Molecular Basis of the Ligand Binding Specificity of αvß8 Integrin.

αvß8 is an integrin that recognizes an Arg-Gly-Asp (RGD) motif and interacts with fibronectin, vitronectin, and latent TGF-ß1. We comprehensively determined the binding activity of the αvß8 integrin toward 25 secreted proteins having an RGD motif. The αvß8 integrin strongly bound to latent TGF-ß1 but showed marginal activity for other RGD-containing proteins, including fibronectin and vitronectin. Site-directed mutagenesis of latent TGF-ß1 demonstrated that the high affinity binding of αvß8 integrin to latent TGF-ß1 was defined by Leu-218 immediately following the RGD motif within the latency-associated peptide of TGF-ß1. Consistent with the critical role of Leu-218 in latent TGF-ß1 recognition by αvß8 integrin, a 9-mer synthetic peptide containing an RGDL sequence strongly inhibited interactions of latent TGF-ß1 with αvß8 integrin, whereas a 9-mer peptide with an RGDA sequence was ∼60-fold less inhibitory. Because αvß3 integrin did not exhibit strong binding to latent TGF-ß1 or distinguish between RGDL- and RGDA-containing peptides, we explored the mechanism by which the integrin ß8 subunit defines the high affinity binding of latent TGF-ß1 by αvß8 integrin. Production of a series of swap mutants of integrin ß8 and ß3 subunits indicated that the high affinity binding of αvß8 integrin with latent TGF-ß1 was ensured by interactions between the Leu-218 residue and the ß8 I-like domain, with the former serving as an auxiliary recognition residue defining the restricted ligand specificity of αvß8 integrin toward latent TGF-ß1. In support of this conclusion, high affinity binding toward the αvß8 integrin was conferred on fibronectin by substitution of its RGDS motif with an RGDL sequence.

Polydom/SVEP1 is a ligand for integrin α9ß1.

A variety of proteins, including tenascin-C and osteopontin, have been identified as ligands for integrin α9ß1. However, their affinities for integrin α9ß1 are apparently much lower than those of other integrins (e.g. α3ß1, α5ß1, and α8ß1) for their specific ligands, leaving the possibility that physiological ligands for integrin α9ß1 still remain unidentified. In this study, we found that polydom (also named SVEP1) mediates cell adhesion in an integrin α9ß1-dependent manner and binds directly to recombinant integrin α9ß1 with an affinity that far exceeds those of the known ligands. Using a series of recombinant polydom proteins with N-terminal deletions, we mapped the integrin-binding site to the 21st complement control protein domain. Alanine-scanning mutagenesis revealed that the EDDMMEVPY sequence (amino acids 2636-2644) in the 21st complement control protein domain was involved in the binding to integrin α9ß1 and that Glu(2641) was the critical acidic residue for the integrin binding. The importance of this sequence was further confirmed by integrin binding inhibition assays using synthetic peptides. Immunohistochemical analyses of mouse embryonic tissues showed that polydom colocalized with integrin α9 in the stomach, intestine, and other organs. Furthermore, in situ integrin α9ß1 binding assays using frozen mouse tissues showed that polydom accounts for most, but not all, of the integrin α9ß1 ligands in tissues. Taken together, the present findings indicate that polydom is a hitherto unknown ligand for integrin α9ß1 that functions as a physiological ligand in vivo.

Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos.

The multiple displacement amplification (MDA) method, which relies on isothermal DNA amplification using the DNA polymerase of the bacteriophage phi29, was recently developed for high-performance, whole-genome amplification (WGA). The objective of the present study was to determine whether a target sequence could be successfully amplified by conventional PCR when the genomic DNA of a single Day-7 porcine blastocyst (derived from SCNT of a gene-engineered fibroblast) was amplified by the MDA method and used as a template. The yield of double-stranded DNA was 103.5 ± 16.0 ng/embryo (range, 75-125), as assessed by a PocoGreen assay. However, non-specific products (20 ± 5 ng/tube) were also generated, even in the negative control. Thus, ∼81% of the 103.5 ng (84 ng) of amplified DNA was estimated to be porcine sequences (2.2 × 10(3)-fold enrichment). In addition, PCR confirmed the presence of transgenes, as well as endogenous α-1,3-galactosyltransferase and homeobox Nanog genes in all embryos. Sequencing of the amplified products verified the fidelity of this system. In conclusion, the MDA-mediated WGA, which was simple, inexpensive, and did not require a thermal cycler, could be a powerful tool for multiple genomic analyses of individual early porcine embryos.

