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.

Biocompatibility of Calcium Phosphate Bone Cement with Optimised Mechanical Properties

The broad aim of this work was to investigate and optimise the properties of calcium phosphate bone cements (CPCs) for use in vertebroplasty to achieve effective primary fixation of spinal fractures. The incorporation of collagen, both bovine and from a marine sponge (Chondrosia reniformis), into a CPC was investigated. The biological properties of the CPC and collagen-CPC composites were assessed in vitro through the use of human bone marrow stromal cells. Cytotoxicity, proliferation and osteoblastic differentiation were evaluated using lactate dehydrogenase, PicoGreen and alkaline phosphatase activity assays respectively. The addition of both types of collagen resulted in an increase in cytotoxicity, albeit not to a clinically relevant level. Cellular proliferation after 1, 7 and 14 days was unchanged. The osteogenic potential of the CPC was reduced through the addition of bovine collagen but remained unchanged in the case of the marine collagen. These findings, coupled with previous work showing that incorporation of marine collagen in this way can improve the physical properties of CPCs, suggest that such a composite may offer an alternative to CPCs in applications where low setting times and higher mechanical stability are important.

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.

Development of an app to support learning in A-level maths

This project involved producing an app for smart devices to enable modernised learning for A-level maths students. Research in a stakeholder school showed that 94% of pupils surveyed within the upper-secondary level owned a smartphone and most owned a tablet also, emphasising the opportunity for using apps to support learning. The app was developed using iBuildApp, an online app-creation programme which requires no programming. Past exam questions and solutions, notes and video tutorials were included and the topic was vectors, identified by teachers as problematic. Pupils generally found the app easy to use and wanted further development. The videos were popular despite this not ranking highly as a preferred method of revision previously. Teachers were happy for pupils to use the app to supplement their learning, both in the classroom and outside.

An Efficient LS-SVM-Based Method for Fuzzy System Construction

This paper proposes an efficient learning mechanism to build fuzzy rule-based systems through the construction of sparse least-squares support vector machines (LS-SVMs). In addition to the significantly reduced computational complexity in model training, the resultant LS-SVM-based fuzzy system is sparser while offers satisfactory generalization capability over unseen data. It is well known that the LS-SVMs have their computational advantage over conventional SVMs in the model training process; however, the model sparseness is lost, which is the main drawback of LS-SVMs. This is an open problem for the LS-SVMs. To tackle the nonsparseness issue, a new regression alternative to the Lagrangian solution for the LS-SVM is first presented. A novel efficient learning mechanism is then proposed in this paper to extract a sparse set of support vectors for generating fuzzy IF-THEN rules. This novel mechanism works in a stepwise subset selection manner, including a forward expansion phase and a backward exclusion phase in each selection step. The implementation of the algorithm is computationally very efficient due to the introduction of a few key techniques to avoid the matrix inverse operations to accelerate the training process. The computational efficiency is also confirmed by detailed computational complexity analysis. As a result, the proposed approach is not only able to achieve the sparseness of the resultant LS-SVM-based fuzzy systems but significantly reduces the amount of computational effort in model training as well. Three experimental examples are presented to demonstrate the effectiveness and efficiency of the proposed learning mechanism and the sparseness of the obtained LS-SVM-based fuzzy systems, in comparison with other SVM-based learning techniques.

Shocks in unmagnetized plasma with a shear flow: Stability and magnetic field generation

A pair of curved shocks in a collisionless plasma is examined with a two-dimensional particle-in-cell simulation. The shocks are created by the collision of two electron-ion clouds at a speed that exceeds everywhere the threshold speed for shock formation. A variation of the collision speed along the initially planar collision boundary, which is comparable to the ion acoustic speed, yields a curvature of the shock that increases with time. The spatially varying Mach number of the shocks results in a variation of the downstream density in the direction along the shock boundary. This variation is eventually equilibrated by the thermal diffusion of ions. The pair of shocks is stable for tens of inverse ion plasma frequencies. The angle between the mean flow velocity vector of the inflowing upstream plasma and the shock's electrostatic field increases steadily during this time. The disalignment of both vectors gives rise to a rotational electron flow, which yields the growth of magnetic field patches that are coherent over tens of electron skin depths.

