Hyaluronic acid pretreatment for Sendai virus-mediated cochlear gene transfer.

Gene therapy with viral vectors is one of the most promising strategies for sensorineural hearing loss. However, safe and effective administration of the viral vector into cochlear tissue is difficult because of the anatomical isolation of the cochlea. We investigated the efficiency and safety of round window membrane (RWM) application of Sendai virus, one of the most promising non-genotoxic vectors, after pretreatment with hyaluronic acid (HA) on the RWM to promote efficient viral translocation into the cochlea. Sendai virus expressing the green fluorescent protein reporter gene was detected throughout cochlear tissues following application combined with HA pretreatment. Quantitative analysis revealed that maximum expression was reached 3 days after treatment. The efficiency of transgene expression was several 100-fold greater with HA pretreatment than that without. Furthermore, unlike the conventional intracochlear delivery methods, this approach did not cause hearing loss. These findings reveal the potential utility of gene therapy with Sendai virus and HA for treatment of sensorineural hearing loss.

Synthesis, crystal structure and phase transition of a Xe-N2 compound at high pressure: experimental indication of orbital interaction between xenon and nitrogen.

The van der Waals compound Xe(N2)2 with a C15 Laves structure was successfully synthesised at pressures greater than 4.4 GPa. We found that, at 10 GPa, the structure reversibly transforms from a cubic to a tetragonal phase. Further compression results in changes of Xe-N compound, which could result in the enhancement of orbital interactions between the xenon and nitrogen atoms.

Isolation and characterization of halophilic lactic acid bacteria acting as a starter culture for sauce fermentation of the red alga Nori (Porphyra yezoensis).

AIMS: A screening test was conducted for environmental samples to isolate halophilic lactic acid bacteria (HLAB) that can act as a starter in a Nori (Porphyra yezoensis)-sauce culture. METHOD AND RESULTS: After 9 months of incubation of enrichment cultures added with 25 kinds of environmental samples, growth of HLAB-like microorganisms was observed in six cultures salted at a 15% w/w level, including culture samples originally from mesopelagic water taken from 321 m-depth and from mountain snow taken at 2450 m-height. Ten strains were isolated and characterized as Tetragenococcus halophilus based on sequence analysis of the 16S rRNA gene. The isolates were inoculated into a newly prepared Nori-sauce culture and were confirmed to be able to act as a starter culture while three reference strains of T. halophilus obtained from a culture collection could not grow in the same culture. CONCLUSIONS: Halophilic lactic acid bacteria strains that can make growth in a highly salted Nori-sauce culture were isolated from environmental samples for the first time. All the isolates were identified as T. halophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated strains are expected to be utilized as a starter culture for manufacturing fermented seaweed-sauce, which will be the first fermented food products obtained from algae.

Genetic relationships between Japanese native and commercial breeds using 70 chicken autosomal SNP genotypes by the DigiTag2 assay.

Recently, single nucleotide polymorphisms (SNPs) have been used to identify genes or genomic regions responsible for economic traits, including genetic diseases in domestic animals, and to examine genetic diversity of populations. In this study, we genotyped 70 chicken autosomal SNPs using DigiTag2 assay to understand the genetic structure of the Japanese native chicken breeds Satsumadori and Ingie, and the relationship of these breeds with other established breeds, Rhode Island Red (RIR), commercial broiler and layer. Five breeds, each consisting of approximately 20 chickens, were subjected to the assay, revealing the following: Average expected heterozygosities of broiler, Satsumadori, RIR, layer and Ingie were 0.265, 0.254, 0.244, 0.179 and 0.176, respectively. Phylogenetic analysis using the concatenated 70 autosomal SNP genotypes distinguished all chickens and formed clusters of chickens belonging to the respective breeds. In addition, the 2-D scatter plot of the first two principal components was consistent with the phylogenic tree. Taken together with the pairwise F(st) distances, broiler and RIR were closely positioned near each other, while Ingie was positioned far from the other breeds. Structure analysis revealed that the probable number of genetic clusters (K) was six and four with maximum likelihood and ΔK values, respectively. The clustering with maximum likelihood revealed that, in addition to the clustering of the other five breeds, the Satsumadori was subdivided into two genetic clusters. The clustering with ΔK value indicated that the broiler and Rhode Island Red were assigned to the same genetic cluster.

