Knockout of a transgene by transcription activator-like effector nucleases (TALENs) in the sawfly, Athalia rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis (Coleoptera).

Transcription activator-like effector nucleases (TALENs) are efficient tools for targeted genome editing and have been utilized in a number of insects. Here, we demonstrate the gene disruption (knockout) caused by TALENs targeting a transgene, 3xP3-driven enhanced green fluorescence protein (EGFP), that is integrated in the genome of two species, the sawfly Athalia rosae (Hymenoptera) and the ladybird beetle Harmonia axyridis (Coleoptera). Messenger RNAs of TALENs targeting the sequences adjacent to the chromophore region were microinjected into the eggs/embryos of each species. In At. rosae, when microinjection was performed at the posterior end of eggs, 15% of G(0) individuals showed a somatic mosaic phenotype for eye EGFP fluorescence. Three-quarters of the somatic mosaics produced EGFP-negative G(1) progeny. When eggs were injected at the anterior end, 63% of the G(0) individuals showed somatic mosaicism, and 17% of them produced EGFP-negative G(1) progeny. In H. axyridis, 25% of posterior-injected and 8% of anterior-injected G(0) individuals produced EGFP-negative G(1) progeny. In both species, the EGFP-negative progeny retained the EGFP gene, and various deletions were detected in the target sequences, indicating that gene disruption was successfully induced. Finally, for both species, 18-21% of G(0) founders produced gene knockout progeny sufficient for establishing knockout strains.

Vestigial and scalloped in the ladybird beetle: a conserved function in wing development and a novel function in pupal ecdysis.

In Drosophila melanogaster, Vestigial (Vg) and Scalloped (Sd) form a transcription factor complex and play a crucial role in wing development. To extend our knowledge of insect wing formation, we isolated vg and sd homologues from two ladybird beetle species, Henosepilachna vigintioctopunctata and Harmonia axyridis. Although the ladybird beetle vg homologues had only low homology with D. melanogaster vg, ectopic expression of H. vigintioctopunctata vg induced wing-like tissues in antennae and legs of D. melanogaster. Subsequent larval RNA interference (RNAi) analysis in H. vigintioctopunctata demonstrated conserved functions of vg and sd in wing development, and an unexpected novel function of sd in pupal ecdysis. Furthermore, our results can be applied to the production of a flightless ladybird beetle for biological control purposes using larval RNAi.

Fractal-feature distance analysis of contrast-detail phantom image and meaning of pseudo fractal dimension and complexity.

The purposes of our studies are to examine whether or not fractal-feature distance deduced from virtual volume method can simulate observer performance indices and to investigate the physical meaning of pseudo fractal dimension and complexity. Contrast-detail (C-D) phantom radiographs were obtained at various mAs values (0.5 - 4.0 mAs) and 140 kVp with a computed radiography system, and the reference image was acquired at 13 mAs. For all C-D images, fractal analysis was conducted using the virtual volume method that was devised with a fractional Brownian motion model. The fractal-feature distances between the considered and reference images were calculated using pseudo fractal dimension and complexity. Further, we have performed the C-D analysis in which ten radiologists participated, and compared the fractal-feature distances with the image quality figures (IQF). To clarify the physical meaning of the pseudo fractal dimension and complexity, contrast-to-noise ratio (CNR) and standard deviation (SD) of images noise were calculated for each mAs and compared with the pseudo fractal dimension and complexity, respectively. A strong linear correlation was found between the fractal-feature distance and IQF. The pseudo fractal dimensions became large as CNR increased. Further, a linear correlation was found between the exponential complexity and image noise SD.

[Cystic thymoma; report of a case].

