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Purpose: Cervical cancer is mainly caused by infections of high-risk human papillomavirus (HR-HPV). Persistent expression of HR-HPV oncogenes E6 and E7 is implicated in malignant transformation. The aim was to provide proof-of-concept data to support use of zinc finger nucleases (ZFN) targeting HPV E7 to treat HPV-related cervical cancer.Experimental Design: We designed and constructed ZFNs that could specifically recognize and cleave HPV16/18 E7 DNA. We tested the cleavage efficiency of selected ZFN16-E7-S2 and ZFN18-E7-S2 by using single-strand annealing (SSA) assay. Cell viability and colony formation assays were used to estimate the inhibition of cell growth that received treatments of ZFNs. Gene disruption of HPV E7 and downstream genes were examined by Western blotting. Cell apoptosis assay was used to test the specificity and efficiency of induction of HPV type-specific apoptosis. We also introduced xenograft formation assays to estimate the potential of inhibition of HPV-related disease.Results: We found ZFN16-E7-S2 and ZFN18-E7-S2 disrupted HPV E7 oncogenes in HPV16/18–positive cervical cancer cells. Both ZFNs effectively led to inhibition of type-specific cervical cancer cell growth, and specifically induced apoptosis of corresponding HPV16- and HPV18-positive cervical cancer cell lines. ZFN16-E7-S2 and ZFN18-E7-S2 also repressed xenograft formation in vivo.Conclusion: ZFNs targeting HPV16/18 E7 could effectively induce disruption of E7 oncogenes and lead to type-specific and efficient growth inhibition and apoptosis of HPV-positive cells. ZFNs targeting HPV16/18 E7 oncogenes could be used as novel therapeutic agents for the treatment of HPV-related cervical cancer. Clin Cancer Res; 20(24); 6495–503. ©2014 AACR.

IHC stainings of PCNA, Caspase-3 and CD31 in the non-necrosis area of xenografts.

Target sequences and construction approaches of ZFNs used in this manuscript.

Supplementary Methods and Supplementary Figure 1-20: Supplementary Figure 1 shows the growth of the tumors after orthotopic transplantation. Supplementary Figure 2 shows that over-expression of SIX1 promotes lymphangiogenesis and lymph-node metastasis of C33a cells in vivo. Supplementary Figure 3 shows the assay of cytokine and growth factor with antibody-based array. Supplementary Figure 4 shows the expression of the genes coding for the lymphangiogenic factors. Supplementary Figure 5 shows that SIX1 promotes VEGF-C expression in C33a cells. Supplementary Figure 6 shows that SIX1 promotes migration and tube-formation of HLECs. Supplementary Figure 7 shows the knock-down of VEGF-C in cervical cancer cells. Supplementary Figure 8 shows that the promotional effect of SIX1-expressing tumor cells on the migration and tube-formation of HLECs in vitro is mediated by VEGF-C. Supplementary Figure 9 shows that the promotional effect of SIX1-expressing tumor cells on lymphangiogenesis in vivo is mediated by VEGF-C. Supplementary Figure 10 shows that the promotional effect of SIX1-expressing C33a cells on lymphatic-vessel is mediated by VEGF-C. Supplementary Figure 11 shows that the promotional effect of SIX1 on lymph-node metastasis in vivo is mediated by VEGF-C. Supplementary Figure 12 shows the differences in gene expression levels between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 13 shows the immunohistochemical analysis of TGF-βs in tissue microarray of human CSCC specimens. Supplementary Figure 14 shows that SIX1 enhances the effect of TGF-β1 on VEGF-C production by tumor cells and tube-formation of HLECs in vitro. Supplementary Figure 15 shows that TGF-β contributes to the differences in VEGF-C expression between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 16 shows that TGF-β signaling is required for SIX1 to promote lymphangiogenesis and lymph-node metastasis in vivo. Supplementary Figure 17 shows that knocking down the expression of SMAD2 or SMAD3 or TβR1 suppresses TGF-β1 induced VEGF-C expression. Supplementary Figure 18 shows that higher expression of SIX1 correlates with higher expression of TβR1 in human CSCC specimens. Supplementary Figure 19 shows the binding of SIX1 to the VEGF-C promoter. Supplementary Figure 20 shows that knocking down the expression of SMAD2 and SMAD3 does not affect the binding of SIX1 to the VEGF-C promoter.

NCG is increasingly being co-injected with steam in heavy oil production systems to reduce heat loss and greenhouse gas emissions, as well as to maintain reservoir pressure. Given increased use of NCG co-injection, the validity of conventional subcool models must be revisited since they assume that the steam chamber is comprised of water alone. The current study makes modifications to the pure-steam hydrostatic subcool model, as well as the Yuan & Nugent (2013) subcool model to account for the presence of NCG in the steam chamber. Using typical values from the Athabasca oilfield, the study then compares the liquid-height predictions made by the original and modified models and proposes rules-of-thumb that correct for the presence of NCG. In general, increasing NCG in the steam chamber results in a reduction in subcool relative to pure steam. According to modified hydrostatic model, to achieve a liquid-pool height equal to that of pure steam injection, the subcool must be increased by 0.60K per 1% increase in the vapor-phase molar fraction. In contrast, over a wide range of production rates and drawdowns, the modified Yuan & Nugent (2013) model predicts that to achieve a liquid-pool height equal to that of the pure steam case, the subcool must be increased by 0.66K per 1% increase in the vapor-phase molar fraction. Despite the rule-of-thumbs being qualitatively in line with expectations, they suffer from the inability to accurately calculate subcool from field data. The final section of the paper reviews limitations of subcool as a well performance metric and proposes an alternative method of assessment that relies on data that are more readily available to operators.