Volume-doubling time of pulmonary nodules with ground glass opacity at multidetector CT: Assessment with computer-aided three-dimensional volumetry.

RATIONALE AND OBJECTIVES: To investigate the volume-doubling time (VDT) of histologically proved pulmonary nodules showing ground glass opacity (GGO) at multidetector CT (MDCT) using computer-aided three-dimensional volumetry. MATERIALS AND METHODS: We retrospectively evaluated 47 GGO nodules (mixed n = 28, pure n = 19) that had been examined by thin-section helical CT more than once. They were histologically confirmed as atypical adenomatous hyperplasia (AAH, n = 13), bronchioloalveolar carcinoma (BAC, n = 22), and adenocarcinoma (AC, n = 12). Using computer-aided three-dimensional volumetry software, two radiologists independently performed volumetry of GGO nodules and calculated the VDT using data acquired from the initial and final CT study. We compared VDT among the three pathologies and also compared the VDT of mixed and pure GGO nodules. RESULTS: The mean VDT of all GGO nodules was 486.4 ± 368.6 days (range 89.0-1583.0 days). The mean VDT for AAH, BAC, and AC was 859.2 ± 428.9, 421.2 ± 228.4, and 202.1 ± 84.3 days, respectively; there were statistically significant differences for all comparative combinations of AAH, BAC, and AC (Steel-Dwass test, P < .01). The mean VDT for pure and mixed GGO nodules was 628.5 ± 404.2 and 276.9 ± 155.9 days, respectively; it was significantly shorter for mixed than pure GGO nodules (Mann-Whitney U-test, P < .01). CONCLUSION: The evaluation of VDT using computer-aided volumetry may be helpful in assessing the histological entities of GGO nodules.

Usefulness of a non-invasive reporter system for monitoring reprogramming state in pig cells: results of a cell fusion experiment.

Dedifferentiation of differentiated cells such as fibroblasts into pluripotent stem cells, so-called iPS cells, was first reported by Yamanaka et al., who successfully employed retroviral gene delivery of four stem-cell-specific transcription factors (Oct-3/4, Klf4, Sox2 and c-myc). Despite the mouse system in which an Oct-3/4 or Nanog promoter-based reporter system has already been established, there is no useful system in pigs for reporting the reprogramming state of gene-engineered cells. In this study, we constructed a pOEIN plasmid carrying a ca. 5.4-kb mouse Oct-3/4 promoter linked to the EGFP cDNA and neomycin expression unit and produced a porcine embryonic cell line stably incorporating it in the genome. Cell fusion with mouse embryonal carcinoma cell line F9 resulted in generation of colonies with distinct EGFP-derived fluorescence around 14 days after fusion. RT-PCR using these colonies also confirmed expression of endogenous porcine pluripotency-specific Oct-3/4, Sox2 and Stat3 mRNA. These findings suggest that mouse-derived components are sufficient to induce dedifferentiation of differentiated pig cells and also that reprogramming proceeds gradually. The present non-invasive reporter system will be useful to better define the reprogramming mechanism and/or to identify novel reprogramming molecules in the pig.

Valproic acid enhances in vitro development and Oct-3/4 expression of miniature pig somatic cell nuclear transfer embryos.

The present study was carried out to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, on in vitro development of miniature pig somatic cell nuclear transfer (SCNT) embryos and on expression of a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) introduced into donor cells for SCNT during their development. The addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.01) increased the blastocyst formation rate of SCNT embryos compared with the control, whereas VPA did not affect their cleavage rate. The rate of SCNT embryos expressing EGFP at 5 days of culture was not affected by the presence or absence of VPA treatment. At 7 days of culture, however, the addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly (p < 0.05) increased the rate of SCNT embryos expressing EGFP compared with the control. The results indicate that VPA enhances the ability of miniature pig SCNT embryos to develop into blastocysts and maintains the ability of them to express Oct-3/4 gene.

Computer-aided volumetry of pulmonary nodules exhibiting ground-glass opacity at MDCT.

