The Toughness-Enhanced Atelocollagen Double-Network Gel for Biomaterials.

A tough gel composed of atelocollagen, which lacks an immunogenetic site, is a promising material for biomedical application. In this study, we created a composite hydrogel composed of atelocollagen gel cross-linked with glutaraldehyde (GA) and poly-(N,N-dimethylacrylamide) gel exhibiting biocompatibility based on the double-network (DN) gel principle. The tensile toughness of atelocollagen gel remained constant regardless of the amount of cross-linker (GA) used. In contrast, tensile tests of the DN gel indicated that mechanical properties, such as fracture stress and toughness, were significantly higher than those of the atelocollagen gel. Moreover, fibroblast cells adhered and spread on the gels, the Schiff bases of which were treated via reductive amination for detoxification from GA. These findings demonstrate the potential of the proposed gel materials as artificial alternative materials to soft tissues with sub-MPa fracture stress.

Defective membrane expression of the Na(+)-HCO(3)(-) cotransporter NBCe1 is associated with familial migraine.

Homozygous mutations in SLC4A4, encoding the electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1, have been known to cause proximal renal tubular acidosis (pRTA) and ocular abnormalities. In this study, we report two sisters with pRTA, ocular abnormalities, and hemiplegic migraine. Genetic analysis ruled out pathological mutations in the known genes for familial hemiplegic migraine, but identified a homozygous 65-bp deletion (Delta65bp) in the C terminus of NBCe1, corresponding to the codon change S982NfsX4. Several heterozygous members of this family also presented glaucoma and migraine with or without aura. Despite the normal electrogenic activity in Xenopus oocytes, the Delta65bp mutant showed almost no transport activity due to a predominant cytosolic retention in mammalian cells. Furthermore, coexpression experiments uncovered a dominant negative effect of the mutant through hetero-oligomer formation with wild-type NBCe1. Among other pRTA pedigrees with different NBCe1 mutations, we identified four additional homozygous patients with migraine. The immunohistological and functional analyses of these mutants demonstrate that the near total loss of NBCe1 activity in astrocytes can cause migraine potentially through dysregulation of synaptic pH.

Synthesis, Characterization, and Potential Application of Cyclodextrin-Based Polyrotaxanes for Reinforced Atelocollagen Threads.

Preparing strong and flexible atelocollagen-based materials for biomedical applications is still a challenging task. To address this challenge, this study describes the synthesis and characterization of water-soluble polyrotaxanes (PRs) with different coverage ratios and molecular weights of axle polymers, and their potential applications for PR-reinforced atelocollagen threads (PRATs). A novel method was established for the syntheses of PRs with relatively low coverage ratio at the sub-gram scale, in which the aldehyde groups were employed as crosslinking sites for preparing the PRATs via reductive amination. The aldehyde groups were successfully quantified by 1H nuclear magnetic resonance spectroscopy using 1,1-dimethylhydrazine as an aldehyde marker. Fourier-transform infrared and thermogravimetric analysis measurements supported the characterization of the PRs. Interestingly, tensile testing demonstrated that coverage ratio affected the mechanical properties of the PRATs more strongly than molecular weight. The insights obtained in this study would facilitate the development of soft materials based on atelocollagens and PRs.

AFM observation of single, functioning ionotropic glutamate receptors reconstituted in lipid bilayers.

BACKGROUND: Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment. METHODS: Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate. RESULTS: Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements. CONCLUSIONS: Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment. GENERAL SIGNIFICANCE: AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications.

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo.

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.

Micro­dimpled surface atelocollagen maintains primary human hepatocytes in culture and may promote their functionality compared with collagen coat culture.

