Complete sequence analysis of human norovirus GII.17 detected in South Korea.

Norovirus, a major cause of gastroenteritis in people of all ages worldwide, was first reported in South Korea in 1999. The most common causal agents of pediatric acute gastroenteritis are norovirus and rotavirus. While vaccination has reduced the pediatric rotavirus infection rate, norovirus vaccines have not been developed. Therefore, prediction and prevention of norovirus are very important. Norovirus is divided into genogroups GI-GVII, with GII.4 being the most prevalent. However, in 2012-2013, GII.17 showed a higher incidence than GII.4 and a novel variant, GII.P17-GII.17, appeared. In this study, 204 stool samples collected in 2013-2014 were screened by reverse transcriptase-polymerase chain reaction; 11 GI (5.39%) and 45 GII (22.06%) noroviruses were identified. GI.4, GI.5, GII.4, GII.6 and GII.17 were detected. The whole genomes of the three norovirus GII.17 were sequenced. The whole genome of GII.17 consists of three open reading frames of 5109, 1623 and 780 bp. Compared with 20 GII.17 strains isolated in other countries, we observed numerous changes in the protruding P2 domain of VP1 in the Korean GII.17 viruses. Our study provided genome information that might aid in epidemic prevention, epidemiology studies and vaccine development.

Whole-genome sequencing analysis of human bocavirus detected in South Korea.

Human bocaviruses (HBoVs) have been detected in human gastrointestinal infections worldwide. In 2005, HBoV was also discovered in infants and children with infections of the lower respiratory tract. Recently, several genotypes of this parvovirus, including HBoV genotype 2 (HBoV2), genotype 3 (HBoV3) and genotype 4 (HBoV4), were discovered and found to be closely related to HBoV. HBoV2 was first detected in stool samples from children in Pakistan, followed by detection in other countries. HBoV3 was detected in Australia and HBoV4 was identified in stool samples from Nigeria, Tunisia and the USA. Recently, HBoV infection has been on the rise throughout the world, particularly in countries neighbouring South Korea; however, there have been very few studies on Korean strains. In this study, we characterised the whole genome and determined the phylogenetic position of CUK-BC20, a new clinical HBoV strain isolated in South Korea. The CUK-BC20 genome of 5184 nucleotides (nt) contains three open-reading frames (ORFs). The genotype of CUK-BC20 is HBoV2, and 98.77% of its nt sequence is identical with those of other HBoVs, namely Rus-Nsc10-N386. Especially, the ORF3 amino acid sequences from positions 212-213 and 454 corresponding to a variable region (VR)1 and VR5, respectively, showed genotype-specific substitutions that distinguished the four HBoV genotypes. As the first whole-genome sequence analysis of HBoV in South Korea, this information will provide a valuable reference for the detection of recombination, tracking of epidemics and development of diagnosis methods for HBoV.

Acute endophthalmitis after cataract surgery: 164 consecutive cases treated at a referral center in South Korea.

PurposeTo identify prognostic factors in patients referred with endophthalmitis after cataract surgery, and to evaluate the efficacy of primary vitrectomy as an initial management.MethodsOver an eight-year study period, we retrospectively reviewed the medical records of 164 patients who were referred with endophthalmitis following cataract surgery. Treatment generally conformed to standard guidelines, although primary vitrectomy was performed in several eyes with a visual acuity of hand motion or better, depending on the patient's status. Using multivariate analysis, we analyzed outcomes to determine the effect on final visual outcome.ResultsA final visual acuity of ≥20/40 was achieved in 92/164 (56.1%) cases after treatment. Bacterial cultures showed bacterial growth in 89/164 cases (54.3%). Among the various baseline characteristics, old age (P=0.028), poor visual acuity at presentation (P=0.004), gram-negative bacterial infection (P=0.030), and short time between cataract surgery and signs of endophthalmitis (P=0.021) were associated with poor visual outcome. The visual outcome showed no significant difference, in terms of initial treatment feature, between the primary vitrectomy with intraocular antibiotics injection (IOAI) and IOAI-only groups. However, reintervention was significantly less frequent in the primary vitrectomy group than in the IOAI group (12.5 and 32.7%, respectively; P=0.002).ConclusionOld age, poor visual acuity at presentation, type of cultured organism (gram-negative bacteria), and early onset of endophthalmitis after cataract surgery were significantly related to poor visual outcome after endophthalmitis treatment. Primary vitrectomy may decrease the need for reintervention to control infection, although the treatment showed no benefits with regard to visual outcome.

