Charge-coupled device operated in a time-delayed integration mode as an approach to high-throughput flow-based single molecule analysis

Single molecule detection (SMD) readouts are particularly attractive for assays geared toward high-throughput processing, because they can potentially reduce assay time by eliminating various processing steps. Unfortunately, most flow-based SMD experiments have generated low throughputs due primarily to the fact that they are configured in single assay formats. The use of a charge-coupled device (CCD) with flow-based SMD can image multiple single molecule assays simultaneously to realize high-throughput processing capabilities. We present, for the first time, the ability to simultaneously track and detect single molecules in multiple microfluidic channels by employing a CCD camera operated in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement. As an example of the technology, we have configured a CCD to operate in a TDI mode to detect single double-stranded DNA molecules (lambda and pBR322) labeled with an intercalating dye (TOTO-3) in a series of microfluidic channels poised on a poly(methyl methacrylate), PMMA, chip. A laser beam was launched into the side of the chip, which irradiated a series of fluidic channels (eight) with the resulting fluorescence imaged onto a CCD. Using this system, we were able to identify single DNA molecules based on the fluorescence burst intensity arising from differences in the extent of dye labeling associated with the DNA molecule length. The CCD/TDI approach allowed increasing sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s (-1) per channel and 2208 molecules s (-1) for an eight channel microfluidic system was demonstrated. Operated in its full capacity, this multichannel format was projected to yield a sample throughput of 1.7 x 10 (7) molecules s (-1), which represents a 170-fold improvement over previously reported single molecule sampling rates.     

Simulation of salinity effects on past, present, and future soil organic carbon stocks

Soil organic carbon (SOC) models are used to predict changes in SOC stocks and carbon dioxide (CO(2)) emissions from soils, and have been successfully validated for non-saline soils. However, SOC models have not been developed to simulate SOC turnover in saline soils. Due to the large extent of salt-affected areas in the world, it is important to correctly predict SOC dynamics in salt-affected soils. To close this knowledge gap, we modified the Rothamsted Carbon Model (RothC) to simulate SOC turnover in salt-affected soils, using data from non-salt-affected and salt-affected soils in two agricultural regions in India (120 soils) and in Australia (160 soils). Recently we developed a decomposition rate modifier based on an incubation study of a subset of these soils. In the present study, we introduce a new method to estimate the past losses of SOC due to salinity and show how salinity affects future SOC stocks on a regional scale. Because salinity decreases decomposition rates, simulations using the decomposition rate modifier for salinity suggest an accumulation of SOC. However, if the plant inputs are also adjusted to reflect reduced plant growth under saline conditions, the simulations show a significant loss of soil carbon in the past due to salinization, with a higher average loss of SOC in Australian soils (55 t C ha(-1)) than in Indian soils (31 t C ha(-1)). There was a significant negative correlation (p < 0.05) between SOC loss and osmotic potential. Simulations of future SOC stocks with the decomposition rate modifier and the plant input modifier indicate a greater decrease in SOC in saline than in non-saline soils under future climate. The simulations of past losses of SOC due to salinity were repeated using either measured charcoal-C or the inert organic matter predicted by the Falloon et al. equation to determine how much deviation from the Falloon et al. equation affects the amount of plant inputs generated by the model for the soils used in this study. Both sets of results suggest that saline soils have lost carbon and will continue to lose carbon under future climate. This demonstrates the importance of both reduced decomposition and reduced plant input in simulations of future changes in SOC stocks in saline soils.     

A Model for Understanding Electromigration-Induced Void Evolution in Dual-Inlaid Cu Interconnect Structures

Electromigration-induced void evolution in various dual-inlaid copper (Cu) interconnect structures was simulated by applying a phenomenological model assisted by Monte Carlo-based simulations, considering the redistribution of heterogeneously nucleated voids and/or pre-existing vacancy clusters at the Cu/dielectric cap interface during electromigration. The results indicate that this model can qualitatively explain the electromigration-induced void evolution observed during experimental in situ secondary-electron microscopy (SEM) investigations as well as in various other reported studies. The electromigration mechanism in Cu interconnect structures and differences in the peculiar electromigration-induced void evolution in various dual-inlaid Cu interconnect structures can be clearly understood based on this model. These findings warrant reinvestigation of technologically important electromigration mechanisms by developing rigorous models based on similar concepts.     