Primary human hepatocytes (PHHs) are the gold standard for drug development procedures; however, maintaining functional PHHs in vitro is challenging in conventional collagen­coated cultures. In the present study, we developed a new scaffold comprising high amounts (≥1 mg/cm2) of atelocollagen exposed to ultraviolet radiation to induce cross­linking and improve stability. Scanning and transmission electron microscopy revealed a micro­dimpled surface (MDS) scaffold composed of randomly arranged atelocollagen fibrils. The scaffold was therefore designated as MDS atelocollagen. PHHs cultured on MDS atelocollagen were round with a compact cytoplasm and exhibited enhanced levels of albumin (ALB) secretion and cytochrome P450 (CYP) 3A4 activity. The expression of hepatocyte­related genes, such as serum proteins, drug metabolism­related CYPs, and nuclear receptors, was enhanced in cells cultured on MDS atelocollagen, but not in those cultured on conventional atelocollagen. Moreover, the abnormal gene expression of cell adhesion molecules observed in conventional atelocollagen culture was suppressed when the cells were grown on MDS atelocollagen, thereby suggesting a cell behavior similar to that of in vivo hepatocytes. These results suggest that MDS atelocollagen functionally preserves PHHs while conserving the simplicity of conventional PHH atelocollagen­coated cultures.

System stability of a 3T-MRI during continuous EPI scan.

The purpose of this study is to evaluate the general stability and image properties of a 3T MRI system newly installed at the ATR-Brain Activity Imaging Center (ATR-BAIC), in addition to a conventional 1.5T system. In this study, we focused on the echo planar imaging (EPI) sequence since continuous EPI with a relatively long duration of up to 30 min is routinely used, and the stabilization of EPI is always a concern. The following five results were obtained: (1) Significant image shifts along the phase direction were observed in the 1.5T data but not in the 3T data, although B0 shifts in both the 1.5T and 3T systems were the same level (1.3 Hz/min); (2) The signal fluctuations were 1/2-1/3 smaller in the 3T system compared with the 1.5T system; (3) The temporal signal-to-noise ratio (TSNR) of the 3T system was 1.7-2.0 (CP-coil) and 2.5-4.0 (12ch-coil) greater than the 1.5T system; (4) We found a low frequency periodic fluctuation (cycles of approximately 30-40 sec), and an increase in noise in the latter half of the long term series, which might originate from the 3T MRI scanner; and (5) Spatial non-uniformity of TSNR and voxels with a linear-trend were observed in the 3T data.

Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy.

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca(2+) from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP(3)R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP(3)R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP(3)R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending approximately 4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP(3)R (Sf9-IP(3)R1). The average diameter of the large protrusions was approximately 32 nm. A specific antibody against a cytosolic epitope close to the IP(3)-binding site enabled us to gold-label the Sf9-IP(3)R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of approximately 30 nm, which should be IP(3)R1-antibody complexes. Authentic IP(3)R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP(3)R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP(3)R1 membrane correspond to the cytosolic domain of IP(3)R1. Our study provides the first 3D representation of individual IP(3)R1 particles in an aqueous solution.

Optimal bovine collagen concentration to achieve tracheal epithelial coverage of collagen sponges.

OBJECTIVES/HYPOTHESIS: Artificial tracheas prepared using a collagen sponge and polypropylene mesh have been implanted in patients who received tracheal resections, but epithelialization in the reconstructed area is slow. We determined the optimal bovine atelocollagen concentration necessary for the rapid and complete tracheal epithelial coverage of collagen sponge implants. STUDY DESIGN: Preliminary animal experiment. METHODS: Collagen sponges were prepared using lyophilizing 0.5%, 0.7%, and 1.0% atelocollagen solutions (0.5%, 0.7%, and 1.0% sponges) and were analyzed using scanning electron microscopy. Partial tracheal defects were prepared in rabbits and reconstructed using sponges. Epithelial regeneration in the reconstructed area was evaluated by endoscopic, histological, and scanning electron microscope analyses. RESULTS: All sponges had a membranous structural framework, and numerous fibrous structures filled the spaces within the framework in the 0.5% sponges. The membranous structure in the 0.7% sponges branched at many points, and intermembrane spaces were frequently observed. Conversely, the membranous structure in the 1.0% sponges was relatively continuous, thick, and closely arranged. Two weeks after implantation, tracheal defects were entirely covered with epithelium in two of the four and three of the four of the 0.5% and 0.7% sponge-implanted rabbits, respectively. The collagen sponges remained exposed to the tracheal lumen in four of the four rabbits in the 1.0% sponge group. Ciliogenesis in the center of the epithelialized region was detected only in the 0.7% sponge group. CONCLUSION: Collagen sponges prepared from various concentrations of bovine atelocollagen have different structures. Complete epithelial coverage was achieved in more rabbits implanted with sponges prepared using the 0.7% bovine atelocollagen solution than in those implanted with sponges prepared from the 0.5% and 1.0% solutions. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E396-E403, 2016.

