Bone Degeneration and Its Recovery in SMP30/GNL-Knockout Mice.

Senescence marker protein-30 (SMP30) decreases androgen-independently with aging and is a lactone-hydrolyzing enzyme gluconolactonase (GNL) that is involved in vitamin C biosynthesis. In the present study, bone properties of SMP30/GNL knockout (KO) mice with deficiency in vitamin C synthesis were investigated to reveal the effects of SMP30/GNL and exogenous vitamin C supplementation on bone formation. Mineral content (BMC) and mineral density (BMD) of the mandible and femur of SMP30/GNL KO and wild-type mice at 2 and 3 months of age with or without vitamin C supplementation were measured by dual-energy X-ray absorptiometry. Body and bone weight of both age groups decreased and became significantly lower than those of wild-type mice. The bones of SMP30/GNL KO mice were rough and porous, with BMC and BMD significantly below wild-type. Oral supplementation with vitamin C eliminated differences in body weight, bone weight, BMC, and BMD between SMP30/GNL KO and wild-type mice at each age. These results indicate that bone degeneration in SMP30/GNL KO mice was caused by lack of vitamin C, and that this mouse strain is an appropriate model for bone metabolism in humans, which have no ability to synthesize vitamin C.

Search for bosonic superweakly interacting massive dark matter particles with the XMASS-I detector.

Bosonic superweakly interacting massive particles (super-WIMPs) are a candidate for warm dark matter. With the absorption of such a boson by a xenon atom, these dark matter candidates would deposit an energy equivalent to their rest mass in the detector. This is the first direct detection experiment exploring the vector super-WIMPs in the mass range between 40 and 120 keV. With the use of 165.9 day of data, no significant excess above background was observed in the fiducial mass of 41 kg. The present limit for the vector super-WIMPs excludes the possibility that such particles constitute all of dark matter. The absence of a signal also provides the most stringent direct constraint on the coupling constant of pseudoscalar super-WIMPs to electrons. The unprecedented sensitivity was achieved exploiting the low background at a level 10(-4) kg-1 keVee-1 day-1 in the detector.

Association between cystoid spaces on indocyanine green hyperfluorescence and optical coherence tomography after vitrectomy for diabetic macular oedema.

PURPOSE: To study retrospectively the characteristics of residual indocyanine green (ICG) fluorescence after ICG-assisted vitrectomy and the association with spectral-domain optical coherence tomography (SD-OCT) findings in diabetic macular oedema (DMO). METHODS: Thirteen consecutive eyes of 12 patients for whom fundus near-infrared fluorescence and 20° retinal sectional images were obtained using HRA2 and Spectralis OCT, respectively, 5 days after vitrectomy combined with ICG-assisted inner limiting membrane peeling for DMO. The relationship between the characteristics of the ICG hyperfluorescence and the cystoid spaces in the outer plexiform layer (OPL) on SD-OCT images was evaluated. RESULTS: A total of 390 well-demarcated areas of ICG hyperfluorescence were delineated on 20° radial OCT scans dissecting the fovea 5 days after vitrectomy. The areas of ICG hyperfluorescence in the parafovea or perifovea were significantly smaller than those at the fovea. Most areas of hyperfluorescence were irregularly shaped in the parafovea and perifovea, whereas 18 of 38 areas of hyperfluorescence were round or oval at the fovea (P<0.001). SD-OCT delineated the cystoid spaces in the OPL in 73 areas of hyperfluorescence that were round or oval and accompanied by dark spots more frequently than that without cystoid spaces on OCT images (P<0.001 and P=0.002). Of the 123 cystoid spaces in the OPL on OCT images, 44 did not have ICG hyperfluorescence, had lower OCT reflectivity, and contained fewer hyperreflective foci than those with ICG hyperfluorescence (P<0.001 and P=0.020). CONCLUSION: The results provided novel interpretations of the ICG hyperfluorescence and its association with OCT characteristics of the cystoid spaces in DMO.

Transverse uterine fundal incision for placenta praevia with accreta, involving the entire anterior uterine wall: a case series.

