Older drivers' insight into their hazard perception ability

Even though the driving ability of older adults may decline with age, there is evidence that some individuals attempt to compensate for these declines using strategies such as restricting their driving exposure. Such compensatory mechanisms rely on drivers’ ability to evaluate their own driving performance. This paper focuses on one key aspect of driver ability that is associated with crash risk and has been found to decline with age: hazard perception. Three hundred and seven drivers, aged 65–96, completed a validated video-based hazard perception test. There was no significant relationship between hazard perception test response latencies and drivers’ ratings of their hazard perception test performance, suggesting that their ability to assess their own test performance was poor. Also, age-related declines in hazard perception latency were not reflected in drivers’ self-ratings. Nonetheless, ratings of test performance were associated with self-reported regulation of driving, as was self-rated driving ability. These findings are consistent with the proposal that, whileself-assessments of driving ability may be used by drivers to determine the degree to which they restrict their driving, the problem is that drivershave little insight into their own driving ability. This may impact on the potential road safety benefits of self-restriction of driving because drivers may not have the information needed to optimally self-restrict. Strategies for addressing this problem are discussed.     

Study of metal barrier deposition-induced damage to porous low-k materials

A unique test structure based on a metal-insulator-semiconductor planar capacitor (Pcap) design was used to investigate several aspects of metal barrier-induced low-k damage. A special term called Effective Damage Thickness was introduced to describe the degree of damage. Ta(N) barrier was deposited on various dielectric films with porosity up to 32%. It has been found that the Effective Damage Thickness increases as the porosity increases. The damage is influenced more by the porosity of low-k films than the film density. Furthermore, the damage was modulated by Ta(N) deposition conditions. More damage was observed when higher target and/or substrate bias power was used, suggesting that the ion energy of the barrier material plays an important role in the low-k damage mechanism. A same degree of damage was observed for Ta barrier as for Ta(N), suggesting that Ta(N) deposition-induced low-k damage was primarily caused by Ta ions not nitrogen. Impact of Ru(Ta) and Cu(Mn) self forming barrier on low-k damage was also investigated. Among all the barriers studied in this work, the Ta-based barriers caused the most damage while the Cu(Mn) self forming barrier had the least damage to the low-k. The atomic masses for Ta, Ru, and Cu are 181, 101, and 64, respectively, corresponding with the observed degree of damage in the low-k material.     

Selection and separation of viable cells based on a cell-lethal assay

A method to select and separate viable cells based on the results of a cell-lethal assay was developed. Cells were plated on an array of culture sites with each site composed of closely spaced, releasable micropallets. Clonal colonies spanning multiple micropallets on individual culture sites were established within 72 h of plating. Adjacent sites were widely spaced with 100% of the colonies remaining sequestered on a single culture site during expansion. A laser-based method mechanically released a micropallet underlying a colony to segment the colony into two genetically identical colonies. One portion of the segmented colony was collected with 90% efficiency while viability of both fractions was 100%. The segmented colonies released from the array were fixed and subjected to immunofluorescence staining of intracellular phospho-ERK kinase to identify colonies that were highly resistant or sensitive to phorbol ester-induced activation of ERK. These resistant and sensitive cells were then matched to the corresponding viable colonies on the array. Sensitive and resistant colonies on the array were released and cultured. When these cultured cells were reanalyzed for phorbol ester-induced ERK activity, the cells retained the sensitive or resistant phenotype of the originally screened subcolony. Thus, cells were separated and collected based using the result of a cell-lethal assay as selection criteria. These microarrays enabling clonal colony segmentation permitted sampling and manipulation of the colonies at very early times and at small cell numbers to reduce reagent, time, and manpower requirements.     

Relative humidity sensors based on an environment-sensitive fluorophore in hydrogel films

A fluorescence-based sensing scheme exploiting an environment-sensitive fluorophore embedded in a hydrogel has been developed for measurement of relative humidity (RH). The fluorophore, dapoxyl sulfonic acid (DSA), is incorporated into two different hydrogel films, agarose and a copolymer of acrylamide and 2-(dimethylamino)ethyl methacrylate (DMAEM) cross-linked with N,N'-methylenebisacrylamide. The swelling and contracting of the hydrogels in response to relative humidity alters the polarity of the environment of DSA, stimulating a shift in the emission wavelength. From 0 to 100% RH, acrylamide-DMAEM sensors exhibited a 40 and 15 nm wavelength shift in still air and flowing gas, respectively. Agarose sensors showed a 40 nm wavelength shift from 0 to 100% RH in still air and a 30 nm shift from 0 to 70% RH in flowing gas. Response times for both sensors were 15 min in still air and less than 5 min in flowing gas. The sensing approach is straightforward and cost-effective, yields sensors with characteristics suitable for commercial measurement of RH (i.e., sensitivity, response times, reproducibility), and allows ease of adaptability to specific RH measurement requirements. The results support the potential extension of the method to a wide variety of analytes in the vapor phase and aqueous solution by incorporation of functionalized "smart" hydrogels.     