Isolation and characterization of an N-linked oligosaccharide that is increased in glioblastoma tissue and cell lines.

We have isolated and characterized N-linked oligo-saccharides that are significantly increased in glioblastoma tissue and cell lines. The structures of N-linked oligosaccharides present in 3 human normal brain tissues, 15 patients with glio-blastoma and 3 glioma cell lines were analyzed by partially automated technique for the isolation and fluorescent labeling of N-linked sugar chains from glycoproteins. Characterization of the sugar chains was achieved with the use of a combination of HPLC columns and a highly sensitive fluorescence detector at femtomole levels. By collecting peaks which accounted for 0.1% or more, sixteen different oligosaccharide structures were characterized from glioblastoma tissue and cell lines. The 16 oligosaccharide structures accounted for 48.9% of the total N-linked oligosaccharides present in glioblastoma tissue. The major components of total oligosaccharides were similar to those of normal brain tissue. The amount of a biantennary bigalactosylated structure with one core fucosylation (A2G2F) was present in increased levels in glioblastoma tissue (mean = 2.90%) and glioma cell lines (mean = 5.60%), while being less than 0.1% in normal brain tissue. Expression of highly branched tetra-antennary N-glycans that are usually detected in lungs or hepatocellular cancer was not observed. Tissue glioma cells and cultured cells also displayed strong LCA-lectin binding, which binds to sugar chains with core fucose (including A2G2F), while normal brain tissue did not. Moreover, LCA lectin inhibited proliferation of glioma cells through induction of apoptosis. A2G2F on glioma specimens may provide a novel marker and target for the diagnosis and treatment of glioblastoma, respectively.

Identification of Akt1 as a potent therapeutic target for oral squamous cell carcinoma.

Oncogene addiction can provide therapeutic opportunities in human malignancies. In this study, we aimed to identify critical oncogenes for oral squamous cell carcinoma (OSCC) development and progression. We determined gene expression profiles in 10 primary OSCCs and 10 human OSCC cell lines using Applied Biosystems Human Genome Survey Arrays. Akt1 was the only gene identified that was expressed in all OSCC tissues and cultured cells, but not in non-neoplastic tissues and cells. Subsequently, western blot analysis showed that Akt1 protein was overexpressed in OSCC tissues and cell lines. Immunohistochemistry also showed Akt1 protein expression in 59 of 63 (94%) primary OSCCs. To clarify the oncogenic function of Akt1 in human OSCC cells, we used RNA interference. We designed and synthesized 5 small interfering RNAs specific for Akt1 (siAkt1). Transfecting human OSCC cells with siAkt1 in vitro markedly suppressed their expression of Akt1 protein and significantly reduced their growth rate. Furthermore, the growth of human OSCC tumors which had been subcutaneously xenografted in athymic nude mice lacking interferon responses was markedly inhibited by atelocollagen-mediated systemic siAkt1 administration. We also found that synthetic siAkt1 had an inhibitory effect on the growth of primary cultured OSCC cells. Finally, we investigated the molecular mechanisms involved in the growth inhibitory effect of Akt1 suppression using microarray analysis of human OSCC cells transfected with siAkt1. Knockdown of Akt1 induced the expression of CDKN2B, a tumor suppressor gene, and reduced the expression of TGFBR1, which supports malignant phenotypes. These results suggest that Akt1 functions as a critical oncogene in human OSCC cells and may therefore be an appropriate target for novel OSCC therapies.

A technique to reduce motion artifact for externally triggered cine-MRI(EC-MRI) based on detecting the onset of the articulated word with spectral analysis.

One issue in externally triggered cine-magnetic resonance imaging (EC-MRI) for the dynamic observation of speech organs is motion artifact in the phase-encoding direction caused by unstable repetitions of speech during data acquisition. We propose a technique to reduce such artifact by rearranging the k-space data used to reconstruct MR images based on the analysis of recorded speech sounds. We recorded the subject's speech sounds during EC-MRI and used post hoc acoustical processing to reduce scanning noise and detect the onset of each utterance based on analysis of the recorded sounds. We selected each line of k-space from several data acquisition sessions and rearranged them to reconstruct a new series of dynamic MR images according to the analyzed time of utterance onset. Comparative evaluation showed significant reduction in motion artifact signal in the dynamic MR images reconstructed by the proposed method. The quality of the reconstructed images was sufficient to observe the dynamic aspects of speech production mechanisms.

