Pre-defined Site Research Articles

Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery.

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8+ T cells and gene targeting of a GFP transgene cassette in >40% of CD8+ and CD4+ T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.

Charge Redistribution and Extraction in Photocatalytically Synthesized Au–ZnO Nanohybrids

ZnO is a promising catalyst for hydrogen and oxygen production due to the position of the conduction and valence bands. Nevertheless, there are some limitations to the efficiency due to its n-type character. Once in contact with the electrolyte, an extraction barrier for electrons is formed. The purpose of this work was to create an extraction site for electrons by synthesizing small pyramidally shaped ZnO nanocrystals (∼10 nm), which consist of a predefined site for the growth of a gold particle at the tip. Photoelectrochemical deposition of gold on ZnO was performed to yield the hybrid structure. Photoluminescence (PL) studies of the relative change of intensities of band gap versus defect state relaxation showed electron transfer from the conduction band of ZnO to Au. Using cyclic voltammetry, Au-mediated charge extraction from Au–ZnO hybrids was demonstrated, which circumvents the electron extraction barrier in ZnO. Thus, this work demonstrates the nanoscale design of hybrid structures for photocataly...

In silico characterization of binding mode of CCR8 inhibitor: homology modeling, docking and membrane based MD simulation study

Human CC-chemokine receptor 8 (CCR8) is a crucial drug target in asthma that belongs to G-protein-coupled receptor superfamily, which is characterized by seven transmembrane helices. To date, there is no X-ray crystal structure available for CCR8; this hampers active research on the target. Molecular basis of interaction mechanism of antagonist with CCR8 remains unclear. In order to provide binding site information and stable binding mode, we performed modeling, docking and molecular dynamics (MD) simulation of CCR8. Docking study of biaryl-ether-piperidine derivative (13C) was performed inside predefined CCR8 binding site to get the representative conformation of 13C. Further, MD simulations of receptor and complex (13C-CCR8) inside dipalmitoylphosphatidylcholine lipid bilayers were performed to explore the effect of lipids. Results analyses showed that the Gln91, Tyr94, Cys106, Val109, Tyr113, Cys183, Tyr184, Ser185, Lys195, Thr198, Asn199, Met202, Phe254, and Glu286 were conserved in both docking and MD simulations. This indicated possible role of these residues in CCR8 antagonism. However, experimental mutational studies on these identified residues could be effective to confirm their importance in CCR8 antagonism. Furthermore, calculated Coulombic interactions represented the crucial roles of Glu286, Lys195, and Tyr113 in CCR8 antagonism. Important residues identified in this study overlap with the previous non-peptide agonist (LMD-009) binding site. Though, the non-peptide agonist and currently studied inhibitor (13C) share common substructure, but they differ in their effects on CCR8. So, to get more insight into their agonist and antagonist effects, further side-by-side experimental studies on both agonist (LMD-009) and antagonist (13C) are suggested.

FUBAR: A Fast, Unconstrained Bayesian AppRoximation for Inferring Selection

Model-based analyses of natural selection often categorize sites into a relatively small number of site classes. Forcing each site to belong to one of these classes places unrealistic constraints on the distribution of selection parameters, which can result in misleading inference due to model misspecification. We present an approximate hierarchical Bayesian method using a Markov chain Monte Carlo (MCMC) routine that ensures robustness against model misspecification by averaging over a large number of predefined site classes. This leaves the distribution of selection parameters essentially unconstrained, and also allows sites experiencing positive and purifying selection to be identified orders of magnitude faster than by existing methods. We demonstrate that popular random effects likelihood methods can produce misleading results when sites assigned to the same site class experience different levels of positive or purifying selection--an unavoidable scenario when using a small number of site classes. Our Fast Unconstrained Bayesian AppRoximation (FUBAR) is unaffected by this problem, while achieving higher power than existing unconstrained (fixed effects likelihood) methods. The speed advantage of FUBAR allows us to analyze larger data sets than other methods: We illustrate this on a large influenza hemagglutinin data set (3,142 sequences). FUBAR is available as a batch file within the latest HyPhy distribution (http://www.hyphy.org), as well as on the Datamonkey web server (http://www.datamonkey.org/).

