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

Mechanisms of Cognitive Impairment in Cerebral Small Vessel Disease: Multimodal MRI Results from the St George's Cognition and Neuroimaging in Stroke (SCANS) Study.

Cerebral small vessel disease (SVD) is a common cause of vascular cognitive impairment. A number of disease features can be assessed on MRI including lacunar infarcts, T2 lesion volume, brain atrophy, and cerebral microbleeds. In addition, diffusion tensor imaging (DTI) is sensitive to disruption of white matter ultrastructure, and recently it has been suggested that additional information on the pattern of damage may be obtained from axial diffusivity, a proposed marker of axonal damage, and radial diffusivity, an indicator of demyelination. We determined the contribution of these whole brain MRI markers to cognitive impairment in SVD. Consecutive patients with lacunar stroke and confluent leukoaraiosis were recruited into the ongoing SCANS study of cognitive impairment in SVD (n = 115), and underwent neuropsychological assessment and multimodal MRI. SVD subjects displayed poor performance on tests of executive function and processing speed. In the SVD group brain volume was lower, white matter hyperintensity volume higher and all diffusion characteristics differed significantly from control subjects (n = 50). On multi-predictor analysis independent predictors of executive function in SVD were lacunar infarct count and diffusivity of normal appearing white matter on DTI. Independent predictors of processing speed were lacunar infarct count and brain atrophy. Radial diffusivity was a stronger DTI predictor than axial diffusivity, suggesting ischaemic demyelination, seen neuropathologically in SVD, may be an important predictor of cognitive impairment in SVD. Our study provides information on the mechanism of cognitive impairment in SVD

Influence of real-world characteristics on outcomes for patients with methicillin-resistant Staphylococcal skin and soft tissue infections:a multi-country medical chart review in Europe

BACKGROUND: Patient-related (demographic/disease) and treatment-related (drug/clinician/hospital) characteristics were evaluated as potential predictors of healthcare resource use and opportunities for early switch (ES) from intravenous (IV)-to-oral methicillin-resistant Staphylococcus aureus (MRSA)-active antibiotic therapy and early hospital discharge (ED). METHODS: This retrospective observational medical chart study analyzed patients (across 12 European countries) with microbiologically confirmed MRSA complicated skin and soft tissue infections (cSSTI), ≥3 days of IV anti-MRSA antibiotics during hospitalization (July 1, 2010-June 30, 2011), and discharged alive by July 31, 2011. Logistic/linear regression models evaluated characteristics potentially associated with actual resource use (length of IV therapy, length of hospital stay [LOS], IV-to-oral antibiotic switch), and ES and ED (using literature-based and expert-verified criteria) outcomes. RESULTS: 1542 patients (mean ± SD age 60.8 ± 16.5 years; 61.5% males) were assessed with 81.0% hospitalized for MRSA cSSTI as the primary reason. Several patient demographic, infection, complication, treatment, and hospital characteristics were predictive of length of IV therapy, LOS, IV-to-oral antibiotic switch, or ES and ED opportunities. Outcomes and ES and ED opportunities varied across countries. Length of IV therapy and LOS (r = 0.66, p < 0.0001) and eligibilities for ES and ED (r = 0.44, p < 0.0001) showed relatively strong correlations. IV-to-oral antibiotic switch patients had significantly shorter length of IV therapy (−5.19 days, p < 0.001) and non-significantly shorter LOS (−1.86 days, p > 0.05). Certain patient and treatment characteristics were associated with increased odds of ES (healthcare-associated/ hospital-acquired infection) and ED (patient living arrangements, healthcare-associated/ hospital-acquired infection, initiating MRSA-active treatment 1–2 days post cSSTI index date, existing ED protocol), while other factors decreased the odds of ES (no documented MRSA culture, ≥4 days from admission to cSSTI index date, IV-to-oral switch, IV line infection) and ED (dementia, no documented MRSA culture, initiating MRSA-active treatment ≥3 days post cSSTI index date, existing ES protocol). CONCLUSIONS: Practice patterns and opportunity for further ES and ED were affected by several infection, treatment, hospital, and geographical characteristics, which should be considered in identifying ES and ED opportunities and designing interventions for MRSA cSSTI to reduce IV days and LOS while maintaining the quality of care. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2334-14-476) contains supplementary material, which is available to authorized users

A trial platform to develop a tailored theory-based intervention to improve professional practice in the disclosure of a diagnosis of dementia: Study protocol [ISRCTN15871014]