We present a case of a 33-year-old man with a noninvasive thymoma undergoing extensive cystic degeneration. The mediastinal tumor was asymptomatic and first noted on a routine chest radiograph. Chest computed tomography (CT) and magnetic resonance imaging (MRI) showed a 11 cm cystic mass with some solid portions in the anterior mediastinum. A cystic thymoma was suggested. The mass and remnants of the thymus were removed by video-assisted thoracoscopic surgery. On cut section, the tumor was predominantly cystic, with several solid nodules randomly attached to the cyst wall. The cystic space was filled with turbid brown fluid. Histopathological examination revealed a World Health Organization (WHO) type B3 thymoma with foci of hemorrhage, necrosis and cystic degeneration, and absence of an epithelial lining of the cyst.

[Intrathoracic chest wall type lipoma with crescent-shaped mass on computed tomography; report of a case].

An asymptomatic 53-year-old woman was referred to our hospital with a slow-growing intrathoracic tumor after 3 years observation. Chest computed tomography (CT) and magnetic resonance imaging (MRI) showed an intrathoracic crescent-shaped mass measuring 13 x 2.5 cm along the left chest wall. Fatty contents were suggested. Surgical resection was successfully performed by video-assisted thracoscopic surgery. At operation, a yellowish, thinly encapsulated, 10 cm tumor was attached with its pedicle 2.5 cm in diameter to the pleura of the 7th intercostal space. The lesion was a soft mass, pedunculated and numerously lobulated, that caused the crescent form of the mass on chest CT and MRI. Histopathological examination revealed mature lipoma.

Molecular cloning and characterization of cDNAs encoding dopamine receptor-1 and -2 from brain-suboesophageal ganglion of the silkworm, Bombyx mori.

In order to better understand the relationship between dopamine and the release of diapause hormone into the blood, we cloned and characterized cDNAs encoding Bombyx mori dopamine receptor-1 and -2 (BmDopR1 and 2) from the pupal brain-suboesophageal ganglion. BmDopR1 and 2 had high similarities to group 1 (Drosophila melanogaster DOP1 and Apis mellifera DOP1) and group 2 (D. melanogaster DopR99B, A. mellifera DOP2 and Papilio xuthus DOP1), respectively. When BmDopR1 and 2 were expressed in human embryonic kidney (HEK) cells, they responded to dopamine by increasing intracellular cAMP levels, thus indicating the presence of D1-like receptors. There were no clear differences in BmDopR1 and 2 mRNA levels between brain-suboesophageal ganglion complexes of diapause and nondiapause egg producers during pupal-adult development. BmDopR1 and 2 mRNAs were concentrated in the mushroom body calyx rather than in the suboesophageal ganglion. Taking into account the results of earlier experiments on excised regions corresponding to mushroom bodies, BmDopR1 and 2 in the mushroom body apparently play a role in the release of diapause hormone.

The Fat-Dachsous signaling pathway regulates growth of horns in Trypoxylus dichotomus, but does not affect horn allometry.

Males of the Asian rhinoceros beetle, Trypoxylus dichotomus, possess exaggerated head and thoracic horns that scale dramatically out of proportion to body size. While studies of insulin signaling suggest that this pathway regulates nutrition-dependent growth including exaggerated horns, what regulates disproportionate growth has yet to be identified. The Fat signaling pathway is a potential candidate for regulating disproportionate growth of sexually-selected traits, a hypothesis we advanced in a previous paper (Gotoh et al., 2015). To investigate the role of Fat signaling in the growth and scaling of the sexually dimorphic, condition-dependent traits of the in the Asian rhinoceros beetle T. dichotomus, we used RNA interference to knock down expression of fat and its co-receptor dachsous. Knockdown of fat, and to a lesser degree dachsous, caused shortening and widening of appendages, including the head and thoracic horns. However, scaling of horns to body size was not affected. Our results show that Fat signaling regulates horn growth in T. dichotomus as it does in appendage growth in other insects. However, we provide evidence that Fat signaling does not mediate the disproportionate, positive allometric growth of horns in T. dichotomus.

claudin-18, a novel downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing.