Influence of systemic administration of atelocollagen on mouse livers: an ideal biomaterial for systemic drug delivery.

Atelocollagen (AC), a biomaterial with low antigenicity and high bioaffinity, has been widely used in implantable materials in clinical practice. Preclinical studies have demonstrated that AC is a potential drug carrier for local and systemic delivery of cytokines, growth factors, plasmid DNA, small interfering RNA, and microRNA. AC is also believed to have low systemic toxicity on the basis of the safety of implant usage; however, this is not enough determined. Therefore, we performed whole genome expression profiling in mouse liver after systemic administration of AC or the cationic liposome carrier DOTAP/cholesterol (LP) and compared the changes of gene expressions associated with hepatotoxicity. Microarray analysis revealed that systemic LP administration significantly increased expression of toxicity-related genes, i.e., those for lipocalin-2, cyclin-dependent kinase inhibitor 1A, serum amyloid A isoforms, chemokine ligands, and granzyme B. Alternatively, AC administration did not alter the expression of any of these genes. Further gene ontology (GO) enrichment analysis highlighted the characteristic annotations extracted from genes upregulated after LP administration, and most of them were related to toxicity annotations such as immune response, inflammatory response, and apoptosis induction. In contrast, GO enrichment analysis of genes induced after AC administration revealed that only three annotations, all of which were unrelated to toxicity. These findings indicate that AC is potentially far less hepatotoxic than LP after systemic administration, suggesting that AC may be an excellent biomaterial for nontoxic drug delivery system carriers.

Effect of fat-saturation pulse on EPI time series in the presence of B0 drift.

PURPOSE: We examined the hypothesis that the fat-saturation pulse in a global off-resonance state caused by magnetic field (B(0)) drift produced signal fluctuation in echo planar imaging (EPI) time series. METHODS: We performed 3 experiments using 2 types of phantoms, one of which was homemade and contained water and a well emulsified fat source. RESULTS: We found that B(0) drift was approximately +30 Hz in the first 30-min EPI time series and +15 Hz in the second series. We experimentally reproduced the signal fluctuations observed during actual measurement in an artificial global off-resonance state using a fat-saturation pulse, the frequency profile of which also affected the pattern of fluctuation. CONCLUSION: These results are direct evidence that the fat-saturation pulse is a source of signal fluctuation in the presence of B(0) drift.

Visualisation of hypopharyngeal cavities and vocal-tract acoustic modelling.

The hypopharyngeal cavities consist of the laryngeal cavity and bilateral piriform fossa, constituting the bottom part of the vocal tract near the larynx. Visualisation of these cavities with magnetic resonance imaging (MRI) techniques reveals that during speech, the laryngeal cavity takes the form of a long-neck flask and the piriform fossa takes the form of a goblet of varying shapes: the former diminishes greatly in whispering and the latter disappears during deep inhalation. These cavities have been shown to exert significant acoustic effects at higher frequency spectra. In this study, acoustic experiments were conducted for male and female mechanical vocal tracts with the results that acoustic effects of those cavities determine the frequency spectra above 2 kHz, giving rise to peaks and zeros. An acoustic model of vowel production was proposed with three components: voice source, hypopharyngeal cavities and vocal tract proper, which provides effective means in controlling voice quality and expressing individual vocal characteristics.

Developmental changes in the expression of glycogenes and the content of N-glycans in the mouse cerebral cortex.

