Chad Walton

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Information theory for atypical sequences

One characteristic of the information age is the exponential growth of information, and the ready availability of this information through networks, including the internet - “Big Data.” The question is what to do with this enormous amount of information. One possibility is to characterize it through statistics - think averages. The perspective in this paper is the opposite, namely that most of the value in the information is in the parts that deviate from the average, that are unusual, atypical. Think of art: the valuable paintings or writings are those that deviate from the norms, that are atypical. The same could be true for venture development and scientific research. The paper first discusses what exactly should be understood by “atypical.” This is by no means straightforward. It has to be a well defined theoretical concept corresponding to some intuitive idea of atypicality, which when applied gives useful results. This is followed by a simple example of iid binary sequences. This example is simple enough that complete algorithms can be developed and analyzed, which give insights into atypicality. We finally develop a more general algorithm based on the Context Tree Weighing algorithm and apply that to heart rate variability.

Tiny Bubbles and Endocytosis

See related article, pages 679–687 Since the initial use of bubbles for echocardiographic contrast, it has been a common clinical observation that these bubbles are disrupted in a sonographic field. It was recognized that the frequency and power of the ultrasound beam were key elements in the efficiency of this disruption, and it was concluded that bubble resonance, leading to cavitation, caused bubble destruction. The combination of ultrasound and microbubbles as a therapeutic modality was described in 1998, as an approach to in vivo arterial clot dissolution.1 Subsequently, this combination has been investigated for delivery of bioactive molecules and has been shown to deliver plasmids with reporter constructs to a variety of target organs, usually with low efficiency. More recently, it has been observed that in the presence of microbubbles, even levels of ultrasound energy that would not be expected to produce cavitation can augment delivery of macromolecules. Thus, there may be 2 distinct processes of cell permeabilization occurring in the presence of ultrasound and microbubbles. High acoustic pressure leads to cavitation, causing the bubbles to implode, with substantial release of energy, driving lipid shell through the cell membrane. (This process has been called UTMD for ultrasound targeted microbubble destruction.) Lower acoustic pressure produces subcavitary oscillation and has been suggested to activate the cell membrane to allow for transfer of macromolecules.2 In the article …

Universal data discovery using atypicality

With the enormous amount of data generated through the internet and sensors, Internet of Things, it becomes too overwhelming for humans to examine it all. One solution is to reduce the data to a set of statistics. The perspective in this paper is the opposite, namely that most of this data is just background noise, and the interesting parts are those that deviate from background noise, the parts that are atypical. In order to find such “interesting” parts of data, universal approaches are required, since it is not known in advance what we are looking for. The paper develops new algorithms for detecting atypical data based on information theory concepts. These are applied to a number of real-world data.

Chromatin Immunoprecipitation Assay: Examining the Interaction of NFkB with the VEGF Promoter

The chromatin immunoprecipitation (ChIP) assay is a versatile technique used to evaluate the association of proteins with specific DNA regions both in vivo and in vitro. This assay can be used to identify proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome associated with a particular protein. The ChIP assay can also be used to analyze binding of transcription factors, transcription cofactors, DNA replication factors, and DNA repair proteins. Here we describe a useful ChIP-qPCR protocol to examine the interaction of NFkB with the VEGF promoter in adult rat primary cardiomyocytes that have been mechanically stretched after attaching to the extracellular matrix protein laminin.

The pydA-pydB fusion gene produces an active dioxygenase-hydrolase that degrades 3-hydroxy-4-pyridone, an intermediate of mimosine metabolism.

The objective of this research was to construct a pydA–pydB hybrid gene that encodes a functional dioxygenase–hydrolase (PydA–PydB) fusion protein for degradation of 3-hydroxy-4-pyridone (HP). HP is an intermediate in both synthesis and degradation of mimosine, a toxic amino acid produced by the tree legume Leucaena leucocephala. Computer-generated models of the fusion proteins suggested that joining of PydA and PydB with 0, 3, or 7 glycine residues as a linker should produce a functional PydA–PydB fusion protein. Accordingly, three hybrid genes, G0, G3, and G7, were constructed in which pydA and pydB were connected with 0, 9, and 21 nucleotides, respectively, encoding the glycine residues of the linker region. When these hybrid genes were expressed in Rhizobium and Escherichia coli, only one of them, G3, produced a functional PydA–PydB fusion protein, having both the dioxygenase and hydrolase activities. The G3 hybrid gene could complement both pydA and pydB mutants of Rhizobium, and E. coli lysate containing the overexpressed G3 protein was able to degrade HP. This hybrid gene may be useful for developing mimosine-free L. leucocephala plants in the future.