Ultrasound Molecular Imaging of Secreted Frizzled Related Protein-2 Expression in Murine Angiosarcoma

Angiosarcoma is a biologically aggressive vascular malignancy with a high metastatic potential. In the era of targeted medicine, knowledge of specific molecular tumor characteristics has become more important. Molecular imaging using targeted ultrasound contrast agents can monitor tumor progression non-invasively. Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. We hypothesize that SFRP2-directed imaging could be a novel approach to imaging the tumor vasculature. To develop an SFRP2 contrast agent, SFRP2 polyclonal antibody was biotinylated and incubated with streptavidin-coated microbubbles. SVR angiosarcoma cells were injected into nude mice, and when tumors were established the mice were injected intravenously with the SFRP2 -targeted contrast agent, or a control streptavidin-coated contrast agent. SFRP2 -targeted contrast agent detected tumor vasculature with significantly more signal intensity than control contrast agent: the normalized fold-change was 1.6±0.27 (n = 13, p = 0.0032). The kidney was largely devoid of echogenicity with no significant difference between the control contrast agent and the SFRP2-targeted contrast agent demonstrating that the SFRP2-targeted contrast agent was specific to tumor vessels. Plotting average pixel intensity obtained from SFRP2-targeted contrast agent against tumor volume showed that the average pixel intensity increased as tumor volume increased. In conclusion, molecularly-targeted imaging of SFRP2 visualizes angiosarcoma vessels, but not normal vessels, and intensity increases with tumor size. Molecular imaging of SFRP2 expression may provide a rapid, non-invasive method to monitor tumor regression during therapy for angiosarcoma and other SFRP2 expressing cancers, and contribute to our understanding of the biology of SFRP2 during tumor development and progression.     

Predictive Factor of Local Recurrence after Balloon-Occluded TACE with Miriplatin (MPT) in Hepatocellular Carcinoma

Background

Miriplatin (MPT) is a novel platinum complex used in TACE that shows promise for the treatment of hepatocellular carcinoma (HCC). However, rapid washout has been reported in some cases. Therefore, various methods of administration with MPT have been attempted to increase its therapeutic efficacy. One hopeful method is balloon-occluded TACE (B-TACE), but the therapeutic efficacy of B-TACE with MPT has not been evaluated.

Aim

To investigate the treatment outcomes and factors involved in local recurrence after B-TACE with MPT in HCC.

Methods

This study included 51 patients (55 nodules) with HCC lesions equal or less than 5 cm in diameter who underwent B-TACE with MPT between January 2012 and June 2013. Local recurrence after B-TACE with MPT and factors associated with local recurrence were evaluated.

Results

The overall local recurrence rate was 11.1% at 6 months and 26.2% at 12 months. The local recurrence rate did differ significantly depending on CT values immediately after B-TACE with MPT. Multivariate analysis also showed that the CT value after B-TACE with MPT was the only factor related to local recurrence after B-TACE.

Conclusions

B-TACE with MPT achieves relatively good local control of HCC. The plain CT value immediately after B-TACE with MPT is a predictive factor for local recurrence. In patients with unsatisfactory CT values, locoregional therapy or additional treatment is required.     

Arf6 Guanine Nucleotide Exchange Factor Cytohesin-2 Binds to CCDC120 and Is Transported Along Neurites to Mediate Neurite Growth

The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth.     

Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound-healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound-healing drug with antibacterial and angiogenic actions.     

Molecular phylogeny of the tribe Candalidini (Lepidoptera: Lycaenidae): systematics,diversification and evolutionary history