OBJECTIVE: The purpose of this study was to investigate the accuracy and reproducibility of results acquired with computer-aided volumetry software during MDCT of pulmonary nodules exhibiting ground-glass opacity. MATERIALS AND METHODS: To evaluate the accuracy of computer-aided volumetry software, we performed thin-section helical CT of a chest phantom that included simulated 3-, 5-, 8-, 10-, and 12-mm-diameter ground-glass opacity nodules with attenuation of -800, -630, and -450 HU. Three radiologists measured the volume of the nodules and calculated the relative volume measurement error, which was defined as follows: (measured nodule volume minus assumed nodule volume / assumed nodule volume) x 100. Two radiologists performed two independent measurements of 59 nodules in humans. Intraobserver and interobserver agreement was evaluated with Bland-Altman methods. RESULTS: The relative volume measurement error for simulated ground-glass opacity nodules measuring 3 mm ranged from 51.1% to 85.2% and for nodules measuring 5 mm or more in diameter ranged from -4.1% to 7.1%. In the clinical study, for intraobserver agreement, the 95% limits of agreement were -14.9% and -13.7% and -16.6% to 15.7% for observers A and B. For interobserver agreement, these values were -16.3% to 23.7% for nodules 8 mm in diameter or larger. CONCLUSION: With computer-aided volumetry of ground-glass opacity nodules, the relative volume measurement error was small for nodules 5 mm in diameter or larger. Intraobserver and interobserver agreement was relatively high for nodules 8 mm in diameter or larger.

Development of a noninvasive monitoring system for evaluation of Oct-3/4 promoter status in miniature pig somatic cell nuclear transfer embryos.

The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei.

Pulmonary nodules: estimation of malignancy at thin-section helical CT--effect of computer-aided diagnosis on performance of radiologists.

PURPOSE: To evaluate the effect of a computer-aided diagnosis (CAD) system on the diagnostic performance of radiologists for the estimation of the malignancy of pulmonary nodules on thin-section helical computed tomographic (CT) scans. MATERIALS AND METHODS: The institutional review board approved use of the CT database; informed specific study-related consent was waived. The institutional review board approved participation of radiologists; informed consent was obtained from all observers. Thirty-three (18 malignant, 15 benign) pulmonary nodules of less than 3.0 cm in maximal diameter were evaluated. Receiver operating characteristic (ROC) analysis with a continuous rating scale was used to compare observer performance for the estimation of the likelihood of malignancy first without and then with the CAD system. The participants were 10 board-certified radiologists and nine radiology residents. RESULTS: For all 19 participants, the mean area under the best-fit ROC curve (A(z)) values achieved without and with the CAD system were 0.843 +/- 0.097 (standard deviation) and 0.924 +/- 0.043, respectively. The difference was significant (P = .021). The mean A(z) values achieved without and with the CAD system were 0.910 +/- 0.052 and 0.944 +/- 0.040, respectively, for the 10 board-certified radiologists (P = .190) and 0.768 +/- 0.078 and 0.901 +/- 0.036, respectively, for the nine radiology residents (P = .009). CONCLUSION: Use of the CAD system significantly (P = .009) improved the diagnostic performance of radiology residents for assessment of the malignancy of pulmonary nodules; however, it did not improve that of board-certified radiologists.

Pulmonary nodules at chest CT: effect of computer-aided diagnosis on radiologists' detection performance.

PURPOSE: To evaluate the effect of computer-aided diagnosis (CAD) on radiologists' detection of pulmonary nodules. MATERIALS AND METHODS: Fifty chest computed tomographic (CT) examination cases were used. The mean nodule size was 0.81 cm +/- 0.60 (SD) (range, 0.3-2.9 cm). Alternative free-response receiver operating characteristic (ROC) analysis with a continuous rating scale was used to compare the observers' performance in detecting nodules with and without use of CAD. Five board-certified radiologists and five radiology residents participated in an observer performance study. First they were asked to rate the probability of nodule presence without using CAD; then they were asked to rate the probability of nodule presence by using CAD. RESULTS: For all radiologists, the mean areas under the best-fit alternative free-response ROC curves (Az) without and with CAD were 0.64 +/- 0.08 and 0.67 +/- 0.09, respectively, indicating a significant difference (P <.01). For the five board-certified radiologists, the mean Az values without and with CAD were 0.63 +/- 0.08 and 0.66 +/- 0.09, respectively, indicating a significant difference (P <.01). For the five resident radiologists, the mean Az values without and with CAD were 0.66 +/- 0.04 and 0.68 +/- 0.04, respectively, indicating a significant difference (P =.02). At observer performance analyses, there were no significant differences in Az values obtained either without (P =.61) or with (P =.88) CAD between the board-certified radiologists and the residents. For all radiologists, in the detection of pulmonary nodules 1.0 cm in diameter or smaller, the mean Az values without and with CAD were 0.60 +/- 0.11 and 0.64 +/- 0.11, respectively, indicating a significant difference (P <.01). CONCLUSION: Use of the CAD system improved the board-certified radiologists' and residents' detection of pulmonary nodules at chest CT.