Development of soluble ester-linked aldehyde polymers for proteomics

High molecular weight hyperbranched polyglycerol (HPG) was selected for development as a soluble polymer support for the targeted selection and release of primary-amine containing peptides from a complex mixture. HPG has been functionalized with ester-linked aldehyde groups that can bind primary-amine containing peptides via a reductive alkylation reaction. Once bound, the high molecular weight of the polymer facilitates separation from a complex peptide mixture by employing either a 30 kDa molecular weight cutoff membrane or precipitation in acetonitrile. Following the removal of unbound peptides and reagents, subsequent hydrolysis of the ester linker releases the bound peptide into solution for analysis by mass spectrometry. Released peptides retain the linker moiety and are therefore characteristically mass-shifted. Four water-soluble cleavable aldehyde polymers (CAP1, CAP2, CAP3, and CAP4) ranging in types of linker groups, length of the linker groups, have been prepared and characterized, each demonstrating the ability to selectively enrich and sequence primary-amine peptides from a complex human proteome containing blocked (dimethylated amine) and unblocked (primary amine) peptides. The polymers have very low nonspecific peptide-binding properties while possessing significantly more reactive groups per milligram of the support than commercially available resins. The polymers exhibit a range of reactivities and binding capacities that depend on the type of linker group between the aldehyde group and the polymer. Using various linker structures, we also probed the mechanism of the observed dehydration of hydrolyzed peptides during matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis.     

Surface plasmon resonance biosensor screening method for paralytic shellfish poisoning toxins: a pilot interlaboratory study

A surface plasmon resonance (SPR) optical biosensor method was developed for the detection of paralytic shellfish poisoning (PSP) toxins in shellfish. This application was transferred in the form of a prototype kit to seven laboratories using Biacore Q SPR optical biosensor instrumentation for interlaboratory evaluation. Each laboratory received 20 shellfish samples across a range of species including blind duplicates for analysis. The samples consisted of 4 noncontaminated samples spiked in duplicate with a low level of PSP toxins (240 μg STXdiHCl equivalents/kg), a high level of saxitoxin (825 μg STXdiHCl/kg), 2 noncontaminated, and 14 naturally contaminated samples. All 7 participating laboratories completed the study, and HorRat values obtained were <1 demonstrating that the method performance was acceptable. Mean recoveries expressed as STXdiHCl equivalents/kg were 94.6 ± 16.8% for the low level PSP toxin mix and 98.6 ± 5.6% for the high level of saxitoxin. Relative standard deviations for within-laboratory variations (RSD(r): repeatability) and between-laboratory variations (RSD(R) = reproducibility) ranged from 1.8 to 9.6% and 2.9 to 18.3% respectively. This first ever reported SPR biosensor interlaboratory study demonstrated this PSP application to be an empowering tool in the drive toward the reduction and replacement of the mouse bioassay within Europe.     

Extraction chromatographic methods in the sample preparation sequence for thermal ionization mass spectrometric analysis of plutonium isotopes