T/EBP/NKX2.1, a member of the NKX family of homeodomain-containing transcription factors, regulates the expression of a number of genes in lung and thyroid. Here we describe the isolation and characterization of a novel target gene, termed claudin-18, that is down-regulated in the lungs of T/ebp/Nkx2.1-null mouse embryos. The gene product exhibits an amino acid sequence similar to those of the claudin multigene family of proteins that constitute tight junction strands in epithelial cells. The gene was localized by fluorescence in situ hybridization to mouse chromosome 9 at region 9E3-F1 and to human chromosome 3 at region 3q21-23. The claudin-18 gene has two promoters, each with its own unique exon 1 that is spliced to common exons 2 through 5. Alternative usage of these promoters leads to production of lung and stomach-specific transcripts. The downstream lung-specific promoter contains two T/EBP/NKX2.1 binding sites responsible for trans activation of the gene by T/EBP/NKX2.1 in lung cells. Only claudin-18 was down-regulated in T/ebp/Nkx2.1-null embryo lungs among 11 claudin transcripts examined. Furthermore, the claudin-18 transcript has an alternative 12-bp insertion derived from the 5' end of intron 4, which produces a C-terminally truncated isoform in lung and stomach. Immunohistochemistry demonstrated complete membrane localization of claudin-18 with small focal dots in the lung and stomach epithelial cells. Immunogold electron microscopy analysis revealed that claudin-18 is concentrated at the cell-cell borders of epithelial cells. These unique features suggest a potentially important role for claudin-18 in the structure and function of tight junctions in lung and stomach.

Prognostic significance of p53 mutations and 3p deletions in primary resected non-small cell lung cancer.

We evaluated the prognostic significance of p53 mutations and an allelic loss of chromosome 3p in 71 patients with non-small cell lung cancer who underwent potentially curative resection. p53 mutations were detected in 35 cases (49%), while 3p deletions were observed in 34 of 70 informative cases (49%). The presence of the p53 mutation was associated with a shortened survival in all patients (P = 0.014 by log rank test), including those in early stages of the disease (stage I or II, n = 48) (P = 0.016 by log rank test). Multivariate analysis by the Cox proportional hazards model also revealed that p53 mutation was an independent yet unfavorable prognostic factor (P = 0.013). Patients with 3p deletion tended to have a poorer prognosis, but not to a statistically significant extent.

Endocrine Control of Exaggerated Trait Growth in Rhinoceros Beetles.

Juvenile hormone (JH) is a key insect growth regulator frequently involved in modulating phenotypically plastic traits such as caste determination in eusocial species, wing polymorphisms in aphids, and mandible size in stag beetles. The jaw morphology of stag beetles is sexually-dimorphic and condition-dependent; males have larger jaws than females and those developing under optimum conditions are larger in overall body size and have disproportionately larger jaws than males raised under poor conditions. We have previously shown that large males have higher JH titers than small males during development, and ectopic application of fenoxycarb (JH analog) to small males can induce mandibular growth similar to that of larger males. What remains unknown is whether JH regulates condition-dependent trait growth in other insects with extreme sexually selected structures. In this study, we tested the hypothesis that JH mediates the condition-dependent expression of the elaborate horns of the Asian rhinoceros beetle, Trypoxylus dichotomus. The sexually dimorphic head horn of this beetle is sensitive to nutritional state during larval development. Like stag beetles, male rhinoceros beetles receiving copious food produce disproportionately large horns for their body size compared with males under restricted diets. We show that JH titers are correlated with body size during the late feeding and early prepupal periods, but this correlation disappears by the late prepupal period, the period of maximum horn growth. While ectopic application of fenoxycarb during the third larval instar significantly delayed pupation, it had no effect on adult horn size relative to body size. Fenoxycarb application to late prepupae also had at most a marginal effect on relative horn size. We discuss our results in context of other endocrine signals of condition-dependent trait exaggeration and suggest that different beetle lineages may have co-opted different physiological signaling mechanisms to achieve heightened nutrient-sensitive weapon growth.

