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Although nuclear power plant accidents have raised concerns about the genetic consequences of radiation exposure, no studies have successfully compared the DNA of wild animals before and after the accident in the same area around a nuclear power plant. Here, we investigated 12 microsatellites of Japanese wild boar in the Fukushima prefecture before and after the Fukushima Daiichi nuclear accident. A population structure analysis of the microsatellites revealed that wild boar of the 4 prefectures of North Kanto were divided into 5 populations before the nuclear accident. Yet, wild boar sampled after the nuclear accident did not belong to any of these 5 populations. The reason for this result was related to significant changes in the composition of microsatellites after the nuclear accident; on average, 90% of post-accident microsatellite alleles were newly emergent in Fukushima prefecture. In samples obtained before the nuclear accident, each microsatellite had either even or odd-length alleles. Yet, in samples obtained after the nuclear accident, most microsatellites had alleles of both even and odd lengths. Moreover, we examined haplotypes and their frequencies in the mitochondrial DNA control region, and found no significant differences before and after the nuclear accident in both Fukushima and Tochigi prefectures, and no haplotypes were distributed only in distant areas suggesting an influx of wild boar after the nuclear accident. This research suggested that the change in microsatellites was a result of microsatellite instability rather than a result of the migration of wild boar from other areas. This research provides important insights into the effects of a nuclear accident on mammals.

Homing peptides to the spinal cord were identified and isolated using phage display technology. In vivo biopanning was performed by intravenous systemic injection of a phage library to screen specific peptides targeting the spinal cord of mice. Analyses of the sequences of targeted phages yielded two candidate peptides targeting the spinal cord: SP1 (C-LHQSPHI-C) and SP2 (C-PTNNPRS-C). These peptides were synthesized and intravenously injected into mice to evaluate their tissue specificity and potential as gene delivery carriers. The complexes between SP1 or SP2 peptides and the plasmid vector expressing the reporter gene could induce gene transduction in the spinal cord through systemic injection without gene expression in the brain, liver, and kidney. In addition, intravenous injection of the complex between SP1 and the vectors induced interleukin-4 expression in the spinal cord, resulting in effective suppression of lipopolysaccharide-induced hyperalgesia. Therefore, intravenously administered spinal cord homing peptides complexed with a plasmid vector provided tissue-specific treatment featuring gene delivery to the CNS through systemic circulation. This novel method of gene delivery is feasible and has great potential for clinical application. Keywords: homing peptides, gene delivery, spinal cord, systemic circulation, targeting

A disintegrin and metalloprotease 17 (ADAM17) catalyzes the cleavage and release of the ectodomains of its substrates at the cell surface in a process termed ectodomain shedding. However, not all ADAM17 substrates have been identified. Here, we used cell surface protein-specific labeling and proteomic approaches to detect and identify ADAM17 substrates. HeLa cell surface proteins were labeled with a fluorescent dye and cultured with or without TAPI-2, an ADAM17 inhibitor. Labeled proteins released into the culture medium were detected by 2-dimensional gel electrophoresis (2DE). Protein spots showing decreased intensity in response to TAPI-2 were selected as substrates of ADAM17 or their binding proteins, and identified by mass spectrometry. ADAM17 knockdown was preformed to examine the behavior of identified proteins. Of 347 proteins detected by 2DE, 49 showed lower intensity in TAPI-2 (+) than in TAPI-2 (-) samples (p < 0.05), and were considered as candidate substrates of ADAM17. Mass spectrometric analysis of 14 protein spots showing >50% decreased intensity identified clusterin as a novel ADAM17 substrate, in addition to known substrates such as desmoglein-2. Western blot analysis showed that ADAM17 knockdown decreased the levels of clusterin fragments cleaved and released from the cell surface. The results identified clusterin as a novel ADAM17 substrate. The method used to identify clusterin could be used to identify the substrates of other sheddases involved in ectodomain shedding.

Astrocyte- and microglia-targeting peptides were identified and isolated using phage display technology. A series of procedures, including three cycles of both in vivo and in vitro biopanning, was performed separately in astrocytes and in M1 or M2 microglia, yielding 50–58 phage plaques in each cell type. Analyses of the sequences of this collection identified one candidate homing peptide targeting astrocytes (AS1[C-LNSSQPS-C]) and two candidate homing peptides targeting microglia (MG1[C-HHSSSAR-C] and MG2[C-NTGSPYE-C]). To determine peptide specificity for the target cell in vitro, each peptide was synthesized and introduced into the primary cultures of astrocytes or microglia. Those peptides could bind to the target cells and be selectively taken up by the corresponding cell, namely, astrocytes, M1 microglia, or M2 microglia. To confirm cell-specific gene delivery to M1 microglia, the complexes between peptide MG1 and siRNA-interferon regulatory factor 5 were prepared and intrathecally injected into a mouse model of neuropathic pain. The complexes successfully suppressed hyperalgesia with high efficiency in this neuropathic pain model. Here, we describe a novel gene therapy for the treatment neuropathic pain, which has a high potential to be of clinical relevance. This strategy will ensure the targeted delivery of therapeutic genes while minimizing side effects to non-target tissues or cells.