Bacteriorhodopsin protein hybrids for chemical and biological sensing

Bacteriorhodopsin (bR), an optoelectric protein found in Halobacterium salinarum, has the potential for use in protein hybrid sensing systems. Bacteriorhodopsin has no intrinsic sensing properties, however molecular and chemical tools permit production of bR protein hybrids with transducing and sensing properties. As a proof of concept, a maltose binding protein-bacteriorhodopsin ([MBP]-bR) hybrid was developed. It was proposed that the energy associated with target molecule binding, maltose, to the hybrid sensor protein would provide a means to directly modulate the electrical output from the MBP-bR bio-nanosensor platform. The bR protein hybrid is produced by linkage between bR (principal component of purified purple membrane [PM]) and MBP, which was produced by use of a plasmid expression vector system in Escherichia coli and purified utilizing an amylose affinity column. These proteins were chemically linked using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which facilitates formation of an amide bond between a primary carboxylic acid and a primary amine. The presence of novel protein hybrids after chemical linkage was analyzed by SDSPAGE. Soluble proteins (MBP-only derivatives and unlinked MBP) were separated from insoluble proteins (PM derivatives and unlinked PM) using size exclusion chromatography. The putatively identified MBP-bR protein hybrid, in addition to unlinked bR, was collected. This sample was normalized for bR concentration to native PM and both were deposited onto indium tin oxide (ITO) coated glass slides by electrophoretic sedimentation. The photoresponse of both samples, activated using 100 Watt tungsten lamp at 10 cm distance, were equal at 175 mV. Testing of deposited PM with 1 mM sucrose or 1 mM maltose showed no change in the photoresponse of the xiv material, however addition of 1 mM maltose to the deposited MBP-bR linked hybrid material elicited a 57% decrease in photoresponse indicating a positive response for targeting of maltose. This chemically linked MBP-bR hybrid protein, with bacteriorhodopsin, as a photoresponsive transducing substrate, shows promise for creation of a universal sensing array by attachment of other pertinent sensing materials, in lieu of the maltose binding protein utilized. This strategy would allow significant reduction in sensor size, while increasing responsiveness and sensitivity at nano and picomolar levels.

SEARCH FOR LONG-LIVED WEAKLY INTERACTING PARTICLES USING THE PIERRE AUGER OBSERVATORY

Data collected with the surface detector array of the Pierre Auger Observatory during the period from January 1, 2004 to March 17, 2015 was examined for evidence of production of long-lived weakly interacting particles in interactions of ultra high energy cosmic rays in the atmosphere. The search was performed using extensive air showers with primary energies more than 10 EeV and arrival directions in the range of 57.5◦ to 77.5◦. There was no evidence of significant production of such particles. An upper limit on the fraction of extensive air showers in which such particles are produced was set.

