A yeast surface display platform for plant hormone receptors: Toward directed evolution of new biosensors

Small-molecule biosensors have major applications in biotechnology and medicine but remain difficult to engineer. Plant hormone receptors represent an attractive platform for engineering such biosensors because their chemically induced dimerization architectures naturally decouple small-molecule sensing and sensor actuation. Rapid biosensor engineering will require quantitative high-throughput screening methods. Here we develop a yeast surface display (YSD) platform for the PYR1/HAB1 abscisic acid sensor of Arabidopsis thaliana. We extensively optimized PYR1 surface display, HAB1 purification, and binding reaction conditions. Our system reproduces previous results with wild-type and engineered receptors, and a mathematical analysis of the PYR1/HAB1 system allows us to infer all binding constants. Critically, we find that a previously engineered PYR1 receptor with altered ligand specificity binds HAB1 with identical affinity, suggesting that substantial reengineering of the PYR1 binding pocket does not compromise sensor actuation. This YSD platform for A. thaliana PYR1/HAB1 will facilitate future biosensor engineering efforts.     

High-Toughness Silicon Carbide as Armor

Silicon carbide, with single-edge precracked beam (SEPB) toughness greater than 7 MPa·m^1/2, was made by hot-pressing using Al–B–C (ABC) or Al–Y₂O₃ (YAG) as additives. The hardness of SiC processed with a liquid phase was always less than SiC densified without a liquid phase despite having a similar or finer grain size. With increasing Al content, the ABC system changed from trans- to intergranular fracture with a drop in hardness and a two- to threefold increase in SEPB toughness. Strength and Weibull modulus for materials processed with a liquid phase were higher than those of solid-state densified SiC. Ballistic testing, however, did not show any improvement over SiC densified with B and C additives. Depth of penetration was controlled by hardness of the SiC-based materials, while V ₅₀ values for 14.5 mm WC–Co cored projectiles were in the range of 720–750 m/s for all materials tested.     

R Curves and Crack-Stability Map: Application to Ce-TZP/Al2O3

The concept of a crack-stability map is developed by considering the interaction between the crack-driving force and the rising crack-growth resistance of a toughened ceramic. The map plots normalized transition crack length as function of the ratio of the crack-initiation fracture toughness and the plateau toughness to delineate regimes of stable and unstable crack growth. The plot is used to analyze R curves and fracture stresses of a transformation-toughened Ce-TZP/Al₂O₃. It is shown that the fracture stress and the small scatter measured for this ceramic are consistent with its R- curve behavior, which enables stable growth of surface cracks from flaws (pores and second-phase particles), leading to a flaw-insensitive ceramic.     

Growth of Low-Density Vertical Quantum Dot Molecules with Control in Energy Emission

In this work, we present results on the formation of vertical molecule structures formed by two vertically aligned InAs quantum dots (QD) in which a deliberate control of energy emission is achieved. The emission energy of the first layer of QD forming the molecule can be tuned by the deposition of controlled amounts of InAs at a nanohole template formed by GaAs droplet epitaxy. The QD of the second layer are formed directly on top of the buried ones by a strain-driven process. In this way, either symmetric or asymmetric vertically coupled structures can be obtained. As a characteristic when using a droplet epitaxy patterning process, the density of quantum dot molecules finally obtained is low enough (2 × 10⁸ cm⁻²) to permit their integration as active elements in advanced photonic devices where spectroscopic studies at the single nanostructure level are required.     

Materials for double patterning strategies: Development and application

Double patterning has become the most promising approach to overcome the 32 nm node challenges. Several schemes have been proposed to simplify the process, each requiring very specific materials that still have to be developed and optimized. The resist platform presented here is an image lock material which has been developed to meet the Litho-Litho-Etch (or 2P1E) approach. In this paper, we present the material development of dedicated polymers for the double imaging technique. The lithographic properties of these materials are evaluated, in term of process window (PW) and Line Edge Roughness (LER). Successful patterning of 50 and 45 nm lines at 90 nm pitch has been obtained.     

In vitro translation of a 2.3-kb splicing variant of the hamster pgp1 gene whose presence in transfectants is associated with decreased drug resistance