Characterization of the secondary structure of an oligonucleotide corresponding to the autocleavage site of a precursor RNA from bacteriophage T4.

We studied the secondary structure of an RNA fragment (GUUUCGUACAAAC) (R1) having the sequence corresponding to the self-cleavage domain in a precursor RNA molecule from bacteriophage T4 infected Escherichia coli cells (p2Sp1 RNA). We synthesized an oligoribonucleotide (CAAACGUACAAAC) (R3) which contained the sequence (CGUACA) proposed for the p2Sp1 RNA self-cleavage site but did not form the hairpin loop structure. The self-cleavage ability of the single stranded RNA (R3) is significantly lower than that of R1. We have also designed a modified RNA fragment (R2), which contained a noncleavable RNA with 2'-O-methylcytidine or 2-O-methyluridine. R3 did not exhibit cleavage. To further investigate the structural requirements in the cleavage reaction, we synthesized mutant RNAs which contained different bases within consensus sequences and the cleavage sites were tested for self-cleavage. Guanosine and adenosine 3'-phosphates seemed not to be susceptible to transesterification at the cleavage site. The data from native gel electrophoresis, the CD spectra and the Tm suggested that the hairpin structure is necessary for the cleavage.

Molecular recognition of the identity-determinant set of isoleucine transfer RNA from Escherichia coli.

Molecular recognition of Escherichia coli tRNA(Ile) by the cognate isoleucyl-tRNA synthetase (IleRS) was studied by analyses of chemical footprinting with N-nitroso-N-ethylurea and aminoacylation kinetics of variant tRNA(Ile) transcripts prepared with bacteriophage T7 RNA polymerase. IleRS binds to the acceptor, dihydrouridine (D), and anticodon stems as well as to the anticodon loop. The "complete set" of determinants for the tRNA(Ile) identity consists of most of the nucleotides in the anticodon loop (G34, A35, U36, t6A37 and A38), the discriminator nucleotide (A73), and the base-pairs in the middle of the anticodon, D and acceptor stems (C29.G41, U12.A23 and C4.G69, respectively). As for the tertiary base-pairs, two are indispensable for the isoleucylation activity, whereas the others are dispensable. Correspondingly, some of the phosphate groups of these dispensable tertiary base-pair residues were shown to be exposed to N-nitroso-N-ethylurea when tRNA(Ile) was bound with IleRS. Furthermore, deletion of the T psi C-arm only slightly impaired the tRNA(Ile) activity. Thus, it is proposed that the recognition by IleRS of all the widely distributed identity determinants is coupled with a global conformational change that involves the loosening of a particular set of tertiary base-pairs of tRNA(Ile).

Major identity determinants in the "augmented D helix" of tRNA(Glu) from Escherichia coli.

By a kinetic analysis of 59 variant transcripts of Escherichia coli tRNA(Glu) with glutamyl-tRNA synthetase (GluRS), the U11.A24 base-pair, the U13.G22..A46 base-triple, and the lack of residue 47 (delta47) were found to serve as major determinants for tRNA(Glu) identity. This is the first system for which major identity determinants are reported to be clustered in the "augmented D helix", consisting of the D stem with some neighboring residues and the variable loop. Other identity determinants are U34, U35, C36 and A37 in the anticodon loop, and G1.C72 and U2.A71 in the acceptor stem. Phosphate-group protection by GluRS from ethylnitrosourea was observed most strongly for the minor groove side of D-stem helix, indicating that GluRs tightly binds to the D stem for recognition, on the minor groove side, of the potent identity-determinant groups of the U11.A24 and U13.G22 base-pairs. A46 is not involved in direct recognition by GluRS; the U13.G22..A46 base-triple is required probably for formation of the structural features that are recognized by GluRS. In this context, the essential role of characteristic delta47 in tRNA(Glu) identity may be to maintain the U13.G22..A46 base-triple.