BACKGROUND: For people with dementia, care should include an explanation of the diagnosis to individuals and their carers, and information about the likely prognosis and possible packages of care. However, this is neither routine nor inevitable, and there is wide variation in the practice of disclosure. The aim of this study is to develop a tailored theory-based intervention to promote appropriate disclosure of diagnosis of dementia. METHODS: There are three objectives. Objective 1 is to define and develop an appropriate model of disclosure; this will be addressed using a multidisciplinary consensus development process. Objective 2 is to identify factors that influence disclosure of diagnosis; a questionnaire based upon theoretical constructs from a range of behavioural theories will be developed and members of old age mental health teams will be surveyed. The analysis will identify those factors that best predict intention to disclose a diagnosis to a person with dementia. Objective 3 is to develop and pilot test a theory-based intervention to promote disclosure of diagnosis that targets attitudes, beliefs and actions most amenable to change. Objective 3 will use the results of Objectives 1&2 to design and pilot test an intervention to improve the process of and increase the proportion of individuals receiving a diagnosis of dementia, for members of old age mental health teams. This work will lead to a proposal for a randomised controlled trial of the intervention

Evolution of viral variants in remdesivir-treated and untreated SARS-CoV-2-infected pediatrics patients

Detailed information on intrahost viral evolution in SARS-CoV-2 with and without treatment is limited. Sequential viral loads and deep sequencing of SARS-CoV-2 from the upper respiratory tract of nine hospitalized children, three of whom were treated with remdesivir, revealed that remdesivir treatment suppressed viral load in one patient but not in a second infected with an identical strain without any evidence of drug resistance found. Reduced levels of subgenomic RNA during treatment of the second patient, suggest an additional effect of remdesivir on viral replication. Haplotype reconstruction uncovered persistent SARS-CoV-2 variant genotypes in four patients. These likely arose from within-host evolution, although superinfection cannot be excluded in one case. Although our dataset is small, observed sample-to-sample heterogeneity in variant frequencies across four of nine patients suggests the presence of discrete viral populations in the lung with incomplete population sampling in diagnostic swabs. Such compartmentalization could compromise the penetration of remdesivir into the lung, limiting the drugs in vivo efficacy, as has been observed in other lung infections

Augmented visual feedback of movement performance to enhance walking recovery after stroke : study protocol for a pilot randomised controlled trial

Increasing evidence suggests that use of augmented visual feedback could be a useful approach to stroke rehabilitation. In current clinical practice, visual feedback of movement performance is often limited to the use of mirrors or video. However, neither approach is optimal since cognitive and self-image issues can distract or distress patients and their movement can be obscured by clothing or limited viewpoints. Three-dimensional motion capture has the potential to provide accurate kinematic data required for objective assessment and feedback in the clinical environment. However, such data are currently presented in numerical or graphical format, which is often impractical in a clinical setting. Our hypothesis is that presenting this kinematic data using bespoke visualisation software, which is tailored for gait rehabilitation after stroke, will provide a means whereby feedback of movement performance can be communicated in a more meaningful way to patients. This will result in increased patient understanding of their rehabilitation and will enable progress to be tracked in a more accessible way. The hypothesis will be assessed using an exploratory (phase II) randomised controlled trial. Stroke survivors eligible for this trial will be in the subacute stage of stroke and have impaired walking ability (Functional Ambulation Classification of 1 or more). Participants (n = 45) will be randomised into three groups to compare the use of the visualisation software during overground physical therapy gait training against an intensity-matched and attention-matched placebo group and a usual care control group. The primary outcome measure will be walking speed. Secondary measures will be Functional Ambulation Category, Timed Up and Go, Rivermead Visual Gait Assessment, Stroke Impact Scale-16 and spatiotemporal parameters associated with walking. Additional qualitative measures will be used to assess the participant's experience of the visual feedback provided in the study. Results from the trial will explore whether the early provision of visual feedback of biomechanical movement performance during gait rehabilitation demonstrates improved mobility outcomes after stroke and increased patient understanding of their rehabilitation

Quantifying the proportion of different cell types in the human cortex using DNA methylation profiles