Markovian Approximations for a Grid Computing Network with a Ring Structure

Optical grid networks allow many computing sites to share their resources by connecting them through high-speed links, providing a more efficient use of the resources and a timely response for incoming jobs. These jobs originate from users connected to each of the sites and, in contrast to traditional queueing networks, a particular job does not have to be processed in a predefined site. Furthermore, a job is always processed locally if there is an available local server. In this paper we propose two different methods to approximate the performance of an optical grid network with a ring topology. The first method is based on approximating the inter-overflow time process, while the second separately characterizes the periods where jobs are overflowed and the periods where they are served locally. Both approaches rely on a marked Markovian representation of the overflow process at each station and on reducing this representation by moment-matching methods. The results show that the methods accurately approximate the rate of locally processed jobs, one of the main performance measures.

Rationalisation of the Differences between APOBEC3G Structures from Crystallography and NMR Studies by Molecular Dynamics Simulations

The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal β-strand, β2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures. In the course of these simulations, we observed a general trend towards increased definition of the β2 strand for those structures that have a distorted starting conformation of β2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the β2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.

Split hybridisation probes for electrochemical typing of single-nucleotide polymorphisms

This paper describes the development of a highly selective single-nucleotide polymorphisms (SNPs) typing method based on the use of split hybridisation probes and demonstrates the concept through the electrochemical analysis of single-base mutations in actual patient samples. The requirement that two probes hybridised adjacent to one another to allow for stabilisation (via base-stacking) and binding of the allele-specific oligonucleotide (ASO), imparted highly stringent selectivity criteria to the assay. Simple rules for tuning the characteristics of such stacking/ASO probe pairs and achieve full mismatch discrimination at ambient conditions (with no need to strictly control the temperature) are provided. All genotyping experiments were indeed performed at room temperature, using the planar surface of disposable probe-modified gold electrodes as the genosensing platform. The ability to detect nanomolar amounts of a synthetic target even within a vast excess of single-base substituted sequences gave strong evidence of the specificity of the split probes assay. Proving the general validity of this genotyping approach, application of the analytical pathway was further demonstrated for clinical targets (amplified from the human TP53 gene) whose mutational site was poorly accessible, being part of a thermodynamically stable hairpin. In combination with use of auxiliary oligonucleotides (which restored the availability of each pre-defined hybridisation site), the assay demonstrated the ability to fully discriminate single-base mutations with detection limits in the high picomolar range (total analysis time: 60 min). Our specific probe design, hybridisation and signal transduction paths make the analytical process remarkably simple, relatively low cost and, thus, well suited for low throughput analysis of clinically relevant samples.

Multifunctional cargo systems for biotechnology

One of the challenges in the field of bio-nanotechnology is the development of nano-sized delivery systems comprising different functionalities. These systems should carry bioactive substances to predefined site and unload them in controlled manner. Capsules assembled layer-by-layer have been intensively studied in the past few years owing to their ability to be modified, their capacity to encapsulate a wide range of chemicals, their responsiveness to different factors, and the variety of functionalities with which they can be enhanced. Current research focuses on the development of carriers with remote guiding and activation (optical, magnetic or ultrasound), thereby addressing unique in vivo applications with multifunctional biomaterials. Submicron-sized capsules are good models to mimic biochemical processes in a confined geometry that imitates cell organelles. Moreover, the cellular and tissue-targeted delivery of the capsules might serve as an intracellular reporter or enzymatic reactor. However, several obstacles still have to be overcome before capsule technology can be implemented. This article discusses possible solutions as well as promising applications.

Site-directed alteration of RNA sequence mediated by an engineered twin ribozyme.

