonstrate that the key mechanism in producing these energetic electrons is through the generation of lower-hybrid turbulence via shock-reflected ions. Our experimental results are analogous to many astrophysical systems, including the interaction of a comet with the solar wind 9 , a setting where electron acceleration via lower-hybrid waves is possible

Varieties of living things: Life at the intersection of lineage and metabolism

publication-status: Publishedtypes: Articl

Linking Hydrothermal Geochemistry to Organismal Physiology: Physiological Versatility in Riftia pachyptila from Sedimented and Basalt-hosted Vents

Much of what is known regarding Riftia pachyptila physiology is based on the wealth of studies of tubeworms living at diffuse flows along the fast-spreading, basalt-hosted East Pacific Rise (EPR). These studies have collectively suggested that Riftia pachyptila and its chemoautotrophic symbionts are physiologically specialized, highly productive associations relying on hydrogen sulfide and oxygen to generate energy for carbon fixation, and the symbiont's nitrate reduction to ammonia for energy and biosynthesis. However, Riftia also flourish in sediment-hosted vents, which are markedly different in geochemistry than basalt-hosted systems. Here we present data from shipboard physiological studies and global quantitative proteomic analyses of Riftia pachyptila trophosome tissue recovered from tubeworms residing in the EPR and the Guaymas basin, a sedimented, hydrothermal vent field. We observed marked differences in symbiont nitrogen metabolism in both the respirometric and proteomic data. The proteomic data further suggest that Riftia associations in Guaymas may utilize different sulfur compounds for energy generation, may have an increased capacity for energy storage, and may play a role in degrading exogenous organic carbon. Together these data reveal that Riftia symbionts are far more physiologically plastic than previously considered, and that -contrary to previous assertions- Riftia do assimilate reduced nitrogen in some habitats. These observations raise new hypotheses regarding adaptations to the geochemical diversity of habitats occupied by Riftia, and the degree to which the environment influences symbiont physiology and evolution