UGRP1, a uteroglobin/Clara cell secretory protein-related protein, is a novel lung-enriched downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor.

A novel gene that is down-regulated in lungs of T/ebp/Nkx2.1-null mouse embryos has been identified using a suppressive-subtractive hybridization method. The gene product is a secreted protein, forms a homodimer, and exhibits an amino acid sequence similar to that seen in the uteroglobin/Clara cell secretory protein family of proteins. This gene, designated Ugrp1 (uteroglobin-related protein 1), consists of three exons and two introns and produces three transcripts by alternative splicing. The Ugrp1 gene was localized by fluorescence in situ hybridization to mouse chromosome 18 at region 18C-D; this region is homologous with human 5q31-34, where one of the asthma susceptibility genes has been assigned. UGRP1 mRNA is predominantly expressed in the lung, with low levels of expression in the thyroid. Expression in the lung is detectable as early as embryonic day 12.5 and increases markedly by embryonic day 16.5. In T/ebp/Nkx2.1-null embryo lungs, UGRP1 expression was significantly reduced as assessed by RT-PCR analysis. Cotransfection assays using a T/EBP/NKX2.1 expression construct with Ugrp1 promoter-luciferase reporter constructs confirmed that T/EBP/NKX2.1 regulates Ugrp1 gene activity at the transcriptional level. Thus, Ugrp1 is a downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor. Changes in UGRP1 mRNA levels in lungs from antigen-sensitized mice suggest the possible involvement of UGRP1 in inflammation.

[Aortic dissection induced by symmetry aortic connector system].

A 74-year-old male with severe triple vessel disease underwent off-pump coronary artery bypass grafting (OPCAB). Preoperative computed tomography (CT) showed severely calcified ascending aorta. We revasculize the left coronary arteries with in situ internal thoracic artery (ITA) graft and the right coronary artery with a saphenous vein graft, which was attached to the disease-free portion of the aortic root, using Symmetry aortic connector system (ACS). Although the operation was uncomplicated, and postoperative course was uneventful until the 5th postoperative day when acute type A aortic dissection occurred. The patient died of aortic rupture on the 7th postoperative day. Necropsy disclosed that the entry located just on the proximal anastomotic site of the vein graft. It is possible placement of ACS device would trigger the dissecting process. With regard to the use of these one-shot devices for diseased aorta, its safety needs further investigation, even though it might be placed on an apparently intact portion.

Differentiation-dependent expression of laminin-8 (alpha 4 beta 1 gamma 1) mRNAs in mouse 3T3-L1 adipocytes.

We report that laminin-8 (alpha 4 beta 1 gamma 1) is the specific isoform of laminin synthesized in adipocytes. Reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from mouse 3T3-L1 cells with paired primers for alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, beta 1, beta 2, beta 3, gamma 1 and gamma 2 laminins yielded amplified fragments only for alpha 4, beta 1 and gamma 1. A polyclonal antibody against mouse laminin-1 (alpha 1 beta 1 gamma 1) precipitated alpha 4 in addition to beta 1 and gamma 1, while the antibody against a deduced peptide sequence of mouse alpha 4 in addition to beta 1 and gamma 1 in addition to alpha 4. Thus, laminin-8 (alpha 4 beta 1 gamma 1) is the only isoform expressed in 3T3-L1 cells. Northern blots showed that the levels of alpha 4, beta 1 and gamma 1 mRNAs increased 2.5-fold during adipose conversion of 3T3-L1 cells. A 1062 bp cDNA fragment cloned by RT-PCR demonstrated a polymorphism in the mouse alpha 4 gene which would lead to five amino acid changes in the domain G.

Distinct roles of mouse laminin beta1 long arm domains for alpha1beta1gamma1 trimer formation.

