Model verification for population detection of counterfeits.

The quality of counterfeit items has increased dramatically, with modern global manufacturing being able to duplicate the materials, construction, and visual features of items. Detection of fraudulent coinage can parallel authentication of food, beverages, and manufactured goods by studying product-inherent features. Counterfeit detection is performed by comparing an Example group with a Questioned group. A model is developed for both groups using standard tests on individual pieces. Coin weight is used here as an illustration. The model should also follow the natural science of the system. In this case, the manufacturing process variation is known and steady, and the underlying distribution is known or can be determined from authentic pieces. The proposed detection method uses testing of many individual pieces, then using reverse-quality-engineering methods to identify possible sources. This strategy looks at the variation between individual pieces to determine the process capability of a machine, assembly line, or plant to create product consistency for a manufacturer. Fraudulent items may be manufactured within specification, but demonstrate a manufacturing process capability different than that of the authentic manufacturer. In this report, we examine the model previously reported and use reconstruction techniques to re-create the evidence set to validate the model, increase model accuracy, and confirm the conclusion previously reached, showing that the Questioned set is likely over 37% non-conforming by weight. In this case, the decision outcome of the analysis was improved by using additional methods not included in the modeling software package originally used.

1D micro-nanopatterned integrin ligand surfaces for directed cell movement.

Cell-extracellular matrix (ECM) adhesion mediated by integrins is a highly regulated process involved in many vital cellular functions such as motility, proliferation and survival. However, the influence of lateral integrin clustering in the coordination of cell front and rear dynamics during cell migration remains unresolved. For this purpose, we describe a novel protocol to fabricate 1D micro-nanopatterned stripes by integrating the block copolymer micelle nanolithography (BCMNL) technique and maskless near UV lithography-based photopatterning. The photopatterned 10 µm-wide stripes consist of a quasi-perfect hexagonal arrangement of gold nanoparticles, decorated with the RGD (arginine-glycine-aspartate) motif for single integrin heterodimer binding, and placed at a distance of 50, 80, and 100 nm to regulate integrin clustering and focal adhesion dynamics. By employing time-lapse microscopy and immunostaining, we show that the displacement and speed of fibroblasts changes according to the nanoscale spacing of adhesion sites. We found that as the lateral spacing of adhesive peptides increased, fibroblast morphology was more elongated. This was accompanied by a decreased formation of mature focal adhesions and stress fibers, which increased cell displacement and speed. These results provide new insights into the migratory behavior of fibroblasts in 1D environments and our protocol offers a new platform to design and manufacture confined environments in 1D for integrin-mediated cell adhesion.

Epithelial cell cluster size affects force distribution in response to EGF-induced collective contractility.

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin ß1 peripheral adhesions and transmitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.

Population identification strategies for counterfeit coin detection.

Advances in manufacturing, 3-d imaging, and globalization have led to a rise in fraudulent coinage and a world-wide interest in coin authentication. Modern manufacturing methods allow the alloy, construction, and struck image of coins to be more readily reproduced. Larger coin denominations and efforts to reduce the cost of coining add additional incentive. Detection of fraudulent coinage can parallel authentication of food, beverages, and manufactured goods by studying product-inherent features. Reverse-quality-engineering provides clues to authenticity. One promising method is in the use of finite mixture models to compare individual measurements of groups of coins to assist in authentication. An example is provided using the coin weights of two groups of coins. Authentication of a Questioned set of coins is explored, comparing the weight population of Example coins drawn from circulation with the weights of a Questioned set drawn from an unknown origin. In the test, just over half of the Questioned coin set matched the distribution of the Example coin set. The other portion, nearly half of the coin sample, did not match the Example coins drawn from circulation. If this were combined with a similar analysis of other coin properties, similar results would help validate the finding. The example shows that groups of coins can be authenticated by using one or more measures of properties of populations of Questioned coins versus Example coins that are largely authentic.

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC). To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Regulation of Mitochondrial Complex I Biogenesis in Drosophila Flight Muscles.

The flight muscles of Drosophila are highly enriched with mitochondria, but the mechanism by which mitochondrial complex I (CI) is assembled in this tissue has not been described. We report the mechanism of CI biogenesis in Drosophila flight muscles and show that it proceeds via the formation of ∼315, ∼550, and ∼815 kDa CI assembly intermediates. Additionally, we define specific roles for several CI subunits in the assembly process. In particular, we show that dNDUFS5 is required for converting an ∼700 kDa transient CI assembly intermediate into the ∼815 kDa assembly intermediate. Importantly, incorporation of dNDUFS5 into CI is necessary to stabilize or promote incorporation of dNDUFA10 into the complex. Our findings highlight the potential of studies of CI biogenesis in Drosophila to uncover the mechanism of CI assembly in vivo and establish Drosophila as a suitable model organism and resource for addressing questions relevant to CI biogenesis in humans.