Automated Fast Screening Method for Cocaine Identification in Seized Drug Samples Using a Portable Fourier Transform Infrared (FT-IR) Instrument.

Quick and presumptive identification of seized drug samples without destroying evidence is necessary for law enforcement officials to control the trafficking and abuse of drugs. This work reports an automated screening method to detect the presence of cocaine in seized samples using portable Fourier transform infrared (FT-IR) spectrometers. The method is based on the identification of well-defined characteristic vibrational frequencies related to the functional group of the cocaine molecule and is fully automated through the use of an expert system. Traditionally, analysts look for key functional group bands in the infrared spectra and characterization of the molecules present is dependent on user interpretation. This implies the need for user expertise, especially in samples that likely are mixtures. As such, this approach is biased and also not suitable for non-experts. The method proposed in this work uses the well-established "center of gravity" peak picking mathematical algorithm and combines it with the conditional reporting feature in MicroLab software to provide an automated method that can be successfully employed by users with varied experience levels. The method reports the confidence level of cocaine present only when a certain number of cocaine related peaks are identified by the automated method. Unlike library search and chemometric methods that are dependent on the library database or the training set samples used to build the calibration model, the proposed method is relatively independent of adulterants and diluents present in the seized mixture. This automated method in combination with a portable FT-IR spectrometer provides law enforcement officials, criminal investigators, or forensic experts a quick field-based prescreening capability for the presence of cocaine in seized drug samples.

Role of insulin receptor and insulin signaling on αPS2CßPS integrins' lateral diffusion.

Integrins are ubiquitous transmembrane receptors with adhesion and signaling properties. The influence of insulin receptor and insulin signaling on αPS2CßPS integrins' lateral diffusion was studied using single particle tracking in S2 cells before and after reducing the insulin receptor expression or insulin stimulation. Insulin signaling was monitored by Western blotting for phospho-Akt expression. The expression of the insulin receptor was reduced using RNA interference (RNAi). After insulin receptor RNAi, four significant changes were measured in integrin diffusion properties: (1) there was a 24% increase in the mobile integrin population, (2) 14% of the increase was represented by integrins with Brownian diffusion, (3) for integrins that reside in confined zones of diffusion, there was a 45% increase in the diameter of the confined zone, and (4) there was a 29% increase in the duration integrins spend in confined zones of diffusion. In contrast to reduced expression of the insulin receptor, which alters integrin diffusion properties, insulin stimulation alone or insulin stimulation under conditions of reduced insulin receptor expression have minimal effects on altering the measured integrin diffusion properties. The differences in integrin diffusion measured after insulin receptor RNAi in the presence or absence of insulin stimulation may be the result of other insulin signaling pathways that are activated at reduced insulin receptor conditions. No change in the average integrin diffusion coefficient was measured for any conditions included in this study.

Development of a comprehensive near infrared spectroscopy calibration model for rapid measurements of moisture content in multiple pharmaceutical products.

Near infrared (NIR) spectroscopy has been widely used for the determination of water content in a wide variety of samples. With few exceptions, all methods employ a calibration model developed and applicable for a single product. The current study describes a NIR method using a single, comprehensive calibration model to predict the water content in tablets containing different active pharmaceutical ingredients (API). The calibration model was developed for water content range of 2-13% (w/w) using tablets containing three different APIs and different formulation compositions. To develop a robust comprehensive model, individual calibration models were sequentially developed starting from a simple model for one product to including tablets from all three projects in the final model using partial least square analysis method. Data pretreatments and spectral region selections were performed during the method development to optimize the number of factors and the correlation coefficients for cross-validation and prediction by the comprehensive model. The model reliably predicted the water content in tablet samples of these three products, and can be updated for water measurements of new drug products by adding to the model two samples of the new product for calibration purpose.

Select cytoplasmic and membrane proteins increase the percentage of immobile integrins but do not affect the average diffusion coefficient of mobile integrins.

Integrins are ubiquitous adhesion receptors that are important for signaling and integrating the extracellular matrix and cytoskeleton. The role of cytoplasmic proteins vinculin, focal adhesion kinase (FAK), integrin-linked kinase (ILK), and membrane proteins epidermal growth factor receptor (EGFR) and Notch in altering αPS2CßPS integrin lateral diffusion was measured using single particle tracking (SPT) and RNA interference (RNAi). SPT measures heterogeneous diffusion properties, and RNAi selectively reduces the concentration of a target protein. After systematically reducing the concentration of vinculin, FAK, ILK, EGFR, or Notch, there was a 31 to 80% increase in the mobile integrin fraction, indicating that these five targeted proteins (or assemblies that contain these proteins) are responsible for immobilizing a fraction of the integrins when all proteins are present at native concentrations. The average diffusion coefficient of all mobile integrins did not change after any of the RNAi treatments, and the percentage of Brownian, directed, or anomalous/constrained trajectories relative to total mobile trajectories did not change after vinculin or EGFR RNAi. However, the fraction of anomalous/constrained trajectories relative to the total mobile trajectories increased 9 to 19% after FAK, ILK, and Notch RNAi, when the concentration of these proteins was reduced. In the case of FAK, ILK, and Notch, native concentrations of these proteins simultaneously increase the immobile fraction of integrins but decrease the diffusion constraints to those integrins that remain mobile. Comparisons of single receptor and ensemble measurements of diffusion and what is known about the effect of these proteins in altering integrin clustering are discussed.

The effect of ligand affinity on integrins' lateral diffusion in cultured cells.

The role of ligand affinity in altering αPS2CßPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CßPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CßPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CßPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CßPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

Unraveling the role of membrane proteins Notch, Pvr, and EGFR in altering integrin diffusion and clustering.

The role of three membrane proteins in altering the diffusion and clustering of integrin receptors has been measured. Integrins are membrane proteins responsible for integrating intracellular and extracellular signaling events and anchoring cells to the extracellular matrix. The methodology used to elucidate the role of other membrane proteins in altering integrin diffusion and clustering combines fluorescence microscopy with RNA interference (RNAi), which is a technique to reduce the expression of a target protein. The three RNAi-targeted membrane proteins were epidermal growth factor receptor (EGFR), platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr), and Notch. Real-time polymerase chain reaction or quantitative immunocytochemistry was used to measure a reduction in mRNA or protein concentration after RNAi treatment, respectively. Fluorescence recovery after photobleaching showed that reducing the concentration of EGFR or Notch results in less constrained integrin diffusion and, in the case of Notch RNAi, 4 % more mobile integrins. Fluorescence resonance energy transfer measurements performed before and after RNAi treatments indicate that clustering decreases for wild-type integrin, but increases for a high-ligand-affinity integrin mutant after reducing the expression of EGFR, Pvr, or Notch. A model to explain the measured changes after reducing the expression of these three membrane proteins involving cholesterol-enriched nanodomains is proposed.