CELL AND PROTEIN-BASED SENSING SYSTEMS FOR THE DETECTION OF ENVIRONMENTALLY AND PHYSIOLOGICALLY RELEVANT MOLECULES

The detection of small molecules in complex sample matrices such as environmental (surface and ground water, sediment, etc.) and biological (blood, serum, plasma, etc.) samples is of paramount importance for monitoring the distribution of environmental pollutants and their patterns of exposure within the population as well as diagnosing and managing diseases. Biosensors have demonstrated a singular ability to sensitively and selectively detect analytes in complex samples without the need for extensive sample preparation and pretreatment. Nature has demonstrated myriad examples of exquisite selectivity in spite of complexity and we seek to take advantage of that attribute in the development of novel biosensing systems. In the work presented here, we have developed both cell- and proteinbased biosensing systems for the detection of hydroxylated polychlorinated biphenyls (OH-PCBs) and protein-based sensing systems for the detection of glucose. In the development of a whole-cell sensing system, the regulatory protein, HbpR, and its associated promoter was used to modulate the expression of luciferase. Additionally, the effector binding domain of HbpR, HbpR-A, was isolated and modified with a solvatochromic fluorophore resulting in a proteinbased sensing system. For the detection of glucose, two different glucose binding proteins were engineered in an effort to tailor their characteristics, such as binding affinity and thermal stability, to develop a rugged, sensitive proteinbased sensing system. We envision that these biosensing systems will find applications in the areas of environmental pollutant monitoring and the management and treatment of diseases such as diabetes

Polypeptides, Systems, and Methods Useful for Detecting Glucose

The presently-disclosed subject matter provides biosensors for detecting molecules of interest. The biosensors include a polypeptide capable of selectively-binding glucose, wherein the polypeptide molecule is selected from: an unnatural analogue of wild type glucose binding protein; a fragment of wild type glucose binding protein; and an unnatural analogue fragment of wild type glucose binding protein

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented

Climate reconstruction from Barrow Island, Western Australia

Barrow Island ( 20.7°S) is ideally situated to register the first coastal occupations in Australia as well as peoples' responses to major changes in sea level, climate and eventual isolation from critical resources on the mainland. Its location in the arid region between monsoonal and extratropical rainfall belts also imply that Barrow Island may have experienced dramatic changes in precipitation over the period of human occupation. Boodie cave has been the focus of Barrow Island Archeological Project and records a rich record of human occupation. Also present at Boodie cave are significant quantities of water-lain cave carbonates (flowstones, stalactites, and stalagmites). Active (modern) deposition of such carbonates is limited to very small encrustations and consists primarily of stalactites that are less than 5 cm in diameter. This situation indicates that deposition of significant carbonates is indicative of wetter conditions at Barrow Island and dating of these carbonates using the U/Th method provides a record of wet intervals at Barrow Island over the last 120 thousand years. In addition to ages from flowstones, three complete speleothems were collected Ledge Cave for climatic reconstruction using stable isotopes. Ledge cave is large subterranean with high relative humidity (>98%) and abundant, but largely inactive speleothems. The wettest interval in our cave carbonate record predates stratigraphic units with cultural material, but indicates that wet intervals on Barrow Island were broadly coincidental with lake expansions on the Australian mainland. In particular, a very wet interval between 120 and 90 ka is recorded in two of the Ledge Cave speleothems. The Barrow Island speleothem record suggests that displacement of the Intertropical Convergence Zone (ITCZ) and the strength of the Indo-Australian monsoon may have been the most important influence on water balance at Barrow Island. Continued development of these climate archives will offer insights into climate that is directly applicable to the unique human occupation record also preserved at this site

Preventing childhood scalds within the home: overview of systematic reviews and a systematic review of primary studies

Objective: To synthesise and evaluate the evidence of the effectiveness of interventions to prevent scalds in children. Methods: An overview of systematic reviews (SR) and a SR of primary studies were performed evaluating interventions to prevent scalds in children. A comprehensive literature search was conducted covering various resources up to October 2012. Experimental and controlled observational studies reporting scald injuries, safety practices and safety equipment use were included. Results: Fourteen systematic reviews and 39 primary studies were included. There is little evidence that interventions are effective in reducing the incidence of scalds in children. More evidence was found that inventions are effective in promoting safe hot tap water temperature, especially when home safety education, home safety checks and discounted or free safety equipment including thermometers and thermostatic mixing valves were provided. No consistent evidence was found for the effectiveness of interventions on the safe handling of hot food or drinks nor improving kitchen safety practices. Conclusion: Education, home safety checks along with thermometers or thermostatic mixing valves should be promoted to reduce tap water scalds. Further research is needed to evaluate the effectiveness of interventions on scald injuries and to disentangle the effects of multifaceted interventions on scald injuries and safety practices