Mouse embryonal carcinoma F9 cells expressing partial mouse laminin beta1 covering either the C-terminal end (delta beta1S) or the whole (delta beta1L) of the long arm were established to study the assembly and interchain disulfide-bonding of beta1 to endogenous laminin alpha1 and gamma1. Both delta beta1S and delta beta1L were disulfide-bonded to gamma1 but only delta beta1L gamma1 dimer formed a disulfide-bonded alpha1 delta beta1L gamma1 trimer which was actively secreted into the medium. Meanwhile, in the cells producing delta beta1S gamma1 dimer, the level of endogenous alpha1 beta1 gamma1 was reduced but the level of monomeric alpha1 was increased, suggesting that alpha1 was recruited to trimer formation with the delta beta1S gamma1 dimer without disulfide-bonding. This shows that the delta beta1S gamma1 dimer can associate with alpha1 but not support the disulfide-bonding at the N-terminus of the long arm of alpha1. While control cells secrete neither monomeric alpha1 nor the beta1gamma1 dimer into the medium, the delta beta1S gamma1 producing cells probably do as alpha1 delta beta1 gamma1 trimer. We thus propose that the N- and C-termini of the long arm of laminin beta1 have distinct roles for trimer formation.

A 15N-1H nuclear magnetic resonance study on the interaction between isoleucine tRNA and isoleucyl-tRNA synthetase from Escherichia coli.

Imino 15N and 1H resonances of Escherichia coli tRNA(lIle) were observed in the absence and presence of E coli isoleucyl-tRNA synthetase. Upon complex formation of tRNA(lIle) with isoleucyl-tRNA synthetase, some imino 15N-1H resonances disappeared, and some others were significantly broadened and/or shifted in the 1H chemical shift, while the others were observed at the same 15N-1H chemical shifts. It was indicated that the binding of tRNA(lIle) with IleRS affect the following four regions: the anticodon stem, the junction of the acceptor and T stems, the middle of the D stem, and the region where the tertiary base pair connects the T, D, and extra loops. This result is consistent with those of chemical footprinting and site-directed mutagenesis studies. Taken together, these three independent results reveal the recognition mechanism of tRNA(lIle) by IleRS: IleRS recognizes all the identity determinants distributed throughout the tRNA(lIle) molecule, which induces changes in the secondary and tertiary structures of tRNA(lIle).

Design and synthesis of non-peptidic inhibitors for the Syk C-terminal SH2 domain based on structure-based in-silico screening.

Structure-based in-silico screening was carried out for the Syk C-terminal SH2 domain. Fragments that could interact with the pY or pY+1 pockets were selected by our in-silico screening. After tethering two fragments bound to these pockets, we have designed and synthesized new compounds that show favorable interaction with the pY+3 pocket. One such compound, having a cyclohexylmalonic acid moiety identified as a novel potent phosphotyrosyl mimetic, exhibited an affinity comparable to that of the monophosphorylated ligand peptide.

Structure of the VAP-peptide (BmACP-6.7) gene in the silkworm, Bombyx mori and a possible regulation of its expression by BmFTZ-F1.

The VAP-peptide (BmACP-6.7) is a hydrophobic peptide localized in adult cuticle of the silkworm, Bombyx mori. We isolated and characterized the VAP-peptide gene as a useful marker gene to analyze molecular mechanisms of terminal differentiation processes in the adult. The gene is composed of two exons interrupted by one intron. The 5' upstream promoter region is shown to bear a nucleotide sequence similar to the cis-element that is recognized and bound by the Bombyx mori FTZ-F1 protein (BmFTZ-F1). Expression of the BmFTZ-F1 gene preceded expression of the VAP-peptide gene and injection of 20-hydroxyecdysone suppressed the expression of both genes. An in vitro binding assay indicated direct interaction of BmFTZ-F1 with the VAP-peptide gene promoter sequence. Therefore, BmFTZ-F1 is proposed to be a possible factor regulating the stage-specific expression of the VAP-peptide gene towards adult life.