Actin exposure upon tissue injury is a targetable wound site-specific protein marker.

BACKGROUND: Identification of wound-specific markers would represent an important step toward damaged tissue detection and targeted delivery of biologically important materials to injured sites. Such delivery could minimize the amount of therapeutic materials that must be administered and limit potential collateral damage on nearby normal tissues. Yet, biological markers that are specific for injured tissue sites remain elusive. METHODS: In this study, we have developed an immunohistological approach for identification of protein epitopes specifically exposed in wounded tissue sites. RESULTS: Using ex-vivo tissue samples in combination with fluorescently-labeled antibodies we show that actin, an intracellular cytoskeletal protein, is specifically exposed upon injury. The targetability of actin in injured sites has been demonstrated in vivo through the specific delivery of anti-actin conjugated particles to the wounded tissue in a lethal rat model of grade IV liver injury. CONCLUSIONS: These results illustrate that identification of injury-specific protein markers and their targetability for specific delivery is feasible. GENERAL SIGNIFICANCE: Identification of wound-specific targets has important medical applications as it could enable specific delivery of various products, such as expression vectors, therapeutic drugs, hemostatic materials, tissue healing, or scar prevention agents, to internal sites of penetrating or surgical wounds regardless of origin, geometry or location.

Chromatin structure analysis enables detection of DNA insertions into the mammalian nuclear genome.

BACKGROUND: Genetically modified organisms (GMOs) have numerous biomedical, agricultural and environmental applications. Development of accurate methods for the detection of GMOs is a prerequisite for the identification and control of authorized and unauthorized release of these engineered organisms into the environment and into the food chain. Current detection methods are unable to detect uncharacterized GMOs, since either the DNA sequence of the transgene or the amino acid sequence of the protein must be known for DNA-based or immunological-based detection, respectively. METHODS: Here we describe the application of an epigenetics-based approach for the detection of mammalian GMOs via analysis of chromatin structural changes occurring in the host nucleus upon the insertion of foreign or endogenous DNA. RESULTS: Immunological methods combined with DNA next generation sequencing enabled direct interrogation of chromatin structure and identification of insertions of various size foreign (human or viral) DNA sequences, DNA sequences often used as genome modification tools (e.g. viral sequences, transposon elements), or endogenous DNA sequences into the nuclear genome of a model animal organism. CONCLUSIONS: The results provide a proof-of-concept that epigenetic approaches can be used to detect the insertion of endogenous and exogenous sequences into the genome of higher organisms where the method of genetic modification, the sequence of inserted DNA, and the exact genomic insertion site(s) are unknown. GENERAL SIGNIFICANCE: Measurement of chromatin dynamics as a sensor for detection of genomic manipulation and, more broadly, organism exposure to environmental or other factors affecting the epigenomic landscape are discussed.

Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase.

Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.

Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins.

In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the alpha-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.