Coexpressing shRNA with fluorescence tags for quantification of cell migration studies

Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.

A Tunable Encryption Scheme and Analysis of Fast Selective Encryption for CAVLC and CABAC in H.264/AVC

Recently, two fast selective encryption methods for context-adaptive variable length coding and context-adaptive binary arithmetic coding in H.264/AVC were proposed by Shahid et al. In this paper, it was demonstrated that these two methods are not as efficient as only encrypting the sign bits of nonzero coefficients. Experimental results showed that without encrypting the sign bits of nonzero coefficients, these two methods can not provide a perceptual scrambling effect. If a much stronger scrambling effect is required, intra prediction modes, and the sign bits of motion vectors can be encrypted together with the sign bits of nonzero coefficients. For practical applications, the required encryption scheme should be customized according to a user's specified requirement on the perceptual scrambling effect and the computational cost. Thus, a tunable encryption scheme combining these three methods is proposed for H.264/AVC. To simplify its implementation and reduce the computational cost, a simple control mechanism is proposed to adjust the control factors. Experimental results show that this scheme can provide different scrambling levels by adjusting three control factors with no or very little impact on the compression performance. The proposed scheme can run in real-time and its computational cost is minimal. The security of the proposed scheme is also discussed. It is secure against the replacement attack when all three control factors are set to one.

A latent feature analysis of the neural representation of conceptual knowledge

Bayesian probabilistic analysis offers a new approach to characterize semantic representations by inferring the most likely feature structure directly from the patterns of brain activity. In this study, infinite latent feature models [1] are used to recover the semantic features that give rise to the brain activation vectors when people think about properties associated with 60 concrete concepts. The semantic features recovered by ILFM are consistent with the human ratings of the shelter, manipulation, and eating factors that were recovered by a previous factor analysis. Furthermore, different areas of the brain encode different perceptual and conceptual features. This neurally-inspired semantic representation is consistent with some existing conjectures regarding the role of different brain areas in processing different semantic and perceptual properties. © 2012 Springer-Verlag.

Documents and Dependencies: an Exploration of Vector Space Models for Semantic Composition

In most previous research on distributional semantics, Vector Space Models (VSMs) of words are built either from topical information (e.g., documents in which a word is present), or from syntactic/semantic types of words (e.g., dependency parse links of a word in sentences), but not both. In this paper, we explore the utility of combining these two representations to build VSM for the task of semantic composition of adjective-noun phrases. Through extensive experiments on benchmark datasets, we find that even though a type-based VSM is effective for semantic composition, it is often outperformed by a VSM built using a combination of topic- and type-based statistics. We also introduce a new evaluation task wherein we predict the composed vector representation of a phrase from the brain activity of a human subject reading that phrase. We exploit a large syntactically parsed corpus of 16 billion tokens to build our VSMs, with vectors for both phrases and words, and make them publicly available.

Reduced phospholipase A2 activity is not accompanied by reduced arachidonic acid release

Arachidonic acid release in cells highly over expressing cytosolic phospholipase A2 has been attributed to mitogen-activated protein kinase phosphorylation of cytosolic phospholipase A2 on serine-505. To investigate the role of cytosolic phospholipase A2 in cellular physiology, we attempted to inhibit cytosolic phospholipase A2 in the intact cell employing an antisense RNA strategy. Swiss 3T3 cells were stably transfected with an antisense cytosolic phospholipase A2 expression vector. A clone of cells with reduced immunodetectable cytosolic phospholipase A2, compared to a vector transfected cell line, was identified by Western blotting and a corresponding decrease in phospholipase A2 activity was confirmed by enzymatic assay in cell free extracts. However, arachidonic acid release from intact cells in response to agonists was not different between antisense and control cell lines. Thus, arachidonic acid release in intact cells with decreased cytosolic phospholipase A2 activity is likely to be modulated by rate limiting factors that are extrinsic to cytosolic phospholipase A2.

Finite element modelling of the formation of pre-load damage in cement mantles of orthopaedic joint replacements

Finite element modeling of the formation of pre-loaded damage in cement mantles of orthopaedic joint replacements was presented. The existence of cracking suggested a high level of residual stress. The direction of maximum principal stress vectors corresponded well with the observed crack orientation. Results suggested that cracking depends upon a combination of residual stress, porosity and temperature rise during polymerization.

