Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging

Small interfering RNA (siRNA) molecules are potent effectors of post-transcriptional gene silencing. Using noninvasive bioluminescent imaging and a mathematical model of siRNA delivery and function, the effects of target-specific and treatment-specific parameters on siRNA-mediated gene silencing are monitored in cells stably expressing the firefly luciferase protein. In vitro, luciferase protein levels recover to pre-treatment values within <1 week in rapidly dividing cell lines, but take longer than 3 weeks to return to steady-state levels in nondividing fibroblasts. Similar results are observed in vivo, with knockdown lasting ~10 days in subcutaneous tumors in A/J mice and 3–4 weeks in the nondividing hepatocytes of BALB/c mice. These data indicate that dilution due to cell division, and not intracellular siRNA half-life, governs the duration of gene silencing under these conditions. To demonstrate the practical use of the model in treatment design, model calculations are used to predict the dosing schedule required to maintain persistent silencing of target proteins with different half-lives in rapidly dividing or nondividing cells. The approach of bioluminescent imaging combined with mathematical modeling provides useful insights into siRNA function and may help expedite the translation of siRNA into clinically relevant therapeutics for disease treatment and management

3D occlusion recovery using few cameras

We present a practical framework for detecting and modeling 3D static occlusions for wide-baseline, multi-camera scenarios where the number of cameras is small. The framework consists of an iterative learning procedure where at each frame the occlusion model is used to solve the voxel occupancy problem, and this solution is then used to update the occlusion model. Along with this iterative procedure, there are two contributions of the proposed work: (1) a novel energy function (which can be minimized via graph cuts) specifically designed for use in this procedure, and (2) an application that incorporates our probabilistic occlusion model into a 3D tracking system. Both qualitative and quantitative results of the proposed algorithm and its incorporation with a 3D tracker are presented for support. 1

Physicochemical and Biological Characterization of Targeted, Nucleic Acid-Containing Nanoparticles

Nucleic acid-based therapeutics have the potential to provide potent and highly specific treatments for a variety of human ailments. However, systemic delivery continues to be a significant hurdle to success. Multifunctional nanoparticles are being investigated as systemic, nonviral delivery systems, and here, we describe the physicochemical and biological characterization of cyclodextrin-containing polycations (CDP) and their nanoparticles formed with nucleic acids including plasmid DNA (pDNA) and small interfering RNA (siRNA). These polycation/nucleic acid complexes can be tuned by formulation conditions to yield particles with sizes ranging from 60 to 150 nm, ζ potentials from 10 to 30 mV, and molecular weights from ∼7 × 10^7 to 1 × 10^9 g mol^(-1) as determined by light scattering techniques. Inclusion complexes formed between adamantane (AD)-containing molecules and the β-cyclodextrin molecules enable the modular attachment of poly(ethylene glycol) (AD−PEG) conjugates for steric stabilization and targeting ligands (AD−PEG−transferrin) for cell-specific targeting. A 70 nm particle can contain ∼10 000 CDP polymer chains, ∼2000 siRNA molecules, ∼4000 AD−PEG_(5000) molecules, and ∼100 AD−PEG_(5000)−Tf molecules; this represents a significant payload of siRNA and a large ratio of siRNA to targeting ligand (20:1). The particles protect the nucleic acid payload from nuclease degradation, do not aggregate at physiological salt concentrations, and cause minimal erythrocyte aggregation and complement fixation at the concentrations typically used for in vivo application. Uptake of the nucleic acid-containing particles by HeLa cells is measured by flow cytometry and visualized by confocal microscopy. Competitive uptake experiments show that the transferrin-targeted particles display enhanced affinity for the transferrin receptor through avidity effects (multiligand binding). Functional efficacy of the delivered pDNA and siRNA is demonstrated through luciferase reporter protein expression and knockdown, respectively. The analysis of the CDP delivery vehicle provides insights that can be applied to the design of targeted nucleic acid delivery vehicles in general

Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with 64Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by {approx}50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics

Phase and micromotion of Bose-Einstein condensates in a time-averaged ring trap

Rapidly scanning magnetic and optical dipole traps have been widely utilised to form time-averaged potentials for ultracold quantum gas experiments. Here we theoretically and experimentally characterise the dynamic properties of Bose-Einstein condensates in ring-shaped potentials that are formed by scanning an optical dipole beam in a circular trajectory. We find that unidirectional scanning leads to a non-trivial phase profile of the condensate that can be approximated analytically using the concept of phase imprinting. While the phase profile is not accessible through in-trap imaging, time-of-flight expansion manifests clear density signatures of an in-trap phase step in the condensate, coincident with the instantaneous position of the scanning beam. The phase step remains significant even when scanning the beam at frequencies two orders of magnitude larger than the characteristic frequency of the trap. We map out the phase and density properties of the condensate in the scanning trap, both experimentally and using numerical simulations, and find excellent agreement. Furthermore, we demonstrate that bidirectional scanning eliminated the phase gradient, rendering the system more suitable for coherent matter wave interferometry.Comment: 10 pages, 7 figure