The butterfly tribe Candalidini is geographically restricted to Australia and mainland New Guinea and its adjacent islands. With 60 species and subspecies, it represents a large radiation of Papilionoidea in the Australian region. Although the species-level taxonomy is relatively well understood, the number of genera is uncertain, varying from two to eight. We reconstructed the phylogeny of the Candalidini based on a 13-locus hybrid enrichment probe set (12.8 Kbp: COI, Thiolase, CAD, CAT, DDC, EF1-a, GAPDH, HCL, IDH, MDH, RPS2, RPS5, Wingless), including all previously recognized genera and 76% (28/37) of the species-level diversity of the tribe. Maximum likelihood analysis recovered the Candalidini as a strongly supported monophyletic group. In conjunction with morphological characters, the phylogeny provided a robust framework for a revised classification in which we recognize four genera, 37 species and 23 subspecies. The genus Nesolycaena Waterhouse & R.E. Turner is considered in synonymy with Candalides Hübner, and four other genera are not recognized, namely, Holochila C. Felder, Adaluma Tindale, Zetona Waterhouse and Microscena Tite. Of the four valid genera, the absimilis group (23 species) is placed in the newly described genus Eirmocides Braby, Espeland & Müller gen. nov. (type species Candalides consimilis Waterhouse). The erinus group (six species) is assigned to Erina Swainson, which is reinstated. Chrysophanus cyprotus Olliff is assigned to Cyprotides Tite, which is also reinstated as a monotypic genus. The remaining seven species are placed in Candalides sensu stricto. Overall, we propose 47 new nomenclatural changes at the species and subspecies levels, including the synonymy of Holochila biaka Tite as Eirmocides tringa biaka (Tite) syn. nov. et comb. nov. and recognition of Candalides hyacinthinus gilesi M.R. Williams & Bollam as a distinct species Erina gilesi (M.R. Williams & Bollam stat. rev. et comb. nov. A dated phylogeny using Bayesian inference in BEAST2 and biogeographical and habitat analyses based on the DEC model in BioGeoBEARS indicated that the ancestor of the Candalidini most likely evolved in rainforest habitats of the mesic biome in situ on the Australian plate of Southern Gondwana during the Eocene (c. 43 Ma). A major period of diversification occurred in the Miocene, which coincided with aridification of the Australian continent, followed by a further episode of radiation in montane New Guinea during the Plio-Pleistocene. This published work has been registered on ZooBank by the authors: Michael Braby: http://zoobank.org/urn:lsid:zoobank.org:author:4D3A7605-EBD0-40F6-A5F2-7F67F59E3D60 ; Marianne Espeland: http://zoobank.org/urn:lsid:zoobank.org:author:00D6F9F9-3902-4A8B-846F-720AB32922A6 ; Chris Müller: http://zoobank.org/urn:lsid:zoobank.org:author:15FE5F26-7596-46C2-9697-1FD92A692D0D ; http://zoobank.org/urn:lsid:zoobank.org:pub:47D5CA34-C294-4FBD-84B6-1C2A82B7CADF .     

Structural kinetics of the allosteric transition of aspartate transcarbamylase produced by physiological substrates

We have studied the kinetics of the quaternary structure change associated with the allosteric transition of aspartate transcarbamylase (ATCase) (E. coli), inducing this change by exposure to the natural substrates (carbamyl phosphate and L-aspartate). The presence of 30% ethylene glycol slowed the quaternary structure change sufficiently for it to be followed by stopped-flow X-ray scattering at -5 degrees C. After adding substrates to the enzyme, the change occurred, with a half-life of a few seconds, yielding a mixture of the two standard quaternary structures (or, conceivably, a state intermediate between them). This mixture persisted until the enzyme reduced the substrate concentration below a threshold value.     

Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.     

Crystal structure analysis of human serum albumin complexed with sodium 4-phenylbutyrate

Sodium 4-phenylbutyrate (PB) is an orphan drug for the treatment of urea cycle disorders. It also inhibits the development of endoplasmic reticulum stress, the action of histone deacetylases and as a regulator of the hepatocanalicular transporter. PB is generally considered to have the potential for use in the treatment of the diseases such as cancer, neurodegenerative diseases and metabolic diseases. In a previous study, we reported that PB is primarily bound to human serum albumin (HSA) in plasma and its binding site is drug site 2. However, details of the binding mode of PB to HSA remain unknown. To address this issue, we examined the crystal structure of HSA with PB bound to it. The structure of the HSA–PB complex indicates that the binding mode of PB to HSA is quite similar to that for octanoate or drugs that bind to drug site 2, as opposed to that for other medium-chain length of fatty acids. These findings provide useful basic information related to drug–HSA interactions. Moreover, the information presented herein is valuable in terms of providing safe and efficient treatment and diagnosis in clinical settings.