Gene Sets Net Correlations Analysis (GSNCA): a multivariate differential coexpression test for gene sets

Motivation: To date, Gene Set Analysis (GSA) approaches primarily focus on identifying differentially expressed gene sets (pathways). Methods for identifying differentially coexpressed pathways also exist but are mostly based on aggregated pairwise correlations, or other pairwise measures of coexpression. Instead, we propose Gene Sets Net Correlations Analysis (GSNCA), a multivariate differential coexpression test that accounts for the complete correlation structure between genes.

Results: In GSNCA, weight factors are assigned to genes in proportion to the genes' cross-correlations (intergene correlations). The problem of finding the weight vectors is formulated as an eigenvector problem with a unique solution. GSNCA tests the null hypothesis that for a gene set there is no difference in the weight vectors of the genes between two conditions. In simulation studies and the analyses of experimental data, we demonstrate that GSNCA, indeed, captures changes in the structure of genes' cross-correlations rather than differences in the averaged pairwise correlations. Thus, GSNCA infers differences in coexpression networks, however, bypassing method-dependent steps of network inference. As an additional result from GSNCA, we define hub genes as genes with the largest weights and show that these genes correspond frequently to major and specific pathway regulators, as well as to genes that are most affected by the biological difference between two conditions. In summary, GSNCA is a new approach for the analysis of differentially coexpressed pathways that also evaluates the importance of the genes in the pathways, thus providing unique information that may result in the generation of novel biological hypotheses.

The influence of the magnetic field on running penumbral waves in the solar chromosphere

We use images of high spatial and temporal resolution, obtained using both ground- and space-based instrumentation, to investigate the role magnetic field inclination angles play in the propagation characteristics of running penumbral waves in the solar chromosphere. Analysis of a near-circular sunspot, close to the center of the solar disk, reveals a smooth rise in oscillatory period as a function of distance from the umbral barycenter. However, in one directional quadrant, corresponding to the north direction, a pronounced kink in the period-distance diagram is found. Utilizing a combination of the inversion of magnetic Stokes vectors and force-free field extrapolations, we attribute this behavior to the cut-off frequency imposed by the magnetic field geometry in this location. A rapid, localized inclination of the magnetic field lines in the north direction results in a faster increase in the dominant periodicity due to an accelerated reduction in the cut-off frequency. For the first time, we reveal how the spatial distribution of dominant wave periods, obtained with one of the highest resolution solar instruments currently available, directly reflects the magnetic geometry of the underlying sunspot, thus opening up a wealth of possibilities in future magnetohydrodynamic seismology studies. In addition, the intrinsic relationships we find between the underlying magnetic field geometries connecting the photosphere to the chromosphere, and the characteristics of running penumbral waves observed in the upper chromosphere, directly supports the interpretation that running penumbral wave phenomena are the chromospheric signature of upwardly propagating magneto-acoustic waves generated in the photosphere.

On transition rates in surface hopping

Trajectory surface hopping (TSH) is one of the most widely used quantum-classical algorithms for nonadiabatic molecular dynamics. Despite its empirical effectiveness and popularity, a rigorous derivation of TSH as the classical limit of a combined quantum electron-nuclear dynamics is still missing. In this work, we aim to elucidate the theoretical basis for the widely used hopping rules. Naturally, we concentrate thereby on the formal aspects of the TSH. Using a Gaussian wave packet limit, we derive the transition rates governing the hopping process at a simple avoided level crossing. In this derivation, which gives insight into the physics underlying the hopping process, some essential features of the standard TSH algorithm are retrieved, namely (i) non-zero electronic transition rate ("hopping probability") at avoided crossings; (ii) rescaling of the nuclear velocities to conserve total energy; (iii) electronic transition rates linear in the nonadiabatic coupling vectors. The well-known Landau-Zener model is then used for illustration. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4770280]

Development of a method to quantify the DNA content in DNA- cationic peptide nanoparticles

Gene therapy has the potential to provide safe and targeted therapies for a variety of diseases. A range of intracellular gene delivery vehicles have been proposed for this purpose. Non-viral vectors are a particularly attractive option and among them cationic peptides have emerged as promising candidates. For the pharmaceutical formulation and application to clinical studies it is necessary to quantify the amount of pDNA condensed with the delivery system. There is a severe deficiency in this area, thus far no methods have been reported specifically for pDNA condensed with cationic peptide to form nanoparticles. The current study seeks to address this and describes the evaluation of a range of disruption agents to extract DNA from nanoparticles formed by condensation with cationic fusogenic peptides RALA and KALA. Only proteinase K exhibited efficient and reproducible results and compatibility with the PicoGreen reagent based quantification assay. Thus we report for the first time a simple and reliable method that can quantify the pDNA content in pDNA cationic peptide nanoparticles.

