CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum

Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin. Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc

Individual and demographic consequences of reduced body condition following repeated exposure to high temperatures

Although the lethal consequences of extreme heat are increasingly reported in the literature, the fitness costs of exposure to sublethal high air temperatures, typically identified in the 30–40°C range, are poorly understood. We examine the effect of high (≥35°C) daily maxima on body condition of a semiarid population of White-plumed Honeyeaters, Ptilotula penicillatus, monitored between 1986 and 2012. During this 26-yr period, temperature has risen, on average, by 0.06°C each year at the site, the frequency of days with thermal maxima ≥35°C has increased and rainfall has declined. Exposure to high temperatures affected body condition of White-plumed Honeyeaters, but only in low-rainfall conditions. There was no effect of a single day of exposure to temperatures ≥35°C but repeated exposure was associated with reduced body condition: 3.0% reduction in body mass per day of exposure. Rainfall in the previous 30 d ameliorated these effects, with reduced condition evident only in dry conditions. Heat-exposed males with reduced body condition were less likely to be recaptured at the start of the following spring; they presumably died. Heat-exposed females, regardless of body condition, showed lower survival than exposed males, possibly due to their smaller body mass. The higher mortality of females and smaller males exposed to temperatures ≥35°C may have contributed to the increase in mean body size of this population over 23 years. Annual survival declined across time concomitant with increasing frequency of days ≥35°C and decreasing rainfall. Our study is one of few to identify a proximate cause of climate change related mortality, and associated long-term demographic consequence. Our results have broad implications for avian communities living in arid and semiarid regions of Australia, and other mid-latitudes regions where daily maximum temperatures already approach physiological limits in regions affected by both decreased precipitation and warming

HOW E-COMMERCE IS CHANGING THE FACILITY MANAGEMENT PRACTICES OF BUILDING OWNERS

"The corporate Ford of 2010 will look more like Cisco, a company that manufactures very little.” "Direct-to-the-assembly-line ordering will enable Ford to custom configure cars much the way that Dell custom configures computers.” --Forbes, July 17, 2000 The above quotation is an indication of how information technology is changing even old-line manufacturing organizations. The research reported in this paper was motivated by our desire to better understand how information technology and, in particular, the recent phenomenon of e-commerce, was influencing the design and construction industry. Since trends in the building industry are often determined by the desires and programs initiated by large building owners, we decided to focus our research on facility management organizations in Fortune-500 companies

The Law of Genetic Privacy: Applications, Implications, and Limitations

Recent advances in technology have significantly improved the accuracy of genetic testing and analysis, and substantially reduced its cost, resulting in a dramatic increase in the amount of genetic information generated, analysed, shared, and stored by diverse individuals and entities. Given the diversity of actors and their interests, coupled with the wide variety of ways genetic data are held, it has been difficult to develop broadly applicable legal principles for genetic privacy. This article examines the current landscape of genetic privacy to identify the roles that the law does or should play, with a focus on federal statutes and regulations, including the Health Insurance Portability and Accountability Act (HIPAA) and the Genetic Information Nondiscrimination Act (GINA). After considering the many contexts in which issues of genetic privacy arise, the article concludes that few, if any, applicable legal doctrines or enactments provide adequate protection or meaningful control to individuals over disclosures that may affect them. The article describes why it may be time to shift attention from attempting to control access to genetic information to considering the more challenging question of how these data can be used and under what conditions, explicitly addressing trade-offs between individual and social goods in numerous applications

Digital Droplet PCR for Influenza Vaccine Development

AbstractDevelopment of influenza vaccine processes requires virus quantification to optimize conditions in cell culture or in the associated downstream purification steps. Modern methods include qPCR, which utilizes TaqMan chemistry to detect and quantify viral RNA by comparison of a RNA standard of known concentration. Digital droplet PCR (ddPCR) is similar to qPCR in that it shares the same chemistry for nucleic acid detection. However, in ddPCR, the sample is diluted into partitions (‘droplets’) in order to separate and isolate single molecules. Upon PCR amplification, the droplet's fluorescent intensity depends on the presence or absence of the target; as such, positive and negative droplets are identified, which allows for absolute quantification of the viral genomes. The digital approach has enabled several key advantages. First, a standard is no longer required. Second, efficiency of the reverse transcription and the kinetics of the amplification, principles in qPCR, have no impact on the final digital PCR quantification. For this reason, the extracted RNA does not need to be purified from the reagents needed to lyse the virus. Also, viral associated RNA released by infected cells can be measured directly, further improving the quality of the data generated. Additional improvements to the approach include duplexing with a second assay that measures host cell DNA concentration. The method has been successfully implemented with automation in support of multiple upstream and downstream process development efforts for influenza vaccine manufacturing