Combining FIB milling and conventional Argon ion milling techniques to prepare high-quality site-specific TEM samples for quantitative EELS analysis of oxygen in molten iron.

This paper reports a procedure to combine the focused ion beam micro-sampling method with conventional Ar-milling to prepare high-quality site-specific transmission electron microscopy cross-section samples. The advantage is to enable chemical and structural evaluations of oxygen dissolved in a molten iron sample to be made after quenching and recovery from high-pressure experiments in a laser-heated diamond anvil cell. The evaluations were performed by using electron energy-loss spectroscopy and high-resolution transmission electron microscopy. The high signal to noise ratios of electron energy-loss spectroscopy core-loss spectra from the transmission electron microscopy thin foil, re-thinned down to 40 nm in thickness by conventional Argon ion milling, provided us with oxygen quantitative analyses of the quenched molten iron phase. In addition, we could obtain lattice-fringe images using high-resolution transmission electron microscopy. The electron energy-loss spectroscopy analysis of oxygen in Fe(0.94)O has been carried out with a relative accuracy of 2%, using an analytical procedure proposed for foils thinner than 80 nm. Oxygen K-edge energy-loss near-edge structure also allows us to identify the specific phase that results from quenching and its electronic structure by the technique of fingerprinting of the spectrum with reference spectra in the Fe-O system.

Non-vitamin K antagonist oral anticoagulants (NOACs) for thromboembolic prevention, are they safe in congenital heart disease? Results of a worldwide study.

BACKGROUND: Current guidelines consider vitamin K antagonists (VKA) the oral anticoagulant agents of choice in adults with atrial arrhythmias (AA) and moderate or complex forms of congenital heart disease, significant valvular lesions, or bioprosthetic valves, pending safety data on non-VKA oral anticoagulants (NOACs). Therefore, the international NOTE registry was initiated to assess safety, change in adherence and quality of life (QoL) associated with NOACs in adults with congenital heart disease (ACHD). METHODS: An international multicenter prospective study of NOACs in ACHD was established. Follow-up occurred at 6 months and yearly thereafter. Primary endpoints were thromboembolism and major bleeding. Secondary endpoints included minor bleeding, change in therapy adherence (≥80% medication refill rate, ≥6 out of 8 on Morisky-8 questionnaire) and QoL (SF-36 questionnaire). RESULTS: In total, 530 ACHD patients (mean age 47 SD 15 years; 55% male) with predominantly moderate or complex defects (85%), significant valvular lesions (46%) and/or bioprosthetic valves (11%) using NOACs (rivaroxaban 43%; apixaban 39%; dabigatran 12%; edoxaban 7%) were enrolled. The most common indication was AA (91%). Over a median follow-up of 1.0 [IQR 0.0-2.0] year, thromboembolic event rate was 1.0% [95%CI 0.4-2.0] (n = 6) per year, with 1.1% [95%CI 0.5-2.2] (n = 7) annualized rate of major bleeding and 6.3% [95%CI 4.5-8.5] (n = 37) annualized rate of minor bleeding. Adherence was sufficient during 2 years follow-up in 80-93% of patients. At 1-year follow-up, among the subset of previous VKA-users who completed the survey (n = 33), QoL improved in 6 out of 8 domains (p ≪ 0.05). CONCLUSIONS: Initial results from our worldwide prospective study suggest that NOACs are safe and may be effective for thromboembolic prevention in adults with heterogeneous forms of congenital heart disease.

Nitric oxide-cyclic guanosine monophosphate signaling in the local circuit of the cricket abdominal nervous system.