Biosynthesis of N-glycans varies significantly among tissues and is strictly regulated spatially and temporally within the tissue. The strict molecular mechanisms that are responsible for control of N-glycan synthesis remain largely unknown. We developed complementary deoxyribonucleic acid (cDNA) macroarray system and analyzed gene expression levels of more than 140 glycosyltransferases and glycosidases in the cerebral cortex from developing and adult mice. We also analyzed the relative amounts of major N-glycans present in the cerebral cortex and examined how the synthesis of N-glycans might be regulated through the expression of these genes. We demonstrated that the content of N-linked oligosaccharides dramatically changed during the course of brain development. Some of these changes could not be explained by alterations in the expression of the corresponding genes. For example, the amount of core fucosylated sugar chains in the early embryonic brain and the expression level of fucosyltransferase VIII, the only gene known to be responsible for core fucosylation, did not change proportionately. This result suggests that post-transcriptional regulation of this gene plays an important role in regulating its enzymatic activity. On the other hand, the amount of beta1,3-galactose residue-containing sugar chains increased postnatally following an increase in the level of beta1,3-galactosyltransferase messenger ribonucleic acid (mRNA). Furthermore, the amount of sugar chains with an outer fucose residue, containing LewisX-BA-2, correlated well with the expression of fusocyltransferase IX mRNA. These findings add to our understanding of the molecular mechanisms responsible for the regulation of N-glycan biosynthesis in the cerebral cortex.

Measurement of temporal changes in vocal tract area function from 3D cine-MRI data.

A 3D cine-MRI technique was developed based on a synchronized sampling method [Masaki et al., J. Acoust. Soc. Jpn. E 20, 375-379 (1999)] to measure the temporal changes in the vocal tract area function during a short utterance /aiueo/ in Japanese. A time series of head-neck volumes was obtained after 640 repetitions of the utterance produced by a male speaker, from which area functions were extracted frame-by-frame. A region-based analysis showed that the volumes of the front and back cavities tend to change reciprocally and that the areas near the larynx and posterior edge of the hard palate were almost constant throughout the utterance. The lower four formants were calculated from all the area functions and compared with those of natural speech sounds. The mean absolute percent error between calculated and measured formants among all the frames was 4.5%. The comparison of vocal tract shapes for the five vowels with those from the static MRI method suggested a problem of MRI observation of the vocal tract: data from static MRI tend to result in a deviation from natural vocal tract geometry because of the gravity effect.

IRBIT, an inositol 1,4,5-trisphosphate receptor-binding protein, specifically binds to and activates pancreas-type Na+/HCO3- cotransporter 1 (pNBC1).

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are IP(3)-gated Ca(2+) channels that are located on intracellular Ca(2+) stores. We previously identified an IP(3)R binding protein, termed IP(3)R binding protein released with IP(3) (IRBIT). Because IRBIT is released from IP(3)R by physiological concentrations of IP(3), we hypothesized that IRBIT is a signaling molecule that is released from IP(3)R and regulates downstream target molecules in response to the production of IP(3). Therefore, in this study, we attempted to identify the target molecules of IRBIT, and we succeeded in identifying Na(+)/HCO(3)(-) cotransporter 1 (NBC1) as an IRBIT binding protein. Of the two major splicing variants of NBC1, pancreas-type NBC1 (pNBC1) and kidney-type NBC1 (kNBC1), IRBIT was found to bind specifically to pNBC1 and not to bind to kNBC1. IRBIT binds to the N-terminal pNBC1-specific domain, and its binding depends on the phosphorylation of multiple serine residues of IRBIT. Also, an electrophysiological analysis in Xenopus oocytes revealed that pNBC1 requires coexpression of IRBIT to manifest substantial activity comparable with that of kNBC1, which displays substantial activity independently of IRBIT. These results strongly suggest that pNBC1 is the target molecule of IRBIT and that IRBIT has an important role in pH regulation through pNBC1. Also, our findings raise the possibility that the regulation through IRBIT enables NBC1 variants to have different physiological roles.

An N-glycan structure correlates with pulmonary metastatic ability of cancer cells.

N-Glycan structures on the surface of cancer cells have diverse structures and play significant roles in metastatic process. However, little is known about their roles in organ-selective metastasis. Our study revealed that an alpha1,6-fucosylated biantennary N-glycan structure designated A2G2F is characteristic of lungs, with far more abundant expression in normal human and murine lungs than in other organs. In this study, we further examined the role of A2G2F in pulmonary metastasis. We stained metastatic cancers by alpha1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed alpha1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific alpha1,6-fucosylated N-glycan structure in pulmonary metastatic cancer was identified to be A2G2F. Using a B16 melanoma cell metastasis model, we showed that A2G2F-rich B16 cells formed more pulmonary metastatic nodules than A2G2F-poor cells. Our results suggest that A2G2F plays a critical role in pulmonary metastasis.