The eclipse-stacked hydrogen-bonded organic framework exhibits ultrafast excited carrier dynamics, thus remarkably accelerates radical annihilation through intrareticular electron coupling pathway for enhanced electrochemiluminescence.

Summary Cyclic steam stimulation (CSS) is one the most effective thermal recovery methods. It is widely used as the primary thermal recovery method to recover heavy oil fields in the Middle East, the Asia-Pacific region, and North and South America. In this paper, a novel dual-directional flow control device (FCD) will be introduced. This FCD technology can allocate accurate steam outflow into the reservoir formation and improve steam quality during the steam injection period and can mitigate steam breakthrough from the neighboring wells during the production period. In the first section, we give a brief introduction on CSS and the main issues encountered in the field operation. A multidirectional flow control nozzle specifically designed for CSS application will be presented. Design philosophy in thermodynamics and hydrodynamics of the nozzle will be discussed in detail. Field performance results, computational fluid dynamics (CFD), and flow loop testing data will be shown to evaluate the performance of the technology. The application of the technology in steam-assisted thermal applications will be introduced. Well-known issues such as erosion and scaling on the FCD tools will be studied in the end.

The singularity cancellation transformations are well-known for calculating weakly singular and near-singular integrals in integral equation solutions. However, some singularity cancellation methods suffer from the shape-dependent problem of inaccuracy and inefficiency for deformed triangles. By the theoretical analysis and numerical verification in this article, the relation between the near-singularity and shape-dependence of the singularity cancellation schemes is discussed. Moreover, a novel framework for devising cancellation transformations of weakly near-singular integrals is presented, which results in fast convergence for both regular and irregular triangular domains. Some numerical results are given to illustrate the validity of the theoretical framework and the efficiency of the proposed transformations.

Reducing water production is the primary problem in the oil and gas industry. There are a few flow control technologies with moving parts available on the market to choke back water. However, the main issue with those technologies is the potential of plugging and scaling. In this paper, we will introduce a novel passive flow control nozzle, which has no moving part inside. All the choking is implemented through its internal geometry. Therefore, the risk of plugging and scaling will be significantly mitigated. In this paper, a passive flow control nozzle designed specifically for water choking will be presented. Design philosophy in fluid mechanics will be introduced in detail. The results of Computational Fluid Dynamics (CFD) and physical flow loop testing will be shown to evaluate the performance of the technology. It is shown that a passive choking nozzle can choke back more than 40% of water compared to an orifice while maintaining oil production rates. We will also perform simulation case studies to compare conventional slotted liner completions with the completions equipped with a passive choking nozzle (PCN). We will show that the nozzle can effectively choke back water and promote oil production in a long horizontal well. Finally, we will briefly discuss how the passive nozzle can mitigate well-known issues such as scaling and plugging.

A boron dipyrromethene (BODIPY)-based metal-organic framework (MOF) nanoemitter was for the first time designed with enhanced electrochemiluminescence (ECL) intensity due to the suppression of non-radiative dissipation originating from the ordered arrangement of BODIPY molecules in the framework. Thus, an ECL biosensor was developed for telomerase detection with excellent performance in real samples.

The source reconstruction method (SRM) is a technique developed for antenna diagnostics and for carrying out near‐field to far‐field transformation. This chapter presents two different methods: one that deals with the equivalent magnetic current and the second that deals with the equivalent electric current. It also presents the examples to illustrate the applicability and accuracy of the proposed methodology using either of the equivalent currents and applied to experimental data. This methodology is used for near‐field to near/far‐field transformations for arbitrary near‐field geometry to evaluate the safe distance for commercial antennas followed by some conclusions and a list of references. Antenna diagnostic methods based on the analysis of the near fields close to the antenna surface are becoming of great interest for antenna manufacturers. The near‐field measurement of a large antenna is not only very time consuming, but cumbersome because of measuring the fields over certain large canonical surfaces with high spatial accuracy.

Chebyshev polynomials are used to extract from the data collected under non‐anechoic environment a radiation pattern for the same antenna radiating in free space conditions. The Cauchy method based on Gegenbauer polynomials is presented for antenna near‐field extrapolation and the far‐field estimation. Some numerical results are given to demonstrate the applicability of the proposed scheme in antenna near‐field extrapolation and far‐field estimation. Total least square (TLS) and singular value decomposition also suppress partially the effects of noise in the known data for extrapolation. The TLS solution for best data fitting shows an excellent performance for minimizing errors of interpolation in. TLS can also work well for extrapolating missing data on a small interval, which is quite similar to an interpolation problem with several sample points removed. However, an interesting result of zenith‐directed reflector antenna configuration is that the hole was able to be expanded significantly without increasing the error of the interpolation.

