Tunable phenotypic variability through an autoregulatory alternative sigma factor circuit.

Genetically identical individuals in bacterial populations can display significant phenotypic variability. This variability can be functional, for example by allowing a fraction of stress prepared cells to survive an otherwise lethal stress. The optimal fraction of stress prepared cells depends on environmental conditions. However, how bacterial populations modulate their level of phenotypic variability remains unclear. Here we show that the alternative sigma factor σV circuit in Bacillus subtilis generates functional phenotypic variability that can be tuned by stress level, environmental history and genetic perturbations. Using single-cell time-lapse microscopy and microfluidics, we find the fraction of cells that immediately activate σV under lysozyme stress depends on stress level and on a transcriptional memory of previous stress. Iteration between model and experiment reveals that this tunability can be explained by the autoregulatory feedback structure of the sigV operon. As predicted by the model, genetic perturbations to the operon also modulate the response variability. The conserved sigma-anti-sigma autoregulation motif is thus a simple mechanism for bacterial populations to modulate their heterogeneity based on their environment

Escherichia coli can survive stress by noisy growth modulation.

Gene expression can be noisy, as can the growth of single cells. Such cell-to-cell variation has been implicated in survival strategies for bacterial populations. However, it remains unclear how single cells couple gene expression with growth to implement these strategies. Here, we show how noisy expression of a key stress-response regulator, RpoS, allows E. coli to modulate its growth dynamics to survive future adverse environments. We reveal a dynamic positive feedback loop between RpoS and growth rate that produces multi-generation RpoS pulses. We do so experimentally using single-cell, time-lapse microscopy and microfluidics and theoretically with a stochastic model. Next, we demonstrate that E. coli prepares for sudden stress by entering prolonged periods of slow growth mediated by RpoS. This dynamic phenotype is captured by the RpoS-growth feedback model. Our synthesis of noisy gene expression, growth, and survival paves the way for further exploration of functional phenotypic variability

Gelungene Versöhnung - unsichere Zukunft? Zur Raison d'être der deutsch-französischen Beziehungen in der neuen Europäischen Union

"Die Frage nach der Notwendigkeit und Legitimität der deutsch-französischen Sonderbeziehung im Europa der 25 stellt sich nach der Ablehnung der Europäischen Verfassung in Frankreich und den Niederlanden sowie dem vorläufigen Scheitern der EU-Finanzverhandlungen umso dringlicher. Mit der Rolle und Perzeption der deutsch-französischen Zusammenarbeit in der erweiterten Europäischen Union setzte sich im Juni ein internationales Kolloquium mit dem Titel 'Gelungene Versöhnung – unsichere Zukunft?' in Erinnerung an Joseph Rovan auseinander, dessen Tagungsergebnisse das Dossier dokumentiert. Neben Beiträgen zur Bewertung der aktuellen Krise und den Herausforderungen an Deutschland und Frankreich, analysieren (weitere Beiträge) den Blick der anderen Mitgliedstaaten auf das 'couple'." (Autorenreferat

The Boston criteria version 2.0 for cerebral amyloid angiopathy:a multicentre, retrospective, MRI–neuropathology diagnostic accuracy study

BACKGROUND: Cerebral amyloid angiopathy (CAA) is an age-related small vessel disease, characterised pathologically by progressive deposition of amyloid β in the cerebrovascular wall. The Boston criteria are used worldwide for the in-vivo diagnosis of CAA but have not been updated since 2010, before the emergence of additional MRI markers. We report an international collaborative study aiming to update and externally validate the Boston diagnostic criteria across the full spectrum of clinical CAA presentations. METHODS: In this multicentre, hospital-based, retrospective, MRI and neuropathology diagnostic accuracy study, we did a retrospective analysis of clinical, radiological, and histopathological data available to sites participating in the International CAA Association to formulate updated Boston criteria and establish their diagnostic accuracy across different populations and clinical presentations. Ten North American and European academic medical centres identified patients aged 50 years and older with potential CAA-related clinical presentations (ie, spontaneous intracerebral haemorrhage, cognitive impairment, or transient focal neurological episodes), available brain MRI, and histopathological assessment for CAA diagnosis. MRI scans were centrally rated at Massachusetts General Hospital (Boston, MA, USA) for haemorrhagic and non-haemorrhagic CAA markers, and brain tissue samples were rated by neuropathologists at the contributing sites. We derived the Boston criteria version 2.0 (v2.0) by selecting MRI features to optimise diagnostic specificity and sensitivity in a prespecified derivation cohort (Boston cases 1994-2012, n=159), then externally validated the criteria in a prespecified temporal validation cohort (Boston cases 2012-18, n=59) and a geographical validation cohort (non-Boston cases 2004-18; n=123), comparing accuracy of the new criteria to the currently used modified Boston criteria with histopathological assessment of CAA as the diagnostic standard. We also assessed performance of the v2.0 criteria in patients across all cohorts who had the diagnostic gold standard of brain autopsy. FINDINGS: The study protocol was finalised on Jan 15, 2017, patient identification was completed on Dec 31, 2018, and imaging analyses were completed on Sept 30, 2019. Of 401 potentially eligible patients presenting to Massachusetts General Hospital, 218 were eligible to be included in the analysis; of 160 patient datasets from other centres, 123 were included. Using the derivation cohort, we derived provisional criteria for probable CAA requiring the presence of at least two strictly lobar haemorrhagic lesions (ie, intracerebral haemorrhages, cerebral microbleeds, or foci of cortical superficial siderosis) or at least one strictly lobar haemorrhagic lesion and at least one white matter characteristic (ie, severe visible perivascular spaces in centrum semiovale or white matter hyperintensities in a multispot pattern). The sensitivity and specificity of these criteria were 74·8% (95% CI 65·4-82·7) and 84·6% (71·9-93·1) in the derivation cohort, 92·5% (79·6-98·4) and 89·5% (66·9-98·7) in the temporal validation cohort, 80·2% (70·8-87·6) and 81·5% (61·9-93·7) in the geographical validation cohort, and 74·5% (65·4-82·4) and 95·0% (83·1-99·4) in all patients who had autopsy as the diagnostic standard. The area under the receiver operating characteristic curve (AUC) was 0·797 (0·732-0·861) in the derivation cohort, 0·910 (0·828-0·992) in the temporal validation cohort, 0·808 (0·724-0·893) in the geographical validation cohort, and 0·848 (0·794-0·901) in patients who had autopsy as the diagnostic standard. The v2.0 Boston criteria for probable CAA had superior accuracy to the current Boston criteria (sensitivity 64·5% [54·9-73·4]; specificity 95·0% [83·1-99·4]; AUC 0·798 [0·741-0854]; p=0·0005 for comparison of AUC) across all individuals who had autopsy as the diagnostic standard. INTERPRETATION: The Boston criteria v2.0 incorporate emerging MRI markers of CAA to enhance sensitivity without compromising their specificity in our cohorts of patients aged 50 years and older presenting with spontaneous intracerebral haemorrhage, cognitive impairment, or transient focal neurological episodes. Future studies will be needed to determine generalisability of the v.2.0 criteria across the full range of patients and clinical presentations. FUNDING: US National Institutes of Health (R01 AG26484)