Synergistic effects on gene delivery - co-formulation of small disulfide-linked dendritic polycations with Lipofectamine 2000 (TM)

This paper describes the application of gene delivery vectors based on connecting together two well-defined low-generation poly(L-lysine) (PLL) dendrons using a disulfide-containing linker unit. We report that the transfection ability of these vectors in their own right is relatively low, because the low-generation number limits the endosomal buffering capacity. Importantly, however, we demonstrate that when applied in combination with Lipofectamine 2000 (TM), a vector from the cationic lipid family, these small cationic additives significantly enhance the levels of gene delivery (up to four-fold). Notably, the cationic additives have no effect on the levels of transfection observed with a cationic polymer, such as DEAE dextran. We therefore argue that the synergistic effects observed with Lipofectamine 2000 (TM) arise as a result of combining the delivery advantages of two different classes of vector within a single formulation, with our dendritic additives providing a degree of pH buffering within the endosome. As such, the data we present indicate that small dendritic structures, although previously largely overlooked for gene delivery owing to their inability to transfect in their own right, may actually be useful well-defined additives to well-established vector systems in order to enhance the gene delivery payload.

Measurements of immune responses for establishing correlates of vaccine protection against HIV

Well-defined correlates of protective immunity are an essential component of rational vaccine development. Despite years of basic science and three HIV vaccine efficacy trials, correlates of immunological protection from HIV infection remain undefined. In December 2010, a meeting of scientists engaged in basic and translational work toward developing HIV-1 vaccines was convened. The goal of this meeting was to discuss current opportunities and optimal approaches for defining correlates of protection, both for ongoing and future HIV-1 vaccine candidates; specific efforts were made to engage young scientists. We discuss here the highlights from the meeting regarding the progress made and the way forward for a protective HIV-1 vaccine.

Towards Organic Electronic Materials for Electrically Stimulated Gene Delivery

The cell-specific delivery of polynucleic acids (e.g., DNA, RNA), gene therapy, has the potential to treat various diseases. In this chapter we discuss the use of organic electronic materials as non-viral gene delivery vectors and the great potential for electrochemically triggered gene delivery. We highlight some examples in this chapter based on fullerenes (bucky balls and carbon nanotubes), graphenes and electroactive polymers, particularly those that include experiments in vivo.

Space-Constrained Massive MIMO: Hitting the Wall of Favorable Propagation

The recent development of the massive multiple-input multiple-output (MIMO) paradigm, has been extensively based on the pursuit of favorable propagation: in the asymptotic limit, the channel vectors become nearly orthogonal and interuser interference tends to zero [1]. In this context, previous studies
have considered fixed inter-antenna distance, which implies an increasing array aperture as the number of elements increases. Here, we focus on a practical, space-constrained topology, where an increase in the number of antenna elements in a fixed total space imposes an inversely proportional decrease in the inter-antenna distance. Our analysis shows that, contrary to existing studies, inter-user interference does not vanish in the massive MIMO regime, thereby creating a saturation effect on the achievable rate.

Deriving meteorological variables across Africa for the study and control of vector-borne disease:a comparison of remote sensing and spatial interpolation of climate

This paper presents the results of an investigation into the utility of remote sensing (RS) using meteorological satellites sensors and spatial interpolation (SI) of data from meteorological stations, for the prediction of spatial variation in monthly climate across continental Africa in 1990. Information from the Advanced Very High Resolution Radiometer (AVHRR) of the National Oceanic and Atmospheric Administration's (NOAA) polar-orbiting meteorological satellites was used to estimate land surface temperature (LST) and atmospheric moisture. Cold cloud duration (CCD) data derived from the High Resolution Radiometer (HRR) onboard the European Meteorological Satellite programme's (EUMETSAT) Meteosat satellite series were also used as a RS proxy measurement of rainfall. Temperature, atmospheric moisture and rainfall surfaces were independently derived from SI of measurements from the World Meteorological Organization (WMO) member stations of Africa. These meteorological station data were then used to test the accuracy of each methodology, so that the appropriateness of the two techniques for epidemiological research could be compared. SI was a more accurate predictor of temperature, whereas RS provided a better surrogate for rainfall; both were equally accurate at predicting atmospheric moisture. The implications of these results for mapping short and long-term climate change and hence their potential for the study anti control of disease vectors are considered. Taking into account logistic and analytical problems, there were no clear conclusions regarding the optimality of either technique, but there was considerable potential for synergy.