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function

Open coast seagrass restoration. Can we do it? Large scale seagrass transplants

Some of the major challenges in seagrass restoration on exposed open coasts are the choice of transplant design that is optimal for coastlines periodically exposed to high water motion, and understanding the survival and dynamics of the transplanted areas on a long time-scale over many years. To contribute to a better understanding of these challenges, we describe here part of a large-scale seagrass restoration program conducted in a Marine Park in Portugal. The goal of this study was to infer if it was possible to recover seagrass habitat in this region, in order to restore its ecosystem functions. To infer which methods would produce better long term persistence to recover seagrass habitat, three factors were assessed: donor seagrass species, transplant season, source location. Monitoring was done three times a year for 8 years, in which areas and densities of the planted units were measured, to assess survival and growth. The best results were obtained with the species Zostera marina transplanted during spring and summer as compared to Zostera noltii and Cymodocea nodosa. Long-term persistence of established (well rooted) transplants was mainly affected by extreme winter storms but there was evidence of fish grazing effects also. Our results indicate that persistence assessments should be done in the long term, as all transplants were successful (survived and grew initially) in the short term, but were not resistant in the long term after a winter with exceptionally strong storms. The interesting observation that only the largest (11 m(2)) transplanted plot of Z marina persisted over a long time, increasing to 103 m(2) in 8 years, overcoming storms and grazing, raised the hypothesis that for a successful shift to a vegetated state it might be necessary to overpass a minimum critical size or tipping point. This hypothesis was therefore tested with replicates from two donor populations and results showed effects of size and donor population, as only the larger planting units (PUs) from one donor population persisted and expanded. It is recommended that in future habitat restoration efforts large PUs are considered.Project BIOMARES LIFE06 NAT/P/192 European Union LIFE Program cement company SECIL, Companhia de Cal e Cimentos S. A., Portugal Fundacao para a Ciencia e a Tecnologia FCT SFRH/BD/81086/2011 Pew Marine Fellowshipinfo:eu-repo/semantics/publishedVersio

‘Video Replay: Families, films and fantasy’ as a transformational text: Commentary on Valerie Walkerdine's ‘Video Replay’.

In this commentary I explore the significance of Valerie Walkerdine's paper ‘Video Replay: Families, Films and Fantasy’. I review its impact in 1986 and then discuss how some of its ideas about subjectivity and popular culture – specifically film - can be developed in the contemporary context. A recurring fantasy of Rocky II and its reception is that of social and psychological transformation. I address this theme by drawing on the work of Christopher Bollas to argue that Walkerdine's psychosocial analysis continues to facilitate, across a range of contexts, some of the transformational processes described in her article

Why health visiting? Examining the potential public health benefits from health visiting practice within a universal service: A narrative review of the literature

INTRODUCTION: There is increasing international interest in universal, health promoting services for pregnancy and the first three years of life and the concept of proportionate universalism. Drawing on a narrative review of literature, this paper explores mechanisms by which such services might contribute to health improvement and reducing health inequalities. OBJECTIVES: Through a narrative review of empirical literature, to identify: DESIGN: The paper draws upon a scoping study and narrative review. REVIEW METHODS: We used three complementary approaches to search the widely dispersed literature: Our key inclusion criterion was information about health visiting practice. We included empirical papers from United Kingdom (UK) from 2004 to February 2012 and older seminal papers identified in search (3), identifying a total of 348 papers for inclusion. A thematic content analysis compared the older (up to 2003) with more recent research (2004 onwards). RESULTS: The analysis revealed health visiting practice as potentially characterized by a particular 'orientation to practice.' This embodied the values, skills and attitudes needed to deliver universal health visiting services through salutogenesis (health creation), person-centredness (human valuing) and viewing the person in situation (human ecology). Research about health visiting actions focuses on home visiting, needs assessment and parent-health visitor relationships. The detailed description of health visitors' skills, attitudes, values, and their application in practice, provides an explanation of how universal provision can potentially help to promote health and shift the social gradient of health inequalities. CONCLUSIONS: Identification of needs across an undifferentiated, universal caseload, combined with an outreach style that enhances uptake of needed services and appropriate health or parenting information, creates opportunities for parents who may otherwise have remained unaware of, or unwilling to engage with such provision. There is a lack of evaluative research about health visiting practice, service organization or universal health visiting as potential mechanisms for promoting health and reducing health inequalities. This paper offers a potential foundation for such research in future

Cingulin Contains Globular and Coiled-Coil Domains and Interacts with Zo-1, Zo-2, Zo-3, and Myosin

We characterized the sequence and protein interactions of cingulin, an Mr 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central α-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of cingulin (1–378) interacts in vitro with ZO-1 (Kd ∼5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton