Utilizing pHluorin-Tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins

Mammalian Cell-Derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly

Developing a timeline for evaluating public health nutrition policy interventions: what are the outcomes and when should we expect to see them?

Objective: To develop a timeline for evaluating public health nutrition policy interventions. Design: Concept mapping, a stakeholder-driven approach for developing an evaluation framework to estimate the ‘time to impact’ for policy interventions. The Schools (Health Promotion and Nutrition) (Scotland) Act 2007 was used as the model to develop the evaluation timeline as it had typical characteristics of government policy. Concept mapping requires stakeholders to generate a list of the potential outcomes, sort and rate the outcomes. Multidimensional scaling and hierarchical cluster data analysis were used to develop an anticipated timeline to impact for the policy. Setting: United Kingdom. Subjects: One hundred and eleven stakeholders representing nutrition, public health, medicine, education and catering in a range of sectors: research, policy, local government, National Health Service and schools. Results: Eighty-five possible outcomes were identified and grouped into thirteen clusters describing higher-level themes (e.g. long-term health, food literacy, economics, behaviour, diet, education). Negative and unintended consequences were anticipated relatively soon after implementation of the policy, whereas positive outcomes (e.g. dietary changes, health benefits) were thought likely to take longer to emerge. Stakeholders responsible for implementing the legislation anticipated that it would take longer to observe changes than those from policy or research. Conclusions: Developing an anticipated timeline provides a realistic framework upon which to base an outcome evaluation for policy interventions and identifies positive and negative outcomes as well as considering possible unintended consequences. It offers benefit to both policy makers and researchers in mapping the progress expected towards long-term health goals and outcomes

Arctic petroleum: Local CSR-perceptions in the Nenets region

PURPOSE: The purpose of this article is to identify and discuss perceptions of petroleum-related corporate social responsibility (CSR) among local and regional authorities, local peoples (indigenous and non-indigenous) and representatives of petroleum companies working or living in the Nenets Autonomous Okrug (NAO) in the Russian Arctic. Although the CSR literature comprises a broad spectrum of approaches, an underrepresentation of perspectives from non-business stakeholders has been suggested. The paper seeks to redress this gap. DESIGN/METHODOLOGY/APPROACH: The data were obtained through 34 in-depth, semi-structured interviews conducted and qualitatively analyzed to extrapolate perceptions, views, and expectations of petroleum-related CSR in NAO. By exploring needs, wants, and expectations, differences are identified between short-term expectations and longer-term perspectives. FINDINGS: A central feature of our findings is the wide variation in the responses, not only between community groups and sectors, but also within them. The complexity identified is an argument in favor of local involvement to understand local contexts and suggests avoiding “one-size fits all” CSR approaches. Challenges and opportunities are identified for the petroleum companies in dealing with different stakeholders and diverging interests. RESEARCH LIMITATIONS/IMPLICATIONS: The importance of local context means that caution is advised when considering the transferability or generalizability of lessons, within NAO and elsewhere in and outside the Russian Arctic. Furthermore, fundamental motivations are not always transparent from interviews. ORIGINALITY/VALUE: Although a rich literature exists on CSR, this is perhaps the first study providing a cross-sectoral analysis of people’s perceptions, including those of non-business stakeholders, in this region

The effects of a tea tree oil-containing gel on plaque and chronic gingivitis

The document attached has been archived with permission from the Australian Dental Association. An external link to the publisher’s copy is included.Background: This clinical study assessed the effects of topically applied tea tree oil (TTO)-containing gel on dental plaque and chronic gingivitis. Methods: This was a double-blind, longitudinal, non-crossover study in 49 medically fit non-smokers (24 males and 25 females) aged 18–60 years with severe chronic gingivitis. Subjects were randomly assigned to three groups and given either TTO-gel (2.5 per cent), chlorhexidine (CHX) gel (0.2 per cent), or a placebo gel to apply with a toothbrush twice daily. Treatment effects were assessed using the Gingival Index (GI), Papillary Bleeding Index (PBI) and plaque staining score (PSS) at four and eight weeks. Results: No adverse reactions to any of the gels were reported. The data were separated into subsets by tooth (anterior and posterior) and tooth surface (buccal and lingual). The TTO group had significant reduction in PBI and GI scores. However, TTO did not reduce plaque scores, which tended to increase over the latter weeks of the study period. Conclusion: Although further studies are required, the anti-inflammatory properties of TTO-containing gel applied topically to inflamed gingival tissues may prove to be a useful non-toxic adjunct to chemotherapeutic periodontal therapy.S Soukoulis and R Hirsc

New insights into the breathing phenomenon in ZIF-4

Structural changes in ZIFs upon adsorption remain a paradigm due to the sensitivity of the adsorption mechanism to the nature of the organic ligands and gas probe molecules. Synchrotron X-ray diffraction under operando conditions clearly demonstrates for the first time that ZIF-4 exhibits a structural reorientation from a narrow-pore (np) to a new expanded-pore (ep) structure upon N2 adsorption, while it does not do so for CO2 adsorption. The existence of an expanded-pore structure of ZIF-4 has also been predicted by molecular simulations. In simulations the expanded structure was stabilized by entropy at high temperatures and by strong adsorption of N2 at low temperatures. These results are in perfect agreement with manometric adsorption measurements for N2 at 77 K that show the threshold pressure for breathing at ∼30 kPa. Inelastic neutron scattering (INS) measurements show that CO2 is also able to promote structural changes but, in this specific case, only at cryogenic temperatures (5 K).The authors would like to acknowledge financial support from the MINECO (MAT2016-80285-p), Generalitat Valenciana (PROMETEOII/2014/004), H2020 (MSCA-RISE-2016/NanoMed Project), Spanish ALBA synchrotron (Projects AV-2017021985 and IH-2018012591) and Oak Ridge beam time availability (Project IPTS-20843.1). JSA and JGL acknowledge financial support from UA (ACIE17-15) to cover all the expenses for INS measurements at Oak Ridge. JGL acknowledges GV (GRISOLIAP/2016/089) for the research contract