In the mathematical field of numerical analysis, model order reduction is the key to processing measured data. This chapter outlines philosophy of model order reduction along with the concepts of total least squares and singular value decomposition. It introduces a critical component in solving a total least squares problem called the singular value decomposition. The singular value decomposition is one of the most important concepts in linear algebra. Eigenvectors and eigenvalues play crucial roles in linear algebra ranging from simplifying matrix algebra such as taking the 500th power of to solving differential equations. Diagonalizing a matrix not only provides a quick way to extract eigenvalues but important parameters such as the rank and dimension of a matrix can be found easily once a matrix is diagonalized. The method of total least squares is a linear parameter estimation technique and is used in wide variety of disciplines such as signal processing, statistics, physics, and the like.

This chapter presents two methods for spherical near‐field to far‐field transformation. The first methodology presents an exact explicit analytical formulation for transforming near‐field data generated over a spherical surface to the far field radiation pattern. The second method presents the equivalent source formulation that was described earlier and illustrates how it can be deployed to the spherical scanning case where one component of the field is missing from the measurements, when the tangential components of the fields on a spherical surface is given. Near‐field scanning over canonical surfaces—planar, cylindrical, and spherical—is often used to obtain the far‐field pattern characteristics of an antenna under test via near‐field to far‐field transformation algorithms. A comparison with the technique based on the spherical wave‐mode expansion has been performed in terms of far‐field results.

This chapter presents the matrix pencil method (MPM) which is a methodology to approximate a given data set by a sum of complex exponentials. The MPM approach is computationally very efficient and quite robust to noise in the data. The MPM has been successfully applied to high‐resolution de‐embedding procedures using band‐limited data prevalent in most microwave and antenna measurements. In general, the measurement of antenna radiation patterns takes place in anechoic chambers, where one attempts to reduce as much as possible the reflection and diffraction contributions from walls and mechanical devices present inside the chamber. In a typical direction of arrival estimation problem, it is often assumed that all the signals are arriving at the antenna array at the same frequency which is assumed to be known. The antenna elements in the array are then placed half wavelength apart at the frequency of operation.

This chapter presents two deconvolution techniques to illustrate how the ill‐posed deconvolution problem has been regularized. The two techniques presented have the built in self‐regularizing schemes. The first method is based on solving the ill‐posed deconvolution problem by the iterative conjugate gradient method. The second method uses the method of total least squares implemented through the singular value decomposition (SVD) technique. The conjugate gradient method essentially performs singular value decomposition without actually computing the eigenvalues and the criteria that one utilizes to stop the iterative process is that the magnitude of the largest element in the residual must be a certain value. Two methods ‐ the conjugate gradient method with the Fast Fourier Transform Technique and the Total Least Squares based on the SVD has been presented to obtain the impulse responses of some electromagnetic systems with measured input and output data.

This chapter presents a nonparametric method for the application of the principle of analytic continuation to interpolate/extrapolate system responses resulting in reduced computations. The Hilbert transform can also be used to speed up the spectral analysis of nonuniformly spaced data samples. One of the properties of the periodogram approach is that the there is a Hilbert transform relationship between the coefficients of the parameters used to evaluate the spectrum. The causality of the time domain signal, denoted as h(t), assures one that the real and imaginary components of the frequency domain response are related through the Hilbert transform. The application of the Hilbert transform for computing the spectrum of a waveform in a fast and efficient way from nonuniformly spaced data is illustrated. The Hilbert transform illustrates that the real and imaginary parts of any nonminimum phase transfer function from a causal system satisfy the relationship.

A fast and accurate method is presented for computing far‐field antenna patterns from planar near‐field measurements. The planar near‐field data can be sampled either using a single moving dipole probe or using an array of dipole probes. Near‐field to far‐field transformation for planar scanning may be efficiently performed under certain conditions by exploiting the block Toeplitz structure of the matrix and using conjugate gradient method and the fast Fourier transform, thereby drastically reducing computation time and storage requirements. The chapter argues that a single scanning moving probe may be replaced by a fixed array of probes to sample the planar near field for the solution of the equivalent magnetic currents on the surface enclosing the antenna under test. The source reconstruction method is a computational technique developed for antenna diagnostics and for carrying out near‐field to far‐field transformation.

This chapter presents the concept of the Cauchy method for the interpolation and extrapolation of data. The Cauchy method is to interpolate and extrapolate the data from calculating coefficients simultaneously of the numerator and denominator polynomials which are used to approximate the data by a ratio of two polynomials. The usefulness of the Cauchy method is demonstrated for the ease in which it can be incorporated into a Method of Moments program to speed up the computation process over a broad band. The Cauchy method can also be used in the analysis of filters over a broad frequency range. An application of the Cauchy method is in creating a data‐base of many devices working in varying operating conditions. A fast and accurate interpolation algorithm is proposed to reconstruct the high resolution amplitude‐only frequency domain response such as radar cross section and antenna radiation patterns from sparse and non‐uniform samples based on the Cauchy method.