The distribution of potential nitric oxide (NO) donor neurons and NO-responsive target neurons was revealed in the terminal abdominal ganglion (TAG) of the cricket. The expression of nitric oxide synthase (NOS) in the nervous system was examined by Western blotting using universal nitric oxide synthase (uNOS) antibody that gave about a 130 kDa protein band. Immunohistochemistry using the uNOS antibody detected neurons whose cell bodies are located at the lateral region of the TAG. These neurons expanded their neuronal branches into the dorsal-median region or the dorsal-lateral region of the TAG. NADPH-diaphorase histochemistry was performed to confirm the distribution of NOS-containing neurons. The distributions of cell bodies and stained neuronal branches were similar to those revealed by uNOS immunohistochemistry. NO-induced cGMP immunohistochemistry was performed to reveal NO-responsive target neurons. Most of the cell bodies stained by immunohistochemistry appeared at the dorsal side of the TAG. At the dorsal-median region, some unpaired neuronal cell bodies were strongly stained. Some efferent neurons whose axon innervate into each nerve root were strongly stained. The generation of NO in the TAG was detected by NO electrode. We found that NO is generally produced to maintain a basal concentration of 70 nM. Hemoglobin scavenged released NO from the ganglion. The concentration of NO was partly recovered when hemoglobin was replaced by normal saline. Application of 10 microM L-arginine that is a substrate of NOS increased NO release by approximately 10 nM. Furthermore, an excitatory neurotransmitter acetylcholine (ACh) also increased NO generation by approximately 40-50 nM in concentration in addition to the basal level of 70 nM. Optical imaging with fluorescent NO-indicator demonstrated that ACh-induced enhancement of NO release was transiently observed in the outer-edge region of TAG, where cell bodies of NOS-immunoreactive neurons were located. These results suggest that ACh accelerates NO production via neuronal events activated by ACh in the TAG.

Effect of chondroitin sulfate C on sperm capacitation and fertilization parameters in vitro in pigs.

The objective of this study was to determine the effects of chondroitin sulfate C (CS-C) on sperm capacitation and fertilization parameters in vitro in pigs. Frozen-thawed ejaculated pig sperm (semen S-484) were incubated with fertilization medium containing CS-C (0-2mg/ml) for 1h and the capacitation rate with chlorotetracycline (CTC) assay was examined, which showed that CS-C increased the rate of incapacitation F pattern spermatozoa converted to capacitation B pattern sperm cell in concentration-dependent manner and mostly increased capacitation B pattern sperm cell and decreased acrosome reaction AR pattern sperm cell in 1mg/ml concentration. When sperm was incubated for 1, 2 and 4h in fertilization medium containing 1mg/ml CS-C, it showed that the capacitated B pattern sperm cell was significantly (p<0.01) increased and the AR pattern sperm cell was significantly decreased at each time point in the presence than in the absence of CS-C. For identifying the validity of CS-C in sperm capacitation, sperm-oocyte was inseminated in fertilization medium containing CS-C (0-2mg/ml) and the rate of fertilized oocytes was examined, which showed that the penetration rates significantly (p<0.05) increased from 0.5 to 1.0mg/ml concentrations (87.4-96.3%) compared with control (74.9%). For identifying the universality of CS-C in sperm capacitation, four different semens (boar S-484, S-454, D-815 and D-748) were incubated in fertilization medium containing CS-C (1mg/ml) for 2h, respectively, which showed that CS-C increased the rate of capacitation B pattern sperm cell and decreased acrosome reaction AR pattern sperm cell in each semen. And it showed that CS-C yielded a higher promote effect (93.9%, 83.9%, 60.7% and 44.9%, respectively) on sperm penetration compared to unaddition control (63.4%, 22.0%, 3.3% and 3.3%, respectively). Sperm-oocyte binding analysis showed that CS-C increased the number of sperm bound to oocyte compared unaddition control in each semen. These results suggested that CS-C is the efficient factor on sperm capacitation in pigs, CS-C may promote sperm from the incapacitated to capacitated state and sequentially prevent sperm from spontaneous acrosome reaction, and thus facilitate the sperm-zona binding and sperm penetration to oocyte.

SOD1 rescues cerebral endothelial dysfunction in mice overexpressing amyloid precursor protein.

Peptides derived from proteolytic processing of the beta-amyloid precursor protein (APP), including the amyloid-beta peptide, are important for the pathogenesis of Alzheimer's dementia. We found that transgenic mice overexpressing APP have a profound and selective impairment in endothelium-dependent regulation of the neocortical microcirculation. Such endothelial dysfunction was not found in transgenic mice expressing both APP and superoxide dismutase-1 (SOD1) or in APP transgenics in which SOD was topically applied to the cerebral cortex. These cerebrovascular effects of peptides derived from APP processing may contribute to the alterations in cerebral blood flow and to neuronal dysfunction in Alzheimer's dementia.

Clinical implication of medroxyprogesterone acetate against advanced ovarian carcinoma: a pilot study.

