Single cell measurements of bacterial physiology traits during exposure to an external stress

The electrochemical gradient of protons, or proton motive force (PMF), is at the heart of bacterial energetics. It consists of two components: the pH difference between a cell cytoplasm and the environment, and the membrane potential. The PMF powers such vital cellular processes as ATP production, motility and active membrane transport. The aim of this doctoral project was to relate the changes in the physiological state of the cell to the PMF in a variety of stressful environments and, using this, uncover the mechanisms through which these stresses induce cellular damage. In this thesis I have shown that by modelling an Escherichia coli cell as an electric circuit, the relationship between bacterial PMF, the electric properties of the cell membrane and the catabolism can be described mathematically. Subsequently, using the bacterial flagellar motor (BFM) as a single-cell "voltmeter" I have quantitatively described the effects of different stresses on the maintenance of cellular free energy. To achieve my goal I developed an assay for simultaneous monitoring of the PMF and the intracellular pH by using a combination of fluorescence and back-focal-plane (BFP) interferometry techniques. I confirmed the accuracy of the proposed approach by applying it to a known stress — indole treatment — and recovering the previously shown functional dependency between indole concentration and the membrane conductance. I then tested a variety of different stresses and found that butanol acts as an ionophore changing membrane conductance linearly with concentration and functionally characterised membrane damage caused by light of shorter wavelengths. I further proved that this light damage was mediated by reactive oxygen species by repeating light damage experiments in anaerobic conditions. Anaerobic conditions were also used for studying an acid challenge response where I demonstrated that the presence of oxygen is required for the maintenance of the cytoplasmic pH. Additionally, in the course of my project I tested and characterised several pH indicators. I demonstrated that the cpYFP sensor, previously used in eukaryotic cells, could be successfully used in E. coli to allow the internal pH measurements in the higher pH range, while the pHRed sensor was shown to form the aggregates in the cell cytoplasm and, consequently, to slow down the growth. The optimal protocol of the pH sensors calibration was established iiand several calibration-related issues discussed. Finally, I revisited the experiments that demonstrate the PMF and BFM speed proportionality. I found that under high load the motor speed saturates with PMF disproving the currently accepted idea of the PMF-motor speed linear relationship holding irrespective of the motor load. I proposed the possible explanation of the observed phenomenon and discussed potential experiments that could test my hypotheses

Single-cell bacterial electrophysiology reveals mechanisms of stress-induced damage

Electrochemical gradient of protons, or proton motive force (PMF), is at the basis of bacterial energetics. It powers vital cellular processes and defines the physiological state of the cell. Here we use an electric circuit analogy of an Escherichia coli cell to mathematically describe the relationship between bacterial PMF, electric properties of the cell membrane and catabolism. We combine the analogy with the use of bacterial flagellar motor as a single-cell "voltmeter" to measure cellular PMF in varied and dynamic external environments, for example, under different stresses. We find that butanol acts as an ionophore, and functionally characterise membrane damage caused by the light of shorter wavelengths. Our approach coalesces non-invasive and fast single-cell voltmeter with a well-defined mathematical framework to enable quantitative bacterial electrophysiology

Active efflux leads to heterogeneous dissipation of proton motive force by protonophores in bacteria

ABSTRACT Various toxic compounds disrupt bacterial physiology. While bacteria harbor defense mechanisms to mitigate the toxicity, these mechanisms are often coupled to the physiological state of the cells and become ineffective when the physiology is severely disrupted. Here, we characterized such feedback by exposing Escherichia coli to protonophores. Protonophores dissipate the proton motive force (PMF), a fundamental force that drives physiological functions. We found that E. coli cells responded to protonophores heterogeneously, resulting in bimodal distributions of cell growth, substrate transport, and motility. Furthermore, we showed that this heterogeneous response required active efflux systems. The analysis of underlying interactions indicated the heterogeneous response results from efflux-mediated positive feedback between PMF and protonophores’ action. Our studies have broad implications for bacterial adaptation to stress, including antibiotics. IMPORTANCE An electrochemical proton gradient across the cytoplasmic membrane, alternatively known as proton motive force, energizes vital cellular processes in bacteria, including ATP synthesis, nutrient uptake, and cell division. Therefore, a wide range of organisms produce the agents that collapse the proton motive force, protonophores, to gain a competitive advantage. Studies have shown that protonophores have significant effects on microbial competition, host-pathogen interaction, and antibiotic action and resistance. Furthermore, protonophores are extensively used in various laboratory studies to perturb bacterial physiology. Here, we have characterized cell growth, substrate transport, and motility of Escherichia coli cells exposed to protonophores. Our findings demonstrate heterogeneous effects of protonophores on cell physiology and the underlying mechanism

Efficacy and safety of oral semaglutide in patients with type 2 diabetes and moderate renal impairment (PIONEER 5): a placebo-controlled, randomised, phase 3a trial