OBJECTIVE: To determine the feasibility and safety of transverse fundal incision with manual placental removal in women with placenta praevia and possible placenta accreta. DESIGN: Case series. SETTING: Four level-three Japanese obstetric centres. POPULATION: Thirty-four women with prior caesarean section and placenta praevia that widely covers the anterior uterine wall, in whom placenta accreta cannot be ruled out. METHODS: A transverse fundal incision was performed at the time of caesarean section and manual placental removal was attempted under direct observation. MAIN OUTCOME MEASURE: Operative fluid loss. RESULTS: The total volume of fluid lost during our operative procedure compares favourably with the volume lost during our routine transverse lower-segment caesarean sections performed in patients without placenta praevia or accreta. The average fluid loss was 1370 g. No patients required transfer to intensive care, and there were no cases of fetal anaemia. CONCLUSIONS: This procedure has the potential to reduce the heavy bleeding that arises from caesarean deliveries in women with placenta praevia and placenta accreta.

Delaying embryo development by storing at 4°C for synchronization to recipients in microinjection technique in rabbits.

Short-term storage of embryos at low temperature induces developmental arrest of the embryo and would appear to be a valuable aid in embryo-transfer techniques to avoid wasting embryos. Embryo storage at 4°C was examined to allow synchronization with embryo-transfer recipients using the microinjection technique. Superovulation was induced in female Japanese White donor rabbits four days before mating with males. At the same time, control recipients were injected with human chorionic gonadotropin (hCG) to allow synchronization (R1); the hCG injections were delayed by 24 h in the experimental group (R2). DNA constructs for expressing human C-reactive protein or apolipoprotein AII were microinjected into the male pronuclei of the ova. The microinjected embryos were immediately transferred to recipients (R1) or stored at 4°C in phosphate-buffered saline containing 10% fetal bovine serum. After 17-20 h, the stored embryos were incubated at 37°C for one hour, and the morphologically normal embryos were transferred to recipients (R2). In the R1 rabbits, 855 embryos were transferred to 29 recipients, and 72.4% of the recipients became pregnant. Seven of the 84 offspring were transgenic. In the R2 rabbits, 478 embryos were transferred to 16 recipients, and 62.5% of the recipients became pregnant. Two of the 39 offspring were transgenic. There were no differences in pregnancy rate, litter size and transgenic integration rate between R1 and R2. These results suggest that the short-term 4°C storage of microinjected embryos can be a valuable method for synchronization with recipients, and reducing wastage of embryos and the sacrifice of rabbits.

First Report of Association of Mucor circinelloides on Noni (Morinda citrifolia) in Hawaii.

Noni (Morinda citrifolia) is a popular medicinal plant found in tropical or subtropical regions of the world. The fruit and juice extracts have properties that are reportedly therapeutic for diabetes, high blood pressure, and certain types of cancer (1,4). In our studies on noni juice produced from fruit collected from the Kohala and Puna districts of the island of Hawaii from 2008 to 2010, Mucor circinelloides f. sp. circinelloides was isolated from 85% of 157 juice samples and observed with up to 75% incidence on fruit surfaces during fermentation processing in glass jars. Fungal growth, appearing 14 to 21 days in storage at 22°C, was pale yellow to tan brown and was associated with wounded surfaces. Single-spore strains, KN 06-2 (2006; ripe fruit puree) and KN 08-08 (2008; fermented juice; CBS 124110), identified by Centraalbureau voor Schimmelcultures by molecular methods were 97.3% similar in internal transcribed spacer sequence to the type strain (CBS 195.68). M. circinelloides f. sp. circinelloides strains (KN 08-08, KN 09-06, or KN 10-02) (2008 to 2010; fermented juice) were inoculated by pipetting an aliquot of 100 µl of fungus strain spore suspension (1 × 105 to 1.33 × 106 spores/ml) onto firm, yellow maturity noni fruit that were washed, surface disinfected, and either wounded (surface cuts) or nonwounded. Controls consisted of no inoculation and sterile distilled water (SDW) inoculation treatments. Ten to twenty each of wounded and nonwounded fruit comprised each inoculation treatment. Fruit were incubated in acrylic bins with a layer of distilled water at the bottom, and sealed with snap-on lids. The bins were incubated on a lab bench at 22 to 23°C under fluorescent lights. Fruits were evaluated for presence of fungal growth and severity of symptoms. To determine viability of spores on inoculated fruit without symptoms, surfaces were swabbed with sterile cotton swabs dipped in SDW, streaked on potato dextrose agar (PDA) plates, and incubated at 22°C under fluorescent lights. The inoculation experiment was conducted twice. Nonwounded fruit inoculated with M. circinelloides f. sp. circinelloides strains did not result in infections (KN 09-06 and KN 10-02) or produced slight mycelial growth (0 to 20%; KN 08-08). Wounded fruit inoculated with any of the three strains resulted in 85 to 100% infection of moderate severity. There were no infections in noninoculated or SDW treatments of nonwounded or wounded fruit. Koch's postulates were fulfilled with the reisolation of M. circinelloides f. sp. circinelloides from selected fruit exhibiting soft tissue, discoloration, and sporulating yellowish green mycelial growth. Swab washes from asymptomatic surfaces of inoculated nonwounded fruit resulted in the growth of M. circinelloides f. sp. circinelloides on PDA, proving viability of the spores and confirmed that the fungus is primarily pathogenic only on wounded fruit surfaces. To our knowledge, this is the first report of M. circinelloides as a wound pathogen of noni fruit. The quality of fermented noni juice may be affected by the presence of M. circinelloides f. sp. circinelloides but can be remedied by pasteurization that does not affect antitumor properties (unpublished data). This fungus is also a reported pathogen of mango (2) and peach (3). References: (1) J. Li et al. Oncol. Rep. 20:1505, 2008. (2) K. Pernezny and G. W. Simone. Phytopathol. News 34:25, 2000. (3) C. Restuccia et al. J. Food Prot. 69:2465, 2006. (4) M. Y. Wang et al. Acta Pharmacol. Sin. 23:1127, 2002.