Adherent macrophage populations derived from monocytes isolated from peripheral blood were evaluated for their ability to "shed" the membrane-associated receptor for TNF-alpha (TNFR) following exposure to a calcium ionophor (A23187) and a synthetic chemotactic peptide (fMLP) reagent. A soluble fraction of TNFR was detected in "cell-free" supernatant produced by stimulated macrophage populations applying 125I-TNF-alpha and biotinylated TNF-alpha ligand-binding analysis (96-well format) in combination with conventional autoradiographic techniques. Approximate molecular weight of the shed TNFR glycoprotein fraction was estimated to be 75 kDa based on interpretation of nondenaturing PAGE gels transferred laterally onto sheets of nitrocellulose membrane subsequently probed by ligand-binding analysis applying 125I-TNF-alpha and biotinylated TNF-alpha as detection modalities. Immunorecognition techniques were also employed to detect TNFR fragments shed from macrophages using biotinylated anti-TNFR Type II (75 kDa) monoclonal antibody in combination with conjugated strepavidin:HRPO and a chemiluminescent substrate reagent. In an effort to identify the class of enzyme directly mediating TNFR Type II (75 kDa) shedding, a spectrum of carboxyl- (e.g., aspartate), hydroxyl- (e.g., serine), thiol (e.g., cysteine), and metalo- (e.g., Ca2+, Mg2+) protease-inhibiting agents were evaluated. Experimental findings implied that a carboxy (aspartate) peptidase, and possibly to a lesser extent, serine (hydroxyl), and thiol (cysteine) peptidases participate in macrophage TNFR Type II (75 kDa) shedding phenomena. Subsequent investigations demonstrated that the carboxy (aspartate) peptidase cathepsin-D promoted liberation of TNFR Type II (75 kDa) in unactivated populations of adherent macrophages. In an effort to complement these observations, a protein fraction with presumed carboxy (aspartate) protease activity was isolated from the cell-free supernatant generated by activated populations of adherent macrophages using immobilized pepstatin-A beaded agarose. Exposure of unstimulated populations of adherent macrophages to the partially purified pepstatin-A binding protein fractions resulted in the liberation of a soluble TNFR Type II (75 kDa) fragment based on interpretation of ligand-binding and immunorecognition analysis of samples developed by SDS-PAGE/PAGE format and transferred onto sheets of nitrocellulose membrane. The molecular weight of the macrophage pepstatin-A binding protein fraction was estimated to be 47-52 kDa with lesser bands also visible at approximately 26-32 kDa, and 100 kDa based on SDS-PAGE analysis. Nondenaturing hemoglobin-PAGE substrate gel analysis of protein fractions possessing pepstatin-A binding-avidity detected a protease with a molecular weight of approximately 47-52 kDa that proteolytically digested hemoglobin, in addition to a synthetic cathepsin-D specific peptide substrate. Collective interpretation of these experimental findings directly corresponds with many of the physical (molecular) and functional (biochemical) characteristics known to be associated with the leukocyte carboxy (aspartate) peptidase cathepsin-D, which is a non-metaloprotease known to exert relatively limited proteolytic activity.     

Coding mammograms using the classification "probably benign finding--short interval follow-up suggested"

OBJECTIVE: Many benign breast lesions revealed by mammography show features indicating that the lesions have a high, but not complete, likelihood of being benign. The Breast Imaging Reporting and Data System (BI-RADS) allows radiologists to classify these mammograms as "probably benign finding-short interval follow-up suggested" (category 3). We explored whether certain factors are associated with the use of category 3 in a national cancer detection program. MATERIALS AND METHODS: We analyzed data from the National Breast and Cervical Cancer Early Detection Program, a comprehensive nationwide program that provides cancer screening for low-income and medically underserved women. The study population included all women at least 40 years old who had undergone mammography on or before September 30, 1996 (n = 372,760). RESULTS: Of the 372,760 mammograms, 7.7% were classified as category 3. The probability of receiving a category 3 classification decreased as patients' ages increased. Women who were symptomatic were nearly twice as likely as women who were asymptomatic to receive a category 3 classification, and women whose clinical breast examinations had abnormal findings were more than twice as likely as women with examinations having normal findings to receive a category 3 classification. The percentage of mammograms classified as category 3 by state or tribal organization ranged from 1.4% to 14.0%. CONCLUSION: Several patient variables, including patient symptomatology, were associated with the probability of having a mammogram classified as category 3. One of the most important determinants was where the patient underwent mammography, which suggests that variability exists among radiologists themselves in using this BI-RADS code for "probably benign" mammographic lesions.     

Double-blind photoarray administration as a safeguard against investigator bias.

[Correction Notice: An erratum for this article was reported in Vol 85(2) of Journal of Applied Psychology (see record 2007-17253-001). On page 947, the graphs within Figure 1 were incorrectly labeled. The corrected figure appears in this erratum.] This experiment examined whether a photoarray administrator's knowledge of a suspect's identity increased false identification rates. Fifty participant–administrators (PAs) presented 50 participant–witnesses (PWs) two perpetrator-absent photoarrays following a live staged crime involving two perpetrators. For one photoarray per trial, the experimenter revealed the suspect's identity to the PA. Each PA presented the photoarrays sequentially or simultaneously in the presence or absence of an observer. When the observer was present, PA knowledge of the suspect's identity had a biasing effect in sequential photoarrays only. This pattern did not emerge when the observer was absent. The experimental manipulations did not affect PAs' and PWs' ratings of photoarray fairness or PWs' ratings of pressure to make an identification. These data suggest that only administrators who are blind to the suspect's identity should present sequential photoarrays. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The effectiveness of early endoscopy for upper gastrointestinal hemorrhage: a community-based analysis

OBJECTIVES: The effectiveness of upper endoscopy in unselected patients with upper gastrointestinal hemorrhage has not been well studied. This study was undertaken to identify factors associated with the performance of early endoscopy (ie, within 1 day of hospitalization) and, after adjusting for these factors, to determine associations between early endoscopy and in-hospital mortality, length of stay, and performance of surgery. METHODS: Subjects in this observational cohort study were 3,801 consecutive admissions with upper gastrointestinal hemorrhage to 30 hospitals in a large metropolitan region. Demographic and clinical data were abstracted from hospital records. A multivariable model based on factors that potentially could relate to the decision to perform endoscopy was developed to determine the propensity (0 to 100%) for early endoscopy in each patient. RESULTS: Early endoscopy was performed in 2,240 patients (59%), and although it was not associated with mortality after adjusting for severity of illness among all patients, it was associated with a higher risk of death for patients in the lowest propensity group. Early endoscopy was associated with a lower likelihood of upper gastrointestinal surgery in all patients and in the two highest propensity groups and with a shorter length of stay in the entire cohort and in all subgroups. CONCLUSIONS: In the absence of specific contraindications, early endoscopy should be considered because of associated reductions in length of stay and surgical intervention. Further studies are needed to identify subgroups in whom the procedure may be associated with adverse effects on survival.