Experimental analysis of a mass storage system

Mass storage systems (MSSs) play a key role in data‐intensive parallel computing. Most contemporary MSSs are implemented as redundant arrays of independent/inexpensive disks (RAID) in which commodity disks are tied together with proprietary controller hardware. The performance of such systems can be difficult to predict because most internal details of the controller behavior are not public. We present a systematic method for empirically evaluating MSS performance by obtaining measurements on a series of RAID configurations of increasing size and complexity. We apply this methodology to a large MSS at Ohio Supercomputer Center that has 16 input/output processors, each connected to four 8 + 1 RAID5 units and provides 128 TB of storage (of which 116.8 TB are usable when formatted). Our methodology permits storage‐system designers to evaluate empirically the performance of their systems with considerable confidence. Although we have carried out our experiments in the context of a specific system, our methodology is applicable to all large MSSs. The measurements obtained using our methods permit application programmers to be aware of the limits to the performance of their codes. Copyright © 2006 John Wiley & Sons, Ltd.     

Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).     

Social identity,safety climate and self-reported accidents among construction workers

The construction industry has one of the highest frequencies of work-related accidents. We examined whether construction workers predominantly identify themselves in terms of their workgroup or in terms of the construction site. In addition, we examined the associations between social identity and safety climate, and how these constructs are associated with work-related accidents. The analyses were based on questionnaire responses from 478 construction workers from two large construction sites, and the methods involved structural equation modeling. Results showed that the workers identified themselves primarily with their workgroup, and to a lesser degree with the construction site. Social identity and safety climate were related both at the workgroup and construction site levels, meaning that social identity may be an antecedent for safety climate. The association between social identity and safety climate was stronger at the workgroup level than at the construction site level. Finally, safety climate at both levels was inversely associated with self-reported accidents, with the strongest association at the workgroup level. A focus on improving safety climate, particularly by integrating initiatives at both the workgroup and management level, may have the potential to improve safety performance and thus decrease the risk of accidents and injuries on construction sites.     

Hybridity in the housing sector: examining impacts on social and private rented sector tenants in Scotland

Abstract

Housing Associations in many countries exhibit increasing levels of ‘hybridity’, as reductions in state financing for social housing, exacerbated by austerity policies since the 2008 crash, have instigated ‘enterprising’ approaches to maintaining income. Alongside this, hybrid organisations have emerged in the Private Rented Sector (PRS), responding to sectoral growth and consequent increases in vulnerable households entering private renting. These developing hybridities have been considered at a strategic level, but there has been little exploration of the impacts on tenants. This article examines two organisations, operating across the social and private rented sectors, to elucidate potential implications for tenants. The research suggests that different forms of hybridity can affect tenant outcomes and, moreover, that examining such impacts is important in understanding hybridity itself. Furthermore, the study suggests that emerging forms of hybridity, particularly in the PRS, may be blurring the boundaries between housing sectors, with implications for policy and research.     

Contact conductivity detection in poly(methyl methacrylate)-based microfluidic devices for analysis of mono- and polyanionic molecules

An on-column contact conductivity detector was developed for the analysis of various mono- and polyanionic compounds separated by electrophoresis chips fabricated in poly(methyl methacrylate) (PMMA) using hot embossing techniques from Ni electroforms. The detector consisted of a pair of Pt wires (127 microm diameter) with an end-to-end spacing of approximately 20 microm and situated within the fluidic channel. The waveform applied to the electrode pair was a bipolar pulse with a frequency of 5.0 kHz and was used to reduce the charging current from measurement so that the current recorded at the end of one pulse is more representative of the solution conductivity. Using the detector, separations of amino acids, peptides, proteins, and oligonucleotides were demonstrated. For the amino acids and peptides, free-solution zone electrophoresis was performed. A calibration plot for the amino acid alanine was found to be linear from approximately 10 to 100 nM in a carrier electrolyte consisting of 10 mM triethylamonium acetate. The concentration detection limit was found to be 8.0 nM, with the corresponding mass detection limit equal to 3.4 amol (injection volume = 425 pL). The protein separations with conductivity detection were performed using MEKC, in which the carrier electrolyte contained the anionic surfactant sodium dodecyl sulfate (SDS) above its cmc. Near baseline resolution was achieved in the PMMA microchip for a solution containing 8 different proteins. In the case of the DNA fragments, capillary electrochromatography was used with a C18-modified PMMA chip and a carrier electrolyte containing an ion-pairing agent.     

Useful matrix theorem with applications to electronic circuit theory

A new matrix theorem is described which has applications to electronic circuit theory and control theory.