Contrasting effects of summer and winter warming onbody mass explain population dynamics in a food-limitedArctic herbivore

-The cumulative effects of climate warming on herbivore vital rates and population dynamics are hard to predict, given that the expected effects differ between seasons. In the Arctic, warmer summers enhance plant growth which should lead to heavier and more fertile individuals in the autumn. Conversely, warm spells in winter with rainfall (rain-on-snow) can cause ‘icing’, restricting access to forage, resulting in starvation, lower survival and fecundity. As body condition is a ‘barometer’ of energy demands relative to energy intake, we explored the causes and consequences of variation in body mass of wild female Svalbard reindeer (Rangifer tarandus platyrhynchus) from 1994 to 2015, a period of marked climate warming. Late winter (April) body mass explained 88% of the between-year variation in population growth rate, because it strongly influenced reproductive loss, and hence subsequent fecundity (92%), as well as survival (94%) and recruitment (93%). Autumn (October) body mass affected ovulation rates but did not affect fecundity. April body mass showed no long-term trend (coefficient of variation, CV = 8.8%) and was higher following warm autumn (October) weather, reflecting delays in winter onset, but most strongly, and negatively, related to ‘rain-on-snow’ events. October body mass (CV = 2.5%) increased over the study due to higher plant productivity in the increasingly warm summers. Density-dependent mass change suggested competition for resources in both winter and summer but was less pronounced in recent years, despite an increasing population size. While continued climate warming is expected to increase the carrying capacity of the high Arctic tundra, it is also likely to cause more frequent icing events. Our analyses suggest that these contrasting effects may cause larger seasonal fluctuations in body mass and vital rates. Overall our findings provide an important ‘missing’ mechanistic link in the current understanding of the population biology of a keystone species in a rapidly warming Arctic. Keywords: climate change, density dependence, extreme events, icing, nutrition, primary production, Rangifer, reindeer, Svalbard, weathe

HENA, heterogeneous network-based data set for Alzheimer's disease.

Alzheimer's disease and other types of dementia are the top cause for disabilities in later life and various types of experiments have been performed to understand the underlying mechanisms of the disease with the aim of coming up with potential drug targets. These experiments have been carried out by scientists working in different domains such as proteomics, molecular biology, clinical diagnostics and genomics. The results of such experiments are stored in the databases designed for collecting data of similar types. However, in order to get a systematic view of the disease from these independent but complementary data sets, it is necessary to combine them. In this study we describe a heterogeneous network-based data set for Alzheimer's disease (HENA). Additionally, we demonstrate the application of state-of-the-art graph convolutional networks, i.e. deep learning methods for the analysis of such large heterogeneous biological data sets. We expect HENA to allow scientists to explore and analyze their own results in the broader context of Alzheimer's disease research

Saliva-based colorimetric test as an index of gingival inflammation in epidemiologic studies

– There is a pressing need for a reliable, low-cost method of assessing the gingival and periodontal status of large population groups. Existing indexes, despite their value in dental public health, are still too subject to examiner variability for use by uncalibrated examiners. This study describes an evaluation of a quick, inexpensive, extraoral colorimetric test for gingival inflammation, based on a reaction between saliva and the test material. It could probably be applied by non-professional personnel. In this study, the test was applied to a population of elementary schoolchildren, denial hygiene students and faculty, and adult inmates of two correctional institutions. These populations were chosen on the assumption that they would exhibit varying intensities of gingival inflammation. Values obtained from the colorimetric test carried out by one researcher were compared with Gingival Index (GI) scores observed by a different examiner. Results suggest that the colorimetric test may be a valid, reliable means of detecting major differences in the prevalence of gingival inflammation in most adult populations, although having little, if any, useful application among children at the mixed dentition stage.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73489/1/j.1600-0528.1978.tb01169.x.pd

An Exploratory Study into the Factors Impeding Ethical Consumption

Although consumers are increasingly engaged with ethical factors when forming opinions about products and making purchase decisions, recent studies have highlighted significant differences between consumers’ intentions to consume ethically, and their actual purchase behaviour. This article contributes to an understanding of this “ethical purchasing gap” through a review of existing literature, and the inductive analysis of focus group discussions. A model is suggested which includes exogenous variables such as moral maturity and age which have been well covered in the literature, together with further impeding factors identified from the focus group discussions. For some consumers, inertia in purchasing behaviour was such that the decision-making process was devoid of ethical considerations. Several manifested their ethical views through post-purchase dissonance and retrospective feelings of guilt. Others displayed a reluctance to consume ethically due to personal constraints, a perceived negative impact on image or quality, or an outright negation of responsibility. Those who expressed a desire to consume ethically often seemed deterred by cynicism, which caused them to question the impact they, as an individual, could achieve. These findings enhance the understanding of ethical consumption decisions and provide a platform for future research in this area