TERPENE AND TERPENOID EMISSIONS AND SECONDARY ORGANIC AEROSOL PRODUCTION

Approximately 90% of fine aerosol in the Midwestern United States has a regional component with a sizable fraction attributed to secondary production of organic aerosol (SOA). The Ozark Forest is an important source of biogenic SOA precursors like isoprene (> 150 mg m-2 d-1), monoterpenes (10-40 mg m-2 d-1), and sesquiterpenes (10-40 mg m-2d-1). Anthropogenic sources include secondary sulfate and nitrate and biomass burning (51-60%), vehicle emissions (17-26%), and industrial emissions (16-18%). Vehicle emissions are an important source of volatile and vapor-phase, semivolatile aliphatic and aromatic hydrocarbons that are important anthropogenic sources of SOA precursors. The short lifetime of SOA precursors and the complex mixture of functionalized oxidation products make rapid sampling, quantitative processing methods, and comprehensive organic molecular analysis essential elements of a comprehensive strategy to advance understanding of SOA formation pathways. Uncertainties in forecasting SOA production on regional scales are large and related to uncertainties in biogenic emission inventories and measurement of SOA yields under ambient conditions. This work presents a bottom-up approach to develop a conifer emission inventory based on foliar and cortical oleoresin composition, development of a model to estimate terpene and terpenoid signatures of foliar and bole emissions from conifers, development of processing and analytic techniques for comprehensive organic molecular characterization of SOA precursors and oxidation products, implementation of the high-volume sampling technique to measure OA and vapor-phase organic matter, and results from a 5 day field experiment conducted to evaluate temporal and diurnal trends in SOA precursors and oxidation products. A total of 98, 115, and 87 terpene and terpenoid species were identified and quantified in commercially available essential oils of Pinus sylvestris, Picea mariana, and Thuja occidentalis, respectively, by comprehensive, two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC × GC-ToF-MS). Analysis of the literature showed that cortical oleoresin composition was similar to foliar composition of the oldest branches. Our proposed conceptual model for estimation of signatures of terpene and terpenoid emissions from foliar and cortical oleoresin showed that emission potentials of the foliar and bole release pathways are dissimilar and should be considered for conifer species that develop resin blisters or are infested with herbivores or pathogens. Average derivatization efficiencies for Methods 1 and 2 were 87.9 and 114%, respectively. Despite the lower average derivatization efficiency of Method 1, distinct advantages included a greater certainty of derivatization yield for the entire suite of multi- and poly-functional species and fewer processing steps for sequential derivatization. Detection limits for Method 1 using GC × GC- ToF-MS were 0.09-1.89 ng μL-1. A theoretical retention index diagram was developed for a hypothetical GC × 2GC analysis of the complex mixture of SOA precursors and derivatized oxidation products. In general, species eluted (relative to the alkyl diester reference compounds) from the primary column (DB-210) in bands according to n and from the secondary columns (BPX90, SolGel-WAX) according to functionality, essentially making the GC × 2GC retention diagram a Carbon number-functionality grid. The species clustered into 35 groups by functionality and species within each group exhibited good separation by n. Average recoveries of n-alkanes and polyaromatic hydrocarbons (PAHs) by Soxhlet extraction of XAD-2 resin with dichloromethane were 80.1 ± 16.1 and 76.1 ± 17.5%, respectively. Vehicle emissions were the common source for HSVOCs [i.e., resolved alkanes, the unresolved complex mixture (UCM), alkylbenzenes, and 2- and 3-ring PAHs]. An absence of monoterpenes at 0600-1000 and high concentrations of monoterpenoids during the same period was indicative of substantial losses of monoterpenes overnight and the early morning hours. Post-collection, comprehensive organic molecular characterization of SOA precursors and products by GC × GC-ToFMS in ambient air collected with ~2 hr resolution is a promising method for determining biogenic and anthropogenic SOA yields that can be used to evaluate SOA formation models.

Development of Vapor Deposited Silica Sol-Gel Particles for a Bioactive Materials System to Direct Osteoblast Behavior

Tissue engineering and regenerative medicine have emerged in an effort to generate replacement tissues capable of restoring native tissue structure and function, but because of the complexity of biologic system, this has proven to be much harder than originally anticipated. Silica based bioactive glasses are popular as biomaterials because of their ability to enhance osteogenesis and angiogenesis. Sol-gel processing methods are popular in generating these materials because it offers: 1) mild processing conditions; 2) easily controlled structure and composition; 3) the ability to incorporate biological molecules; and 4) inherent biocompatibility. The goal of this work was to develop a bioactive vaporization system for the deposition of silica sol-gel particles as a means to modify the material properties of a substrate at the nano- and micro- level to better mimic the instructive conditions of native bone tissue, promoting appropriate osteoblast attachment, proliferation, and differentiation as a means for supporting bone tissue regeneration. The size distribution, morphology and degradation behavior of the vapor deposited sol-gel particles developed here were found to be dependent upon formulation (H2O:TMOS, pH, Ca/P incorporation) and manufacturing (substrate surface character, deposition time). Additionally, deposition of these particles onto substrates can be used to modify overall substrate properties including hydrophobicity, roughness, and topography. Deposition of Ca/P sol particles induced apatite-like mineral formation on both two- and three-dimensional materials when exposed to body fluids. Gene expression analysis suggests that Ca/P sol particles induce upregulation osteoblast gene expression (Runx2, OPN, OCN) in preosteoblasts during early culture time points. Upon further modification-specifically increasing particle stability-these Ca/P sol particles possess the potential to serve as a simple and unique means to modify biomaterial surface properties as a means to direct osteoblast differentiation.