Structural changes of fovea during remission of Behçet's disease as imaged by spectral domain optical coherence tomography.

PURPOSE: To acquire the image of the fovea during remission of ocular Behçet's disease using spectral domain optical coherence tomography (SD-OCT) and to further assess the correlation between best-corrected visual acuity (BCVA) and SD-OCT findings. METHODS: A total of 24 eyes of 14 patients with ocular Behçet's disease were examined with SD-OCT. The relationships between BCVA and other clinical characteristics, including foveal thickness and status of external limiting membrane (ELM) or the junction between photoreceptor inner and outer segments (IS/OS), were analysed for statistical significance. RESULTS: BCVA in eyes with intact IS/OS was significantly better than that in eyes with ill-defined IS/OS (P=0.0001). Foveal thickness (P=0.0001) and male sex (P=0.03) were also significantly different between eyes with and without complete IS/OS line. CONCLUSIONS: Definition of the IS/OS line by SD-OCT, but not status of the ELM line, appears to be associated with BCVA in patients with ocular Behçet's disease. Accordingly, SD-OCT may prove useful in the evaluation of visual function after ocular attacks of Behçet's disease.

Randomised controlled trial of posterior sub-Tenon triamcinolone as adjunct to panretinal photocoagulation for treatment of diabetic retinopathy.

AIMS: To evaluate the efficacy of a single posterior sub-Tenon capsule injection of triamcinolone acetonide (PSTA) before panretinal photocoagulation (PRP). METHODS: This 6-month study involved the randomisation of 82 eyes of 41 patients, with bilateral severe non-proliferative diabetic retinopathy or proliferative diabetic retinopathy to a single PSTA 20 mg or to no injection before PRP. The primary end-point was change in best-corrected visual acuity (BCVA) at 6 months compared with that at baseline using the logarithm of the minimum angle of resolution (logMAR). Secondary end-points were changes in retinal thickness and intraocular pressure. RESULTS: The mean changes in logMAR BCVA at 6 months compared with that at baseline were a worsening of 0.010 (SD 0.029) in the control group (no injection) and an improvement of 0.072 (0.028) in the PSTA group (p = 0.04). The mean changes in foveal thickness at 6 months compared with baseline measurements were an increase of 32.8 (82.8) mum in the control group and a lessening of 9.7 (85.6) mum in the PSTA group (p = 0.03). CONCLUSIONS: PSTA before PRP appears to be beneficial in preventing PRP-induced visual loss in eyes with diabetic retinopathy by reducing the chance of macular thickening.

Atypical Internal Yellowing of Papaya Fruit in Hawaii Caused by Enterobacter sakazakii.

