In Vivo Behavior of Ultrasmall Spherical Nucleic Acids

The biological properties of spherical nucleic acids (SNAs) are largely independent of nanoparticle core identity but significantly affected by oligonucleotide surface density. Additionally, the payload-to-carrier (i.e., DNA-to-nanoparticle) mass ratio of SNAs is inversely proportional to core size. While SNAs with many core types and sizes have been developed, all in vivo analyses of SNA behavior have been limited to cores >10 nm in diameter. However, “ultrasmall” nanoparticle constructs (<10 nm diameter) can exhibit increased payload-to-carrier ratios, reduced liver accumulation, renal clearance, and enhanced tumor infiltration. Therefore, we hypothesized that SNAs with ultrasmall cores exhibit SNA-like properties, but with in vivo behavior akin to traditional ultrasmall nanoparticles. To investigate, we compared the behavior of SNAs with 1.4-nm Au₁₀₂ nanocluster cores (AuNC-SNAs) and SNAs with 10-nm gold nanoparticle cores (AuNP-SNAs). Significantly, AuNC-SNAs possess SNA-like properties (e.g., high cellular uptake, low cytotoxicity) but show distinct in vivo behavior. When intravenously injected in mice, AuNC-SNAs display prolonged blood circulation, lower liver accumulation, and higher tumor accumulation than AuNP-SNAs. Thus, SNA-like properties persist at the sub-10-nm length scale and oligonucleotide arrangement and surface density are responsible for the biological properties of SNAs. This work has implications for the design of new nanocarriers for therapeutic applications.     

Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: plant-specific mononuclear blue copper proteins

The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arabidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant-specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arabidopsis, while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper-binding amino acids. The recombinant copper binding domain of Arabidopsis uclacyanin 1 was expressed, purified, and shown to bind a copper atom in a fashion known as "blue" or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin 1 rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild-type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron-transfer proteins participating in long-range electron-transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation.     

The Team Role Test: Development and validation of a team role knowledge situational judgment test.

The main objectives in this research were to introduce the concept of team role knowledge and to investigate its potential usefulness for team member selection. In Study 1, the authors developed a situational judgment test, called the Team Role Test, to measure knowledge of 10 roles relevant to the team context. The criterion-related validity of this measure was examined in 2 additional studies. In a sample of academic project teams (N = 93), team role knowledge predicted team member role performance (r = .34). Role knowledge also provided incremental validity beyond mental ability and the Big Five personality factors in the prediction of role performance. The results of Study 2 revealed that the predictive validity of role knowledge generalizes to team members in a work setting (N = 82, r = .30). The implications of the results for selection in team environments are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Mass spectrometric techniques for label-free high-throughput screening in drug discovery

High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.     

Measurement of acetaldehyde in exhaled breath using a laser absorption spectrometer

A high-resolution liquid-nitrogen-free mid-infrared tunable diode laser absorption spectroscopy (TDLAS) system was used to perform real-time measurement of acetaldehyde concentrations in human exhaled breath following ingestion of an alcoholic beverage. Acetaldehyde absorption features were measured near 5.79 mum (1727 cm(-1)) using a IV-VI semiconductor laser, a 100 m long path optical gas cell, and second- harmonic detection coupled with wavelength modulation. Acetaldehyde levels were measured with a minimum detection limit of 80 ppb for 5 s integration time. The variations in exhaled acetaldehyde levels over time were analyzed prior to and following ingestion of two different amounts of white wine. A method to calibrate acetaldehyde measurements internally using water vapor absorption lines was investigated to eliminate the need for system calibration with gas standards. The potential of a TDLAS system to be used as a noninvasive clinical tool for measurements of large volatile compounds with possible applications in cancer detection is demonstrated.     

Compact multimodal adaptive-optics spectral-domain optical coherence tomography instrument for retinal imaging

We have developed a compact, multimodal instrument for simultaneous acquisition of en face quasi-confocal fundus images and adaptive-optics (AO) spectral-domain optical coherence tomography (SDOCT) cross-sectional images. The optical system including all AO and SDOCT components occupies a 60x60 cm breadboard that can be readily transported for clinical applications. The AO component combines a Hartmann-Shack wavefront sensor and a microelectromechanical systems-based deformable mirror to sense and correct ocular aberrations at 15 Hz with a maximum stroke of 4 microm. A broadband superluminescent diode source provides 4 mum depth resolution for SDOCT imaging. In human volunteer testing, we observed up to an 8 dB increase in OCT signal and a corresponding lateral resolution of <10 microm as a result of AO correction.     

Oligonucleotide loading determines cellular uptake of DNA-modified gold nanoparticles

The cellular internalization of oligonucleotide-modified nanoparticles is investigated. Uptake is dependent on the density of the oligonucleotide loading on the surface of the particles, where higher densities lead to greater uptake. Densely functionalized nanoparticles adsorb a large number of proteins on the nanoparticle surface. Nanoparticle uptake is greatest where a large number of proteins are associated with the particle.