PURPOSE OF INVESTIGATION: The present study was performed to identify the effects of medroxyprogesterone acetate (MPA) plus adjuvant chemotherapy on advanced epithelial ovarian carcinoma (FIGO Stage III/IV). METHODS: A total of 50 patients were enrolled in this study. A relatively low dose of MPA (200 mg/day) after surgery was administered in combination with platinum-based chemotherapy and the treatment was continued for two years. Patients' backgrounds were also analyzed. RESULTS: Relapse-free survival (p < 0.05) and overall survival (p < 0.001) rates in FIGO Stage III/IV ovarian cancer patients with MPA combined chemotherapy were significantly longer than the control group. The effect was more prominent in the higher progesterone receptor expression group. The chemotherapy regimens (cyclophosphamide, doxorubicin and cisplatin vs paraplatin plus cyclophosphamide or paclitaxel) did not affect prognosis. CONCLUSION: MPA with platinum-based chemotherapy as an adjuvant therapy might improve the prognosis in FIGO Stage III/IV epithelial ovarian cancer cases. A randomized controlled study is still needed for further analyses.

Effects of selective estrogen receptor modulators and genistein on the expression of ERalpha/beta and COX-1/2 in ovarectomized mouse uteri.

This study was performed to examine the effects of selective estrogen receptor modulators [tamoxifen (TAM) and toremifene (TOR)] and pure anti-estrogen, ICI-182780 (ICI, Faslodex) and soybean isoflavone, genistein (GE) on the expression of estrogen-stimulated c-fos/jun, ERalpha/beta and COX-1/2 in the uteri of ovarectomized mice. TAM, TOR, ICI and GE treatment significantly decreased the levels of estradiol (E2)-induced c-fos. ICI and GE treatment significantly decreased the levels of E2-induced c-jun and ERalpha expressions. High doses of TOR treatment significantly increased the E2-induced ERbeta expression. In contrast, ICI and GE treatment significantly decreased the levels of E,-induced COX-2 expression, thus suggesting that TOR and GE might prevent E2-related endometrial carcinogenesis.

Pseudomyxoma peritonei and mucinous pyometral fluid arising from an ovarian borderline mucinous tumor: case report.

An extremely rare case of a pseudomyxoma peritonei (PMP) and mucinous pyometral fluid, possibly arising from an ovarian borderline mucinous tumor is reported. A 68-year-old Japanese patient received an expolatory laparatomy under a working diagnosis of a PMP, left ovarian cystic tumor and an umbilical hernia. Surgery and platinum-based chemotherapy induced a 15-month disease-free condition.

Primary endodermal sinus tumor of the vulva in a 52-year-old woman with long-term survival: a case report.

A postmenopausal 52-year-old Japanese woman with a 5-month history of a right labial tumor was referred to the Department of Obstetrics and Gynecology. The resected tumor had been diagnosed as a primary endodermal sinus tumor of the vulva with pT2N0M0 in 2001. Six courses of adjuvant chemotherapy using bleomycin, etoposide and cisplatin were administered. The patient was free of recurrence or metastasis 67 months after the initial treatment.

Primary lymphoma of the uterine corpus: an unusual location for a common disease--case report.

A case of a primary uterine corpus lymphoma in a 75-year-old woman is described. Immunohistochemical studies showed diffuse large B-cell type one. Primary lymphoma of the uterine corpus is considered to be an unusual location for a common disease.

Absolute optical responsivity down to the photon counting level with a photomultiplier tube.

A novel method is presented for evaluation of the absolute optical responsivity of a photomultiplier tube (PMT) at optical powers down to the photon counting level under visible light. This method is based on a combination of the calibrated responsivity and nonlinearity corrections using a silicon photodiode and a PMT. The evaluation results with the PMT enable accurate determination of the absolute optical responsivity over a wide power range of 10-11 W to 10-16 W under visible light. This method provides validation of both the photon counting efficiency of a PMT and of accurate optical measurement with attenuators at the photon counting level.

Pyrite form of group-14 element pernitrides synthesized at high pressure and high temperature.

The chemical reaction of group-14 elements with molecular nitrogen at pressures above 60 GPa successfully leads to the formation of pyrite-type pernitrides. These new crystalline group-14 elemental pernitrides were found to show a bulk modulus higher than the other known group-14 elemental nitrides. Our results offer significant new progress in the solid-state chemistry of nitrides.