Repair of deletion mutations at the RNA level: A small synthetic twin ribozyme supports the insertion of four additional nucleotides into a predefined site of an arbitrarily chosen substrate RNA (see scheme). This method of RNA repair constitutes a novel approach to the therapy of medically relevant mutations.

A Virtual Brain for Stereotactic Planning and Supporting Neurosurgery

Visualization has becoming a powerful assistive technology for neurosurgery. This paper introduces a system for stereotactic neurosurgical planning and support. Using visualization technology the system reconstructs and displays a 3D model of the interior structure of the patient's brain. Thus the surgeons can plan for surgery using a computer model. Marker registration is used to create the mapping between the patient's head and the brain model reconstructed in the computer. During the operation a robot arm is used as a navigator to locate the pre-defined incision site and the orientation of incision route. When the robot arm locates at the pre-defined site on the patient's head, it is fixed. Various medical instruments can be installed on the tip of the robot arm. The surgeon can insert a medical instrument into the pre-defined site of the patient's head, and the surgery can be implemented successfully with the help of this system. Using a virtual environment his system can also be used to teach and train new surgeons.

Manipulation and Dynamics at the Atomic Scale: A Dual Use of the Scanning Tunneling Microscopy

Atomic scale modification of surfaces may trigger off dynamical processes. This is shown here by using the tip of a scanning tunneling microscope to extract an individual atom in a controlled manner from a predefined site of the reconstructed Ge(111)- $c$(2 $\ifmmode\times\else\texttimes\fi{}$8) surface. By thermally activated hopping of neighboring adatoms to the vacant site, the produced single-atom vacancy diffuses on the surface. From statistical analysis of the adatom configurations observed around the vacancy, we determine tiny ( $<$0.15 eV) differences in free energy between the configurations. A linear increase of the configuration free energy with the number of adatoms located at metastable ${T}_{4}$ sites is reported.

A novel method for nonfluoroscopic catheter-based electroanatomical mapping of the heart. In vitro and in vivo accuracy results.

Cardiac mapping is essential for understanding the mechanisms of arrhythmias and for directing curative procedures. A major limitation of the current methods is the inability to accurately relate local electrograms to their spatial orientation. The objective of this study was to present and test the accuracy of a new method for nonfluoroscopic, catheter-based, endocardial mapping. The method is based on using a new locatable catheter connected to an endocardial mapping and navigating system. The system uses magnetic technology to accurately determine the location and orientation of the catheter and simultaneously records the local electrogram from its tip. By sampling a plurality of endocardial sites, the system reconstructs the three-dimensional geometry of the chamber, with the electrophysiological information color-coded and superimposed on the anatomy. The accuracy of the system was tested in both in vitro and in vivo studies and was found to be highly reproducible (SD, 0.16 +/- 0.02 [mean +/- SEM] and 0.74 +/- 0.13 mm) and accurate (mean errors, 0.42 +/- 0.05 and 0.73 +/- 0.03 mm). In further studies, electroanatomical mapping of the cardiac chambers was performed in 34 pigs. Both the geometry and activation sequence were repeatable in all pigs. The new mapping method is highly accurate and reproducible. The ability to combine electrophysiological and spatial information provides a unique tool for both research and clinical electrophysiology. Consequently, the main shortcomings of conventional mapping-namely, prolonged x-ray exposure, low spatial resolution, and the inability to accurately navigate to a predefined site-can all be overcome with this new method.

Genetic engineering of surface attachment sites yields oriented protein monolayers

There has been considerable interest in the use of self-assembly and Langmuir-Blodgett techniques to generate ordered macromolecular monolayers. We describe a general method of using genetic engineering to produce a unique chemical group on the surface of a protein at a predefined site which can be used to orient the macromolecule in a self-assembled film. Using the heme protein cytochrome b 5 , produced from a totally synthetic gene, allows direct determination of orientation through measurement of absorbance linear dichroism