This is the final version. Available from BMC via the DOI in this record. Availability of data and materials: All data generated or analysed during this study are included in this published article, its supplementary information files and publicly available repositories. Data generated for this project are available at NCBI Gene Express Omnibus (GEO) under accession number GSE234520 [64]. We also reanalysed data previously made available via GEO (via accession numbers GSE74193 [65], GSE59685 [66], GSE80970 [67], GSE88890 [68], GSE43414 [69]) and the synapse platform (syn7072866 [70], syn8263588 [71]). Code for the analyses presented here can be found on GitHub and Zenodo https://github.com/ejh243/BrainFANS/tree/master/array/DNAm/preprocessing (https://doi.org/https://doi.org/10.5281/zenodo.10402167). Specifically, code for the quality control of the DNAm data can be found at https://github.com/ejh243/BrainFANS/tree/master/array/DNAm/preprocessing and the code for the statistical analyses can be found at https://github.com/ejh243/BrainFANS/tree/master/array/DNAm/analysis/neuralCellComposition. Our new trained deconvolution models for brain are made available to the wider research community via our R package CETYGO available on GitHub (https://github.com/ds420/CETYGO; https://doi.org/10.5281/zenodo.10418430).Background: Due to interindividual variation in the cellular composition of the human cortex, it is essential that covariates that capture these differences are included in epigenome-wide association studies using bulk tissue. As experimentally derived cell counts are often unavailable, computational solutions have been adopted to estimate the proportion of different cell types using DNA methylation data. Here, we validate and profile the use of an expanded reference DNA methylation dataset incorporating two neuronal and three glial cell subtypes for quantifying the cellular composition of the human cortex. Results: We tested eight reference panels containing different combinations of neuronal- and glial cell types and characterised their performance in deconvoluting cell proportions from computationally reconstructed or empirically derived human cortex DNA methylation data. Our analyses demonstrate that while these novel brain deconvolution models produce accurate estimates of cellular proportions from profiles generated on postnatal human cortex samples, they are not appropriate for the use in prenatal cortex or cerebellum tissue samples. Applying our models to an extensive collection of empirical datasets, we show that glial cells are twice as abundant as neuronal cells in the human cortex and identify significant associations between increased Alzheimer’s disease neuropathology and the proportion of specific cell types including a decrease in NeuNNeg/SOX10Neg nuclei and an increase of NeuNNeg/SOX10Pos nuclei. Conclusions: Our novel deconvolution models produce accurate estimates for cell proportions in the human cortex. These models are available as a resource to the community enabling the control of cellular heterogeneity in epigenetic studies of brain disorders performed on bulk cortex tissue.Engineering and Physical Sciences Research CouncilMedical Research CouncilAlzheimer's Research UKMedical Research Counci

The Functional DRD3 Ser9Gly Polymorphism (rs6280) Is Pleiotropic, Affecting Reward as Well as Movement

Abnormalities of motivation and behavior in the context of reward are a fundamental component of addiction and mood disorders. Here we test the effect of a functional missense mutation in the dopamine 3 receptor (DRD3) gene (ser9gly, rs6280) on reward-associated dopamine (DA) release in the striatum. Twenty-six healthy controls (HCs) and 10 unmedicated subjects with major depressive disorder (MDD) completed two positron emission tomography (PET) scans with [11C]raclopride using the bolus plus constant infusion method. On one occasion subjects completed a sensorimotor task (control condition) and on another occasion subjects completed a gambling task (reward condition). A linear regression analysis controlling for age, sex, diagnosis, and self-reported anhedonia indicated that during receipt of unpredictable monetary reward the glycine allele was associated with a greater reduction in D2/3 receptor binding (i.e., increased reward-related DA release) in the middle (anterior) caudate (p<0.01) and the ventral striatum (p<0.05). The possible functional effect of the ser9gly polymorphism on DA release is consistent with previous work demonstrating that the glycine allele yields D3 autoreceptors that have a higher affinity for DA and display more robust intracellular signaling. Preclinical evidence indicates that chronic stress and aversive stimulation induce activation of the DA system, raising the possibility that the glycine allele, by virtue of its facilitatory effect on striatal DA release, increases susceptibility to hyperdopaminergic responses that have previously been associated with stress, addiction, and psychosis

Model-based parametric study of frontostriatal abnormalities in schizophrenia patients

<p>Abstract</p> <p>Background</p> <p>Several studies have suggested that the activity of the prefrontal cortex (PFC) and the dopamine (DA) release in the striatum has an inverse relationship. One would attribute this relationship primarily to the circuitry comprised of the glutamatergic projection from the PFC to the striatum and the GABAergic projection from the striatum to the midbrain DA nucleus. However, this circuitry has not characterized satisfactorily yet, so that no quantitative analysis has ever been made on the activities of the PFC and the striatum and also the DA release in the striatum.</p> <p>Methods</p> <p>In this study, a system dynamics model of the corticostriatal system with dopaminergic innervations is constructed to describe the relationships between the activities of the PFC and the striatum and the DA release in the striatum. By taking published receptor imaging data from schizophrenia patients and healthy subjects into this model, this article analyzes the effects of striatal D2 receptor activation on the balance of the activity and neurotransmission in the frontostriatal system of schizophrenic patients in comparison with healthy controls.</p> <p>Results</p> <p>The model predicts that the suppressive effect by D2 receptors at the terminals of the glutamatergic afferents to the striatum from the PFC enhances the hypofrontality-induced elevation of striatal DA release by at most 83%. The occupancy-based estimation of the 'optimum' D2 receptor occupancy by antipsychotic drugs is 52%. This study further predicts that patients with lower PFC activity tend to have greater improvement of positive symptoms following antipsychotic medication.</p> <p>Conclusion</p> <p>This model-based parametric study would be useful for system-level analysis of the brains with psychiatric diseases. It will be able to make reliable prediction of clinical outcome when sufficient data will be available.</p