Background: Oral semaglutide is the first oral glucagon-like peptide-1 (GLP-1) receptor agonist for glycaemic control in patients with type 2 diabetes. Type 2 diabetes is commonly associated with renal impairment, restricting treatment options. We aimed to investigate the efficacy and safety of oral semaglutide in patients with type 2 diabetes and moderate renal impairment. Methods: This randomised, double-blind, phase 3a trial was undertaken at 88 sites in eight countries. Patients aged 18 years and older, with type 2 diabetes, an estimated glomerular filtration rate of 30–59 mL/min per 1·73 m2, and who had been receiving a stable dose of metformin or sulfonylurea, or both, or basal insulin with or without metformin for the past 90 days were eligible. Participants were randomly assigned (1:1) by use of an interactive web-response system, with stratification by glucose-lowering medication and renal function, to receive oral semaglutide (dose escalated to 14 mg once daily) or matching placebo for 26 weeks, in addition to background medication. Participants and site staff were masked to assignment. Two efficacy-related estimands were defined: treatment policy (regardless of treatment discontinuation or rescue medication) and trial product (on treatment without rescue medication) in all participants randomly assigned. Endpoints were change from baseline to week 26 in HbA1c (primary endpoint) and bodyweight (confirmatory secondary endpoint), assessed in all participants with sufficient data. Safety was assessed in all participants who received at least one dose of study drug. This trial is registered on ClinicalTrials.gov, number NCT02827708, and the European Clinical Trials Registry, number EudraCT 2015-005326-19, and is now complete. Findings: Between Sept 20, 2016, and Sept 29, 2017, of 721 patients screened, 324 were eligible and randomly assigned to oral semaglutide (n=163) or placebo (n=161). Mean age at baseline was 70 years (SD 8), and 168 (52%) of participants were female. 133 (82%) participants in the oral semaglutide group and 141 (88%) in the placebo group completed 26 weeks on treatment. At 26 weeks, oral semaglutide was superior to placebo in decreasing HbA1c (estimated mean change of −1·0 percentage point (SE 0·1; −11 mmol/mol [SE 0·8]) vs −0·2 percentage points (SE 0·1; −2 mmol/mol [SE 0·8]); estimated treatment difference [ETD]: −0·8 percentage points, 95% CI −1·0 to −0·6;

How to create individual educational trajectories in the informational educational environment

The article considers the issues of designing individual educational trajectories for University students in the informational educational environment from the perspective of personal-oriented, environmental and professionally oriented approaches. The leading research methods are comparative statistical analysis and modeling, the first of which helps to analyze quantitative data that reflect parameters that are important for students’ academic performance and progress in the information educational environment, while the latter allows to model linguistic profiles designed for the individual educational trajectories. The informational educational environment as a unit of the educational space of the discipline “Foreign language” is a set of the following components: resource, informational components and socio-methodological, subject-operational and didactic-methodological sub-components, as well as subject-activity and procedural components

Corpus-based translation studies’ view on peculiarities of Russian translated discourse

В статье рассматривается эмпирическое исследование русскоязычного переводного дискурса с позиций дескриптивного подхода в переводоведении с акцентом на корпусную методологию. Кратко описываются цели и задачи данного направления исследований перевода. Приводится характеристика понятия «переводческие универсалии». Дается краткий обзор основных этапов исследования в рамках корпусного переводоведения. На примере пользовательского сравнительного корпуса переводных и непереводных текстов иллюстрируется квантитативный подход к определению свойств переводного дискурса в сравнении с дискурсом непереводным, т. е. изначально порожденным на рассматриваемом языке перевода. В качестве лингвистического индикатора используются частотные характеристики лексики, а именно осуществляется сравнение частотных списков подкорпусов с применением инструментов статистики. Поскольку в исследовании реализуется принцип управления исследования корпусом, перечисляются перспективы дальнейшего рассмотрения полученных . The article presents an empirical study of Russian translated discourse within the framework of descriptive translation studies with an emphasis on corpus-based methodology. A short description of the mentioned translation studies branch is given. The concept of translation universals is discussed. An overview of the main steps of a corpus-based translation research is presented. Quantitative approach is applied to the study of a DIY comparative corpus of translated and non-translated texts, originally composed in the language under consideration. Lexical frequency is used as a linguistic indicator. The study uses statistical methods to compare the subcorpora lexis frequency profiles. Since the study under consideration is corpus-driven, a number of directions for the future research are outlined, building upon the acquired results

Some areas of research on the capital management of a construction firm

В статье рассматриваются некоторые направления управления капиталом, а также вопросы роли кредита в функционировании и развитии строительного предприятия, обеспечивающего эффективную хозяйственную деятельность. Рассмотрена значимость собственных оборотных средств, их состав и источники. Показана роль кредита в формировании оборотных средств в деятельности современного строительного предприятия.The article deals with the role of credit in the functioning and development of a construction company, ensuring effective economic activity. The significance of own working capital, its sources and the role of credit in the formation of working capital are considered. The main directions of the financial and credit impact on the strengthening of the integration of the production and trade sectors in the construction industry are shown

Single-cell bacterial electrophysiology reveals mechanisms of stress induced damage - additional data (Figures 2D, 3D, 4D)

Dataset supporting figures 2D, 3D and 4D of the paper "Single-cell bacterial electrophysiology reveals mechanisms of stress induced damage" (Krasnopeeva et al, 2018). The archive contains excel spreadsheets with the data points plotted in figures 2D, 3D, 4D. For each point the mean and the standard error of the mean are given. The values are calculated as described in Krasnopeeva et al, 2018. Additional DataShare items related to this paper can be accessed: https://doi.org/10.7488/ds/2381, https://doi.org/10.7488/ds/2382, https://doi.org/10.7488/ds/2383.Pilizota, Teuta; Krasnopeeva, Ekaterina. (2019). Single-cell bacterial electrophysiology reveals mechanisms of stress induced damage - additional data (Figures 2D, 3D, 4D), [dataset]. University of Edinburgh. Centre for Synthetic and Systems Biology. https://doi.org/10.7488/ds/2567