Combining single-molecule super-resolved localization microscopy with fluorescence polarization imaging to study cellular processes

Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used as common reporters on the localization of proteins in cellular processes. These enable measurement of spatial location to a super-resolved precision better than the diffraction-limited optical resolution, as well as estimation of molecular stoichiometry based on the brightness of individual fluorophores. In this innovation we have adapted a millisecond timescale microscope used for single-molecule detection to enable splitting of fluorescence polarization emissions into two separate imaging channels for s- and p- polarization signals, which are imaged onto separate halves of the same high sensitivity back-illuminated CMOS camera detector. We applied this fluorescence polarization super-resolved imaging modality to a range of test fluorescent samples relevant to the study of biological processes, including purified monomeric green fluorescent protein, single combed DNA molecules, and protein assemblies and complexes from live Escherichia coli and Saccharomyces cerevisiae cells. Our findings are qualitative but demonstrate promise in showing how fluorescence polarization and super-resolved localization microscopy can be combined on the same sample to enable simultaneous measurements of polarization and stoichiometry of tracked molecular complexes, as well as the translational diffusion coefficient

The role and scope of practice of midwives in humanitarian settings:a systematic review and content analysis

Abstract Background Midwives have an essential role to play in preparing for and providing sexual and reproductive health (SRH) services in humanitarian settings due to their unique knowledge and skills, position as frontline providers and geographic and social proximity to the communities they serve. There are considerable gaps in the international guidance that defines the scope of practice of midwives in crises, particularly for the mitigation and preparedness, and recovery phases. We undertook a systematic review to provide further clarification of this scope of practice and insights to optimise midwifery performance. The review aimed to determine what SRH services midwives are involved in delivering across the emergency management cycle in humanitarian contexts, and how they are working with other professionals to deliver health care. Methods Four electronic databases and the websites of 33 organisations were searched between January and March 2017. Papers were eligible for inclusion if they were published in English between 2007 and 2017 and reported primary research pertaining to the role of midwives in delivering and performing any component of sexual and/or reproductive health in humanitarian settings. Content analysis was used to map the study findings to the Minimum Initial Service Package (MISP) for SRH across the three phases of the disaster management cycle and identify how midwives work with other members of the health care team. Results Fourteen studies from ten countries were included. Twelve studies were undertaken in conflict settings, and two were conducted in the context of the aftermath of natural disasters. We found a paucity of evidence from the research literature that examines the activities and roles undertaken by midwives across the disaster management cycle. This lack of evidence was more apparent during the mitigation and preparedness, and recovery phases than the response phase of the disaster management cycle. Conclusion Research-informed guidelines and strategies are required to better align the scope of practice of midwives with the objectives of multi-agency guidelines and agreements, as well as the activities of the MISP, to ensure that the potential of midwives can be acknowledged and optimised across the disaster management cycle

Human Pathogen Shown to Cause Disease in the Threatened Eklhorn Coral Acropora palmata

Coral reefs are in severe decline. Infections by the human pathogen Serratia marcescens have contributed to precipitous losses in the common Caribbean elkhorn coral, Acropora palmata, culminating in its listing under the United States Endangered Species Act. During a 2003 outbreak of this coral disease, called acroporid serratiosis (APS), a unique strain of the pathogen, Serratia marcescens strain PDR60, was identified from diseased A. palmata, human wastewater, the non-host coral Siderastrea siderea and the corallivorous snail Coralliophila abbreviata. In order to examine humans as a source and other marine invertebrates as vectors and/or reservoirs of the APS pathogen, challenge experiments were conducted with A. palmata maintained in closed aquaria to determine infectivity of strain PDR60 from reef and wastewater sources. Strain PDR60 from wastewater and diseased A. palmata caused disease signs in elkhorn coral in as little as four and five days, respectively, demonstrating that wastewater is a definitive source of APS and identifying human strain PDR60 as a coral pathogen through fulfillment of Koch's postulates. A. palmata inoculated with strain PDR60 from C. abbreviata showed limited virulence, with one of three inoculated fragments developing APS signs within 13 days. Strain PDR60 from non-host coral S. siderea showed a delayed pathogenic effect, with disease signs developing within an average of 20 days. These results suggest that C. abbreviata and non-host corals may function as reservoirs or vectors of the APS pathogen. Our results provide the first example of a marine “reverse zoonosis” involving the transmission of a human pathogen (S. marcescens) to a marine invertebrate (A. palmata). These findings underscore the interaction between public health practices and environmental health indices such as coral reef survival

Bacteria-inducing legume nodules involved in the improvement of plant growth, health and nutrition

Bacteria-inducing legume nodules are known as rhizobia and belong to the class Alphaproteobacteria and Betaproteobacteria. They promote the growth and nutrition of their respective legume hosts through atmospheric nitrogen fixation which takes place in the nodules induced in their roots or stems. In addition, rhizobia have other plant growth-promoting mechanisms, mainly solubilization of phosphate and production of indoleacetic acid, ACC deaminase and siderophores. Some of these mechanisms have been reported for strains of rhizobia which are also able to promote the growth of several nonlegumes, such as cereals, oilseeds and vegetables. Less studied are the mechanisms that have the rhizobia to promote the plant health; however, these bacteria are able to exert biocontrol of some phytopathogens and to induce the plant resistance. In this chapter, we revised the available data about the ability of the legume nodule-inducing bacteria for improving the plant growth, health and nutrition of both legumes and nonlegumes. These data showed that rhizobia meet all the requirements of sustainable agriculture to be used as bio-inoculants allowing the total or partial replacement of chemicals used for fertilization or protection of crops

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

State-dependent system identification for control of a hydraulically-actuated nuclear decommissioning robot

This article considers the identification of state-dependent parameter~(SDP) models for the hydraulically actuated dual-manipulators of a mobile robot used for nuclear decommissioning tasks. A unified framework for calibration, data collection and system identification is developed, and utilized to investigate potential state-dependencies. The latter are associated with nonlinear system dynamics and can cause irregular joint movements when the device is controlled using linear control algorithms. The analysis suggests that a univariate SDP model is suitable for control design. The model has a state-dependent gain, characterized directly from experimental data using a numerically optimized polynomial function of the delayed input variable. In order to demonstrate the practical utility of the SDP model, closed-loop results using a novel non-minimal regulator for joint control are briefly considered

Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify chains of interlinked ~200- to 300-nm-wide chromatin domains (CDs) composed of aggregated nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin compartment. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications toward the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization