In vivo gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation.

Therapeutics based on short interfering RNAs (siRNAs), which act by inhibiting the expression of target transcripts, represent a novel class of potent and highly specific next-generation treatments for human skin diseases. Unfortunately, the intrinsic barrier properties of the skin combined with the large size and negative charge of siRNAs make epidermal delivery of these macromolecules quite challenging. To help evaluate the in vivo activity of these therapeutics and refine delivery strategies we generated an innovative reporter mouse model that predominantly expresses firefly luciferase (luc2p) in the paw epidermis--the region of murine epidermis that most closely models the tissue architecture of human skin. Combining this animal model with state-of-the-art live animal imaging techniques, we have developed a real-time in vivo analysis work-flow that has allowed us to compare and contrast the efficacies of a wide range nucleic acid-based gene silencing reagents in the skin of live animals. While inhibition was achieved with all of the reagents tested, only the commercially available "self-delivery" modified Accell-siRNAs (Dharmacon) produced potent and sustained in vivo gene silencing. Together, these findings highlight just how informative reliable reporter mouse models can be when assessing novel therapeutics in vivo. Using this work-flow, we developed a novel clinically-relevant topical formulation that facilitates non-invasive epidermal delivery of unmodified and "self-delivery" siRNAs. Remarkably, a sustained >40% luc2p inhibition was observed after two 1-hour treatments with Accell-siRNAs in our topical formulation. Importantly, our ability to successfully deliver siRNA molecules topically brings these novel RNAi-based therapeutics one-step closer to clinical use.

What lies beneath? Scanning probe tomography may have the answer.

Scanning probe microscopy facilitates high-resolution noninvasive imaging of surface topography on even the most delicate of biological structures. Moreover, the local probe nature of the instrument architecture lends itself to the measurement of many important physical properties. To date, biological investigations have largely been constrained to imaging surface (membrane)-borne phenomena; however, the advent of extremely high aspect-ratio 'needle' probe tips, as reported by Beard et al. (2013), suggests that the approach can now be extended to address the particular challenges associated with measuring subsurface microscopic targets, including the intracellular components of the stratum corneum.

The spontaneously adhesive leukocyte function-associated antigen-1 (LFA-1) integrin in effector T cells mediates rapid actin- and calmodulin-dependent adhesion strengthening to ligand under shear flow.

Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5-20-s contact time). The maximum unbinding force for this interaction was ∼140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.

Keystroke dynamics in the pre-touchscreen era.

Biometric authentication seeks to measure an individual's unique physiological attributes for the purpose of identity verification. Conventionally, this task has been realized via analyses of fingerprints or signature iris patterns. However, whilst such methods effectively offer a superior security protocol compared with password-based approaches for example, their substantial infrastructure costs, and intrusive nature, make them undesirable and indeed impractical for many scenarios. An alternative approach seeks to develop similarly robust screening protocols through analysis of typing patterns, formally known as keystroke dynamics. Here, keystroke analysis methodologies can utilize multiple variables, and a range of mathematical techniques, in order to extract individuals' typing signatures. Such variables may include measurement of the period between key presses, and/or releases, or even key-strike pressures. Statistical methods, neural networks, and fuzzy logic have often formed the basis for quantitative analysis on the data gathered, typically from conventional computer keyboards. Extension to more recent technologies such as numerical keypads and touch-screen devices is in its infancy, but obviously important as such devices grow in popularity. Here, we review the state of knowledge pertaining to authentication via conventional keyboards with a view toward indicating how this platform of knowledge can be exploited and extended into the newly emergent type-based technological contexts.

On the accuracy of framing-rate measurements in ultra-high speed rotating mirror cameras.

Rotating mirror systems based on the Miller Principle are a mainstay modality for ultra-high speed imaging within the range 1-25 million frames per second. Importantly, the true temporal accuracy of observations recorded in such cameras is sensitive to the framing rate that the system directly associates with each individual data acquisition. The purpose for the present investigation was to examine the validity of such system-reported frame rates in a widely used commercial system (a Cordin 550-62 model) by independently measuring the framing rate at the instant of triggering. Here, we found a small but significant difference between such measurements: the average discrepancy (over the entire spectrum of frame rates used) was found to be 0.66 ± 0.48%, with a maximum difference of 2.33%. The principal reason for this discrepancy was traced to non-optimized sampling of the mirror rotation rate within the system protocol. This paper thus serves three purposes: (i) we highlight a straightforward diagnostic approach to facilitate scrutiny of rotating-mirror system integrity; (ii) we raise awareness of the intrinsic errors associated with data previously acquired with this particular system and model; and (iii), we recommend that future control routines address the sampling issue by implementing real-time measurement at the instant of triggering.