Concentration of norms and eigenvalues of random matrices

We prove concentration results for $\ell_p^n$ operator norms of rectangular random matrices and eigenvalues of self-adjoint random matrices. The random matrices we consider have bounded entries which are independent, up to a possible self-adjointness constraint. Our results are based on an isoperimetric inequality for product spaces due to Talagrand.Comment: 15 pages; AMS-LaTeX; updated one referenc

Cholinergic suppression: A postsynaptic mechanism of long-term associative learning

Food avoidance learning in the mollusc Pleurobranchaea entails reduction in the responsiveness of key brain interneurons in the feeding neural circuitry, the paracerebral feeding command interneurons (PCNs), to the neurotransmitter acetylcholine (AcCho). Food stimuli applied to the oral veil of an untrained animal depolarize the PCNs and induce the feeding motor program (FMP). Atropine (a muscarinic cholinergic antagonist) reversibly blocks the food-induced depolarization of the PCNs, implicating AcCho as the neurotransmitter mediating food detection. AcCho applied directly to PCN somata depolarizes them, indicating that the PCN soma membrane contains AcCho receptors and induces the FMP in the isolated central nervous system preparation. The AcCho response of the PCNs is mediated by muscariniclike receptors, since comparable depolarization is induced by muscarinic agonists (acetyl-ß -methylcholine, oxotremorine, pilocarpine), but not nicotine, and blocked by muscarinic antagonists (atropine, trifluoperazine). The nicotinic antagonist hexamethonium, however, blocked the AcCho response in four of six cases. When specimens are trained to suppress feeding behavior using a conventional food-avoidance learning paradigm (conditionally paired food and shock), AcCho applied to PCNs in the same concentration as in untrained animals causes little or no depolarization and does not initiate the FMP. Increasing the concentration of AcCho 10-100 times, however, induces weak PCN depolarization in trained specimens, indicating that learning diminishes but does not fully abolish AcCho responsiveness of the PCNs. This study proposes a cellular mechanism of long-term associative learning -- namely, postsynaptic modulation of neurotransmitter responsiveness in central neurons that could apply also to mammalian species

Triptycene Structure-Directing Agents in Aluminophosphate Synthesis

The synthesis of aluminophosphates is investigated using a number of triptycene-based organic structure-directing agents (OSDA). These OSDAs are designed to synthesize extra-large pore and/or large cavity-containing molecular sieves. Starting from the hydrophobic triptycene molecule, OSDAs are prepared by introducing three amine-based centers that can be charged either by protonation in the acidic aluminophosphate reaction media or through quaternization. VPI-5 is synthesized using these tripytycene OSDAs, and the OSDAs are occluded inside the pores. This synthesis marks the first time VPI-5 has been made as a single phase with an OSDA occluded inside the framework of the as-made material that is not removed by simple washing with water or other solvents. Additionally, several other aluminophosphates with unknown structures are synthesized using these new OSDAs

An Analysis of the Factors Affecting the Career Orientation of Federal Civilian Engineers

This study was conducted to analyze the factors that affect the career orientation of federal civilian engineers at the Naval Avionics Center in Indianapolis, Indiana. One hundred and sixty-seven scientists and engineers from several engineering divisions were surveyed regarding turnover intentions. Based on literature reviews in the area of turnover with this particular population, a model was developed containing several factors related to intent to remain in the organization. Results indicated that this model predicts the turnover intention with 87.5 percent accuracy. Findings are interpreted in light of ongoing efforts on an organization-wide basis to introduce change in the Center's culture through a quality management program. Keywords: Career orientation, Job satisfaction turnover. (sdw)Naval Avionics Center, Civilian Personnel Departmenthttp://archive.org/details/analysisoffactor00robeN0016389WR90118NAApproved for public release; distribution is unlimited

Constitutional Implications of School Punishment for Cyber Bullying

In this article, we address the scope of student free speech rights as it relates to cyber bullying. We provide a review of legal theories under which school administrators can address cyber bullying while still respecting student free speech rights and the First Amendment. Additionally, we address the jurisdiction of administrators to deal with off-campus bullying conduct