A sample preparation sequence for actinide isotopic analysis by thermal ionization mass spectrometry (TIMS) is described that includes column-based extraction chromatography as the first separation step, followed by anion-exchange column separations. The sequence is designed to include a wet ashing step after the extraction chromatography to prevent any leached extractant or oxalic acid eluent reagents from interfering with subsequent separations, source preparation, or TIMS ionization. TEVA resin and DGA resin materials, containing extractants that consist only of C, N, O, and H atoms, were investigated for isolation of plutonium. Radiotracer level studies confirmed expected high yields from column-based separation procedures. Femtogram-level studies were carried out with TIMS detection, using multiple monoisotopic spikes applied sequentially throughout the separation sequence. Pu recoveries were 87% and 86% for TEVA and DGA resin separations, respectively. The Pu recoveries from 400 μL anion-exchange column separation sequences were 89% and 93% for trial sequences incorporating TEVA and DGA resin. Thus, a prior extraction chromatography step in the sequence did not interfere with the subsequent anion-exchange separation when a simple wet ash step was carried out in between these column separations. The average measurement efficiency for Pu, encompassing the chemical separation recoveries and the TIMS ionization efficiency, was 2.73% ± 0.77% (2σ) for the DGA resin trials and 2.67% ± 0.54% for the TEVA resin trials, compared to 3.41% and 2.37% (average 2.89%) for two control trials. These compare with an average measurement efficiency of 2.78% ± 1.70%, n = 33 from process benchmark analyses using Pu spikes processed through a sequence of oxalate precipitation, wet ash, iron hydroxide precipitation, and anion-exchange column separations. We conclude that extraction chromatography can be a viable separation procedure as part of a multistep sequence for TIMS sample preparation.     

Photothermal nanoblade for large cargo delivery into mammalian cells

It is difficult to achieve controlled cutting of elastic, mechanically fragile, and rapidly resealing mammalian cell membranes. Here, we report a photothermal nanoblade that utilizes a metallic nanostructure to harvest short laser pulse energy and convert it into a highly localized explosive vapor bubble, which rapidly punctures a lightly contacting cell membrane via high-speed fluidic flows and induced transient shear stress. The cavitation bubble pattern is controlled by the metallic structure configuration and laser pulse duration and energy. Integration of the metallic nanostructure with a micropipet, the nanoblade generates a micrometer-sized membrane access port for delivering highly concentrated cargo (5 × 10(8) live bacteria/mL) with high efficiency (46%) and cell viability (>90%) into mammalian cells. Additional biologic and inanimate cargo over 3-orders of magnitude in size including DNA, RNA, 200 nm polystyrene beads, to 2 μm bacteria have also been delivered into multiple mammalian cell types. Overall, the photothermal nanoblade is a new approach for delivering difficult cargo into mammalian cells.     

Valence parity to distinguish c' and z• ions from electron capture dissociation/electron transfer dissociation of peptides: effects of isomers, isobars, and proteolysis specificity

Valence parity provides a way to distinguish between N-terminal and C-terminal electron capture dissociation/electron transfer dissociation (ECD/ETD) product ions based on their number of hydrogen plus nitrogen atoms determined by accurate mass measurement and forms a basis for de novo peptide sequencing. The effect of mass accuracy (0.1-1 ppm error) on c'/z(?) overlap and unique elemental composition overlap is evaluated for a database of c'/z(?) product ions each based on all possible amino acid combinations and four subset databases containing the same c' ions but with z(?) ions determined by in silico digestion with trypsin, Glu-C, Lys-C, or chymotrypsin. High mass accuracy reduces both c'/z(?) overlap and unique elemental composition overlap. Of the four proteases, trypsin offers slightly better discrimination between N- and C-terminal ECD/ETD peptides. Interestingly, unique elemental composition overlap curves for c'/c' and z(?)/z(?) peptide ions exhibit discontinuities at certain nominal masses for 0.1-1.0 ppm mass error. Also, as noted in the companion article (Polfer et al. Anal. Chem.2011, DOI: 10.1021/ac201624t), the number of ECD/ETD product ion amino acid compositions as a function of nominal mass increases exponentially with mass but with a superimposed modulation due to higher prevalence of certain elemental compositions.     

Immuno-hybridization chain reaction for enhancing detection of individual cytokine-secreting human peripheral mononuclear cells

We present here a new method to enhance the detection of secreted cytokines and chemokines from single human mononuclear cells. The technique uses a hybridization chain reaction (HCR) to amplify signals resulting from sandwich immunoassays. This immuno-HCR employs oligonucleotide-based initiators covalently linked to antibodies to propagate a chain reaction of hybridization events involving a pair of complementary hairpin oligomers bearing fluorescent labels. Integrating this strategy for signal amplification with microengraving (a soft lithographic method for printing arrays of secreted proteins from thousands of single cells) improves both the limits of detection and sensitivity for cytokines and chemokines captured from individual cells by an average of 200-fold relative to methods for direct detection by fluoresence. This approach should enhance the utility of microengraving for defining the immunological signatures of diseases and responses to interventional therapies based on multiplexed single-cell analysis.