Cyclooxygenase-1 participates in selected vasodilator responses of the cerebral circulation.

Cyclooxygenase (COX) is a prostanoid-synthesizing enzyme present in 2 isoforms: COX-1 and COX-2. Although it has long been hypothesized that prostanoids participate in cerebrovascular regulation, the lack of adequate pharmacological tools has led to conflicting results and has not permitted investigators to define the relative contribution of COX-1 and COX-2. We used the COX-1 inhibitor SC-560 and COX-1-null (COX-1(-/-)) mice to investigate whether COX-1 plays a role in cerebrovascular regulation. Mice were anesthetized (urethane and chloralose) and equipped with a cranial window. Cerebral blood flow (CBF) was measured by laser Doppler flowmetry or by the (14)C-iodoantipyrine technique with quantitative autoradiography. In wild-type mice, SC-560 (25 micromol/L) reduced resting CBF by 21+/-4% and attenuated the CBF increase produced by topical application of bradykinin (-59%) or calcium ionophore A23187 (-49%) and by systemic hypercapnia (-58%) (P<0.05 to 0.01). However, SC-560 did not reduce responses to acetylcholine or the increase in somatosensory cortex blood flow produced by vibrissal stimulation. In COX-1(-/-) mice, resting CBF assessed by (14)C-iodoantipyrine was reduced (-13% to -20%) in cerebral cortex and other telencephalic regions (P<0.05). The CBF increase produced by bradykinin, A23187, and hypercapnia, but not acetylcholine or vibrissal stimulation, were attenuated (P<0.05 to 0.01). The free radical scavenger superoxide dismutase attenuated responses to bradykinin and A23187 in wild-type mice but not in COX-1(-/-) mice, suggesting that COX-1 is the source of the reactive oxygen species known to mediate these responses. The data provide evidence for a critical role of COX-1 in maintaining resting vascular tone and in selected vasodilator responses of the cerebral microcirculation.

Norethindrone scatters silver-stained nucleolar organizer regions of Ishikawa cells.

We investigated the effects of sex steroids on silver-stained nucleolar organizer regions (Ag-NORs) and DNA/RNA kinetics in Ishikawa cells. Norethindrone and its isomer norethynodrel exclusively caused Ag-NORs to scatter in the nuclear matrix, from the nucleolus. No such effect occurred with the other sex steroids tested, including progestational, androgenic, and estrogenic compounds. Nuclear argyrophilic substances induced by norethindrone, as well as nucleolar ones, were neither DNA nor RNA but protein. Electron microscopy showed that norethindrone caused nucleolar segregation, in which the fibrillar components disappeared, and it produced islets, consisting of dense fibrillar materials, in the nucleoplasm. Ag-NORs observed on the fibrillar components in control nucleoli were translocated onto the dense fibrillar materials in the nucleoplasm. Although scattering was preferentially found in the cells synthesizing DNA, the scintillation assay of DNA/RNA kinetics suggested that scattering was related to the inhibition of RNA synthesis. These results imply that norethindrone preferentially interacts with intranucleolar DNA when its duplication is occurring and then interferes with rRNA synthesis. Scattering of Ag-NORs might not be caused by the hormonal activity of these agents but by a pharmacological effect derived from their molecular structures.

Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of telomerase RNA in endometrial carcinoma cells.

Telomerase activity is found in almost all carcinoma cells but not in most somatic cells, suggesting that telomerase is an excellent target for cancer therapy. We designed hammerhead ribozymes against human telomerase RNA and studied their possible use as a tool for cancer therapy. Three ribozymes targeting the 3' end of the GUC sequence at 33-35 (the template region), 168-170, and 313-315 from the 5' end of telomerase RNA were designed. In a cell-free system, these three hammerhead ribozymes efficiently cleaved the RNA substrate. When these ribozyme RNAs were introduced into Ishikawa cells, which are endometrial carcinoma cells, only a ribozyme targeting the RNA template region could diminish the telomerase activity. Next we subcloned the ribozyme sequence into an expression vector and introduced this into AN3CA cells, which are endometrial carcinoma cells. The clones that were obtained showed reduced telomerase activity and telomerase RNA with expression of the ribozyme. These data suggest that the ribozyme against the RNA template region is a good tool to repress telomerase activity in cancer cells.