Internal yellowing (IY) caused by Enterobacter cloacae and characterized by yellow discolored tissue surrounding the papaya (Carica papaya L.) seed cavity, diffuse margins, and the presence of a distinctly rotten odor was first reported in 1987 (3). Here we report the formation of atypical internal yellowing (AIY) in ripe papaya caused by the bacterium Enterobacter sakazakii. In surveys conducted from 2006 to 2007, 'Kapoho Solo' papayas grown in the Puna District of Hawaii Island were obtained from various packinghouses. After incubation at 27°C, the papayas were bisected and examined for symptoms of IY. Among papayas that were asymptomatic for IY, a dull, greenish yellow discoloration of the flesh with a distinct margin extending from the seed cavity into the pericarp was noted, along with a pungent odor. These symptoms occurred in 5 of the 500 fruit surveyed and bacterial populations were 102 to 103 CFU/g. Discolored tissue was aseptically excised, weighed, macerated, serially diluted in sterile distilled water (SDW), and plated onto modified peptone yeast extract medium (PT-M4) (4). The plates were incubated at 30°C for 24 to 48 h until single colonies were evident. After 48 h, colonies on PT-M4 were orange-red, convex and circular, and surrounded by a somewhat opaque 1-mm margin. After single colony purification, five strains were obtained. The strains, inoculated into oxidation/fermentation-glucose tubes and API 20E strips (bioMerieux, Inc., Durham, NC) incubated at 30°C, were shown to be facultative anaerobes and identified as E. sakazakii with a 98.4% certainty. Colonies plated onto tryptic soy agar (TSA) and incubated for 72 h at 25°C produced yellow pigmentation, indicative of E. sakazakii. Amplification by PCR with E. sakazakii-specific primers (2) yielded a 929-bp fragment, which was absent with E. cloacae and Pseudomonas aeruginosa template DNA. To confirm pathogenicity, cell suspensions at 109 CFU/ml of putative E. sakazakii strains RK07-05, RK07-06, and RK07-07 and E. cloacae (3) were inoculated by injection (0.5 ml per site) into one-third-ripe 'Kapoho Solo' papayas (six fruit per strain, inoculated at duplicate sites) and incubated at 27°C for 4 days. Control sites were injected with 0.5 ml of SDW. Fruit inoculation experiments were repeated. E. cloacae-inoculated sites produced typical IY as previously described (3), while the sites inoculated with the three E. sakazakii strains produced greenish yellow tissue (26% mean incidence), symptomatic of AIY. Control sites did not produce IY or AIY. Koch's postulates were fulfilled, and the identification of reisolated bacterial strains was confirmed with API 20E, PCR, and pigment production on TSA. Although less prevalent (1% incidence) than the typical IY produced by E. cloacae (3), E. sakazakii has the potential to affect quality and food safety of fresh and processed papaya products. E. sakazakii has been implicated in a severe form of neonatal meningitis, sepsis, and necrotizing enterocolitis (1). Research into the transmission and infection of papaya of this cross-domain pathogen merits further study. References: (1) D. H. Adamson. Clin. Microbiol. Newsl. 3:19, 1981. (2) A. Lehner et al. BMC Microbiol. 4:43, 2004. (3) K. A. Nishijima et al. Plant Dis. 71:1029, 1987. (4) K. A. Nishijima et al. Plant Dis. 88:1318, 2004.

Demonstrating Pathogenicity of Enterobacter cloacae on Macadamia and Identifying Associated Volatiles of Gray Kernel of Macadamia in Hawaii.

Gray kernel is an important disease of macadamia (Macadamia integrifolia) that affects the quality of kernels, causing gray discoloration and a permeating, foul odor. Gray kernel symptoms were produced in raw, in-shell kernels of three cultivars of macadamia that were inoculated with strains of Enterobacter cloacae. Koch's postulates were fulfilled for three strains, demonstrating that E. cloacae is a causal agent of gray kernel. An inoculation protocol was developed to consistently reproduce gray kernel symptoms. Among the E. cloacae strains studied, macadamia strain LK 0802-3 and ginger strain B193-3 produced the highest incidences of disease (65 and 40%, respectively). The other macadamia strain, KN 04-2, produced gray kernel in 21.7% of inoculated nuts. Control treatments had 1.7% gray kernel symptoms. Some abiotic and biotic factors that affected incidence of gray kernel in inoculated kernels were identified. Volatiles of gray and nongray kernel samples also were analyzed. Ethanol and acetic acid were present in nongray and gray kernel samples, whereas volatiles from gray kernel samples included the additional compounds, 3-hydroxy-2-butanone (acetoin), 2,3-butanediol, phenol, and 2-methoxyphenol (guaiacol). This is believed to be the first report of the identification of volatile compounds associated with gray kernel.

In vivo evaluation of ocular inflammatory responses in experimental diabetes.