Investigation of Bacillus subtilis Sigma Factor Dynamics Using Improved Single Cell Tools

Bacteria can quickly adapt to changing environmental conditions by activating alternative sigma factors. It has been shown previously that single cell approaches can reveal hidden dynamics in sigma factor activation. Here, we investigate the single cell response dynamics of the \textit{B. subtilis} extracytoplasmic function sigma factors, which are an important part of the cell envelope stress response, under their specific stresses. To do this we use transcriptional reporters of sigma factors, quantitative single cell snapshots, time-lapse microscopy, and microfluidics.\\ By developing an improved microfluidics setup for single cell time-lapse microscopy, as well as improved single cell analysis code, we are able to observe new sigma factor dynamics. First, we observe heterogeneous entry into a higher $\sigma^{V}$ activity state in response to lysozyme, which displays a memory, as the heterogeneity is lost on removal and reapplication of the stress. Next, we observe a pulse amplitude and duration modulated sigma factor response of $\sigma^{M}$ to bacitracin. Finally, for $\sigma^{M}$ under ethanol and acidic stress, and for $\sigma^{Y}$ under ethanol stress, we observe a noisy increase in activity to a new steady state level, where the degree of variability between cells depends on the stress condition.\\ This thesis also discusses efforts on building a single cell microfluidic device based on the ”mother machine” design, for the rod-shaped cyanobacterium, \textit{S. elongatus}, which forces the cells to grow in a straight line. Growing this organism in a traditional mother machine device has, so far, proved challenging. To adapt the mother machine for cyanobacteria we modify the channel geometry using electron beam lithography, and improve the loading protocol.\\ The research presented here reveals the range of regulatory dynamics possible for ECF sigma factors in \textit{B. subtilis}, and provides improved microfluidics and analysis code that will enable easier quantification of bacterial gene circuits at the single cell level in the future

A Probabilistic Vehicle Diagnostic System Using Multiple Models

In addition to being accurate, it is important that diagnostic systems for use in automobiles also have low development and hardware costs. Model-based methods have shown promise at reducing hardware costs since they use analytical redundancy to reduce physical redundancy. In addition to requiring no extra sensors, the diagnostic system presented in this paper also allows for high accuracy and low development costs by using information from multiple simple models. This is made possible by the use of a Bayesian network to process model residuals. A hybrid, dynamic Bayesian network is used to model the temporal behavior of the faults and determine fault probabilities. A prototype of the system has been implemented and tested on a Mercedes-Benz E320 sedan. This paper describes the prototype system and presents results demonstrating the system’s advantages over traditional residual threshold techniques

Estimating reliability: A comparison of Cronbach's α, McDonald's ωt and the greatest lower bound

Cronbach's alpha as a reliability estimate for a set of items has been repeatedly criticised. Using McDonald's omega or the Greatest Lower Bound (GLB) has been suggested instead. A simulation study using a single scale was performed on data with n = 60, 80, 120, 200, 300, 500 and 900 cases utilizing 4, 8, 16 or 32 items. A total of 10% of each item had missing values vs. there were no missing data. Skewness of the items were 0, 1 and 2. Correlations were homogenous vs. heterogeneous. Standard deviations were homogenous vs. heterogeneous. Underlying constructs were homogenous vs. heterogenous. Conditions were combined in a block design, and 1000 replications for each one were calculated. As a result, alpha slightly underestimated reliability in some cases, omega minimally overestimated in large samples, the GLB overestimated strongly in small samples. Additionally, GLB estimates had less precision within the 1000 replications, i.e., standard deviations were almost double as large as for alpha and omega. Interestingly, the performances of alpha and omega were basically similar. However, various programs showed different results particularly in cases with missing data on the items. Out of the programs examined, the R package MBESS displayed best results