Climate-driven changes in body condition

Although the lethal consequences of extreme heat are increasingly reported in the literature, the fitness costs of exposure to sub-lethal high air temperatures, typically identified in the 30- 40°C range, are poorly understood. We examine the effect of high (≥35°C) daily maxima on body condition of a semi-arid population of white-plumed honeyeaters Ptilotula penicillatus monitored between 1986 and 2012. During this 26 year period temperature has risen, on average, by 0.06°C each year at the site, the frequency of days with thermal maxima ≥35°C has increased and rainfall has declined. Exposure to high temperatures affected body condition of white-plumed honeyeaters, but only in low rainfall conditions. There was no effect of a single day of exposure to temperatures ≥35°C but repeated exposure was associated with reduced body condition: 3.0% reduction in body mass per day of exposure. Rainfall in the previous 30 days ameliorated these effects, with reduced condition evident only in dry conditions. Heat-exposed males with reduced body condition were less likely to be recaptured at the start of the following spring; they presumably died. Heat-exposed females, regardless of body condition, showed lower survival than exposed males, possibly due to their smaller body mass. The higher mortality of females and smaller males exposed to temperatures ≥35°C may have contributed to the increase in mean body size of this population over 23 years. Annual survival declined across time concomitant with increasing frequency of days ≥35°C and decreasing rainfall. Our study is one of few to identify a proximate cause of climate change related mortality, and associated long-term demographic consequence. Our results have broad implications for avian communities living in arid and semi-arid regions of Australia, and other mid-latitudes regions where daily maximum temperatures already approach physiological limits in regions affected by both decreased precipitation and warming.The work was conducted under permits SL100167 and SL100825 issued by the Office of Environment and Heritage, National Parks and Wildlife Service (NPWS), NSW and bands were supplied by the Australian Bird and Bat Banding Scheme. We thank the many volunteer banders and NPWS staff for support. Jim Thompson provided initial statistical advice and Loeske Kruuk, Peter Marsack and Katherine Selwood useful discussion or comments on the manuscript. The work received funding from the Norman Wettenhall Foundation and the Australian Research Council (ARC, Discovery grant DP120102651). Sutherland is funded by Arcadia, Peters by an ARC Future Fellowship (FT110100505) and Amano by a Marie Curie International Incoming Fellowship.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1890/15-064

Towards the Application of Uncertainty Analysis in Architectural Design Decision-Making

To this day, proper handling of uncertainties -including unknown variables in primary stages of a design, an actual climate data, occupants` behavior, and degradation of material properties over the time- remains as a primary challenge in an architectural design decision-making process. For many years, conventional methods based on the architects' intuition have been used as a standard approach dealing with uncertainties and estimating the resulting errors. However, with buildings reaching great complexity in both their design and material selections, conventional approaches come short to account for ever-existing but unpredictable uncertainties and prove incapable of meeting the growing demand for precise and reliable predictions. This study aims to develop a probability-based framework and associated prototypes to employ uncertainty analysis and sensitivity analysis in architectural design decision-making. The current research explores an advanced physical model for thermal energy exchange characteristics of a hypothetical building and uses it as a test case to demonstrate the proposed probability-based analysis framework. The proposed framework provides a means to employ uncertainty and sensitivity analysis to improve reliability and effectiveness in a buildings design decision-making process

Translating Building Information Modeling to Building Energy Modeling Using Model View Definition

This paper presents a new approach to translate between Building Information Modeling (BIM) and Building Energy Modeling (BEM) that uses Modelica, an object-oriented declarative, equation-based simulation environment. The approach (BIM2BEM) has been developed using a data modeling method to enable seamless model translations of building geometry, materials, and topology. Using data modeling, we created a Model View Definition (MVD) consisting of a process model and a class diagram. The process model demonstrates object-mapping between BIM and Modelica-based BEM (ModelicaBEM) and facilitates the definition of required information during model translations. The class diagram represents the information and object relationships to produce a class package intermediate between the BIM and BEM. The implementation of the intermediate class package enables system interface (Revit2Modelica) development for automatic BIM data translation into ModelicaBEM. In order to demonstrate and validate our approach, simulation result comparisons have been conducted via three test cases using (1) the BIM-based Modelica models generated from Revit2Modelica and (2) BEM models manually created using LBNL Modelica Buildings library. Our implementation shows that BIM2BEM (1) enables BIM models to be translated into ModelicaBEM models, (2) enables system interface development based on the MVD for thermal simulation, and (3) facilitates the reuse of original BIM data into building energy simulation without an import/export process

Structural basis of severe acute respiratory syndrome coronavirus ADP-ribose-1''-phosphate dephosphorylation by a conserved domain of nsP3.

The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 A resolution. The structure of this "X" domain, seen in many single-stranded RNA viruses, reveals a three-layered alpha/beta/alpha core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1''-phosphate (Appr-1''-p). The SARS nsP3 domain readily removes the 1'' phosphate group from Appr-1''-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1''-p

Hard X-Rays From Supernova 1993J

The OSSE experiment on the Compton Observatory observed SN 1993J during three intervals, approximately 9--15, 23--36, and 93--121 days after outburst. There is evidence for continuum emission below 200 keV in the first two of these periods. Power-law fits yield intensities at 100 keV of (1.82+/-0.39)*E(-3) photons cm(-2) s(-1) MeV(-1) and (0.89+/-0.35)*E(-3) photons cm(-2) s(-1) MeV(-1) , and photon indices of -2.3+/-0.5 and -2.2+/-0.9, respectively. There is no evidence for any emission in the longer, more sensitive, third observation. These continua are too bright and too steep to be entirely due to Comptonized gamma-rays from radioactive (56) Ni and (56) Co alone. A thermal bremsstrahlung spectrum, for example, also adequately describes the OSSE data, with kT =~ 75 keV. These continua extrapolate well above nearly contemporaneous measurements at lower energies. Instead, a power-law of fixed photon index -1.2 fit to the first OSSE observation extrapolates approximately to the total luminosity measured by ASCA (Tanaka IAU Circ. 5753) from 1--10 keV, one day earlier. For a thermal spectrum a higher temperature, near 200 keV, can also fit both data sets---but only marginally. This emission cannot be unambiguously attributed to SN 1993J. Because of the large OSSE field of view, SN 1993J cannot be separated from other sources such as the nucleus of M81 or even M82. However, OSSE did observe this region twice earlier, 597 and 443 days before SN 1993J and no continuum emission was detected at either time. The apparent decline of the emission does seem to correlate well with those of SN 1993J as seen by ASCA and ROSAT. No evidence for line emission is seen in any observation. This work is supported by NASA DPR S-10987C