AIMS: Diabetic patients may have abnormal inflammatory reactions to foreign or endogenous stimuli. This study was designed to evaluate inflammatory reactions in the diabetic eye through retinal leucocyte dynamics in the inflamed eyes of diabetic rats. METHODS: Three weeks after diabetes induction in Long-Evans rats, endotoxin induced uveitis was produced by footpad injection of lipopolysaccharide (LPS). After LPS injection, leucocyte behaviour was evaluated in vivo by acridine orange digital fluorography. RESULTS: The number of rolling leucocytes increased in a biphasic manner at 12 hours and 48 hours. The number of leucocytes accumulating in the retina reached a peak at 72 hours. The maximal numbers of rolling and accumulating leucocytes in the diabetic retina decreased by 56.3% (p<0.01) and 46.7% (p<0.0001), respectively, compared with the non-diabetic retina. The levels of mRNA expression of adhesion molecules in the retina, which were upregulated after LPS injection, were also lower in diabetic rats than in non-diabetic rats. CONCLUSION: This study is the first to show that endotoxin induced inflammation is disturbed in the diabetic eye, based on evidence that the leucocyte-endothelial cell interactions stimulated by LPS were suppressed in the diabetic retina. These findings support the theory that ocular inflammatory reactions are impaired in diabetic patients.

Transverse fundal uterine incision in a patient with placenta increta.

In cases with placenta accreta occurring on the previous cesarean scar, a longitudinal incision is generally appropriate. However, the placenta may prevent both a longitudinal and lower transverse incision. We report a case of placenta increta on the previous cesarean scar, and discuss the role of transverse fundal uterine incision.

Oxidative stress-induced DNA damage in the synovial cells of the temporomandibular joint in the rat.

Synovial hyperplasia is a feature of degenerative temporomandibular joint (TMJ) disease. However, the mechanism by which hyperplasia progresses in the TMJ is unknown. Based on the hypothesis that the oxidative stress generated by mechanical loading causes degenerative changes in the TMJ synovium, we investigated the generation of the highly reactive species, peroxynitrite, and the occurrence of DNA damage in the synovium. After condylar hypermobility of rat TMJs, a marker of peroxynitrite, nitrotyrosine, was localized to the nuclei and cytoplasm of the synovial lining cells and fibroblasts in synovitis-induced TMJ. DNA single-strand breaks were found in the nuclei of the synovial cells only after enzyme treatment, whereas DNA double-strand breaks were not detected. These findings indicate that condylar hypermovement induces the proliferation of synovial cells, and suggest that oxidative stress leads to the progression of synovial hyperplasia via DNA damage of the synovial cells in TMJs after mechanical loading.

Preparation and pharmaceutical evaluation for clinical application of high specific activity S-(-)[11C]CGP-12177, a radioligand for beta-adrenoreceptors.

BACKGROUND: Although S-(-)[C]CGP-12177 is a useful positron emission tomography (PET) ligand for beta-adrenoreceptors, the difficulty in radiolabelling the compound has prevented its extensive clinical application. Recently, we have developed a simple synthesis method for S-(-)[C]CGP-12177. In the present study, we attempted to prepare S-(-)[C]CGP-12177 with a high specific activity for intravenous injection which is feasible for the clinical evaluation of beta-adrenoreceptors. METHODS: The [C]methane produced during irradiation of a N2--H2 (95/5) mixture with an 18 MeV proton beam (20 microA, 30 min) was chlorinated using Cl2 to yield [C]carbon tetrachloride. S-(-)[C]CGP-12177 was synthesized by reacting the diamino precursor with [C]phosgene produced by oxidizing [C]carbon tetrachloride on a Fe--Fe2O3 column. The product was purified by using reversed phase, high-performance liquid chromatography (RP-HPLC) and the radioactive fraction containing S-(-)[C]CGP-12177 was collected and evaporated to dryness. S-(-)[C]CGP-12177 dissolved in physiological saline was sterilized through a 0.22 microm membrane filter. The radiochemical purity and the mass of the compound were determined with RP-HPLC. The residual organic solvents were determined with GC. Tests for sterility and the presence of bacterial endotoxins were also performed. RESULTS: S-(-)[C]CGP-12177 for intravenous injection was prepared in 25 min after the end of bombardment with a yield of 1.5+/-0.2 GBq. Specific activity was found to be 385.4+/-133.0 GBq/ micromol at the end of synthesis (EOS) (n=3). Radiochemical purity was found to be more than 99%. Toluene was not detected in the solution. The ethanol concentration was determined to be 60.3+/-52.5 ppm. Tests for sterility and bacterial endotoxins showed negative results. CONCLUSION: We successfully prepared S-(-)[C]CGP-12177 formulated for intravenous injection with high purity and high specific activity, which is feasible for the clinical evaluation of beta-adrenoreceptors.