Clinical potential of gene-directed enzyme prodrug therapy to improve radiation therapy in prostate cancer patients

Despite the advances in prostate cancer diagnosis and treatment, current therapies are not curative in a significant proportion of patients. Gene-directed enzyme prodrug therapy (GDEPT), when combined with radiation therapy, could improve the outcome of treatment for prostate cancer, the second leading cause of cancer death in the western world. GDEPT involves the introduction of a therapeutic transgene, which can be targeted to the tumour cells. A prodrug is administered systemically and is converted to its toxic form only in those cells containing the transgene, resulting in cell kill. This review will discuss the clinical trials which have investigated the potential of GDEPT at various stages of prostate cancer progression. The advantages of using GDEPT in combination with radiotherapy will be examined, as well as some of the recent advances which enhance the potential utility of GDEPT.

Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system:a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro

BACKGROUND: We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells.

METHODS: We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro.

RESULTS: The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses.

CONCLUSIONS: This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.

Gene-based therapy in prostate cancer

Prostate cancer is one of the commonest causes of illness and death from cancer. Radical prostatectomy, radiotherapy, and hormonal therapy are the main conventional treatments. However, gene therapy is emerging as a promising adjuvant to conventional strategies, and several clinical trials are in progress. Here, we outline several approaches to gene therapy for prostate cancer that have been investigated. Methods of gene delivery are described, particularly those that have commonly been used in research on prostate cancer. We discuss efforts to achieve tissue-specific gene delivery, focusing on the use of tissue-specific gene promoters. Finally, the present use of gene therapy for prostate cancer is evaluated. The ability to deliver gene-therapy vectors directly to prostate tissue, and to regulate gene expression in a tissue-specific manner, offers promise for the use of gene therapy in prostate cancer.

Towards a Cyber-physical Resilience Framework for Smart Grids

As modern power grids move towards becoming a smart grid, there is an increasing reliance on the data that is transmitted and processed by ICT systems. This reliance introduces new digital attack vectors. Many of the proposed approaches that aim to address this problem largely focus on applying well-known ICT security solutions. However, what is needed are approaches that meet the complex concerns of the smart grid as a cyber-physical system. Furthermore, to support the automatic control loops that exist in a power grid, similarly automatic security and resilience mechanisms are needed that rely on minimal operator intervention. The research proposed in this paper aims to develop a framework that ensures resilient smart grid operation in light of successful cyber-attacks.

Seasonality of Plasmodium falciparum transmission: a systematic review

Background: Although Plasmodium falciparum transmission frequently exhibits seasonal patterns, the drivers of malaria seasonality are often unclear. Given the massive variation in the landscape upon which transmission acts, intra-annual fluctuations are likely influenced by different factors in different settings. Further, the presence of potentially substantial inter-annual variation can mask seasonal patterns; it may be that a location has "strongly seasonal" transmission and yet no single season ever matches the mean, or synoptic, curve. Accurate accounting of seasonality can inform efficient malaria control and treatment strategies. In spite of the demonstrable importance of accurately capturing the seasonality of malaria, data required to describe these patterns is not universally accessible and as such localized and regional efforts at quantifying malaria seasonality are disjointed and not easily generalized. 

Methods: The purpose of this review was to audit the literature on seasonality of P. falciparum and quantitatively summarize the collective findings. Six search terms were selected to systematically compile a list of papers relevant to the seasonality of P. falciparum transmission, and a questionnaire was developed to catalogue the manuscripts. 

Results and discussion: 152 manuscripts were identified as relating to the seasonality of malaria transmission, deaths due to malaria or the population dynamics of mosquito vectors of malaria. Among these, there were 126 statistical analyses and 31 mechanistic analyses (some manuscripts did both). 

Discussion: Identified relationships between temporal patterns in malaria and climatological drivers of malaria varied greatly across the globe, with different drivers appearing important in different locations. Although commonly studied drivers of malaria such as temperature and rainfall were often found to significantly influence transmission, the lags between a weather event and a resulting change in malaria transmission also varied greatly by location. 

Conclusions: The contradicting results of studies using similar data and modelling approaches from similar locations as well as the confounding nature of climatological covariates underlines the importance of a multi-faceted modelling approach that attempts to capture seasonal patterns at both small and large spatial scales.