Intravitreal anti-vascular endothelial growth factor monotherapy for large submacular hemorrhage secondary to neovascular age-related macular degeneration.

PURPOSE: To evaluate the efficacy of anti-vascular endothelial growth factor (VEGF) monotherapy for large submacular hemorrhage (SMH) secondary to neovascular age-related macular degeneration (nAMD). METHODS: A total of 49 treatment-naive patients (49 eyes) with large SMH (more than five disc areas (DAs)) secondary to nAMD were retrospectively included. All patients were treated with an initial series of 3 monthly intravitreal anti-VEGF injections, followed by as-needed injections. At the 12-month follow-up, changes in best-corrected visual acuity (BCVA), hemorrhage area, central foveal thickness, and development of vitreous hemorrhage after treatment were evaluated. RESULTS: The mean SMH area was 13.9 ± 8.8 disk areas (DAs) and mean symptom duration was 7.25 ± 5.9 days at baseline. The mean number of injections was 4.49 ± 1.61. Twelve months after treatment, the mean BCVA significantly improved from 1.14 ± 0.61 logarithm of the minimum angle of resolution (logMAR; 20/276, Snellen equivalent) to 0.82 ± 0.53 logMAR (20/132; P = 0.002). Twenty-four eyes (49%) showed improvement of more than three lines of BCVA at 12 months after treatment. Baseline BCVA (odds ratio (OR), 5.119; 95% confidence interval (CI), 1.993-9.545; P = 0.004), duration of symptoms (OR, 0.727; 95% CI, 0.332-0.952; P = 0.024), hemorrhage area (OR, 0.892; 95% CI, 0.721-0.965; P = 0.011), and baseline central foveal thickness (OR, 0.881; 95% CI, 0.722-0.945; P = 0.032) were significantly associated with good visual acuity 12 months after treatment. CONCLUSIONS: Intravitreal anti-VEGF monotherapy is a valuable treatment option for large SMH secondary to nAMD.

Alteration in E-cadherin/ß-catenin expression in canine melanotic tumors.

ß-Catenin, encoded by the ctnnb1 gene, plays a critical role in intercellular adhesion, and its altered expression has been implicated in tumor progression in humans and animals. The aims of this study were to examine the alterations in ß-catenin expression in canine melanoma as well as the causes of these changes (eg, E-cadherin or exon 3 mutations) and to compare identified changes between skin and oral melanomas. Forty-two primary canine skin and oral melanoma tissue samples were used in the study. The expression levels of ctnnb1 and the levels of E-cadherin/ß-catenin complex in the tissues were determined by semiquantitative RT-PCR and immunohistochemistry, respectively. The mutational status of ß-catenin exon 3 was examined by DNA sequencing. RT-PCR revealed higher levels of ctnnb1 expression in oral melanoma tissues compared with normal melanocytes, irrespective of sex or histopathological appearance of the tissue (ie, amelanotic vs melanotic). Immunohistochemistry revealed simultaneous loss of membrane E-cadherin/ß-catenin complex and cytoplasmic accumulation of both proteins in 37 cases (84%). Intranuclear ß-catenin was also detected in all tissues with reduced membrane ß-catenin expression. In mutational analyses, one amelanotic oral melanoma showed 13 single nucleotide polymorphisms (SNPs); however, after protein translation, all the SNPs were silent mutations. The present study demonstrates that dysregulation of E-cadherin/ß-catenin complexes is involved in both types of canine melanotic tumors and that the disruption of E-cadherin/ß-catenin complexes and increased ß-catenin may induce tumor progression and malignancy.

Dysregulation of the Wnt/beta-catenin signaling pathway in canine cutaneous melanotic tumor.

The Wnt/beta-catenin signal transduction pathway is important in many developmental processes and during tumorigenesis. beta-Catenin acts as a signal transducer. To investigate whether the Wnt/beta-catenin signal transduction pathway is involved in canine cutaneous melanomagenesis, 18 formalin-fixed paraffin-embedded canine cutaneous melanotic tumor tissues were examined. For cloning and sequencing of the full-length canine ctnnb1 gene encoding beta-catenin, conserved sequences of the human and mouse ctnnb1 gene were used to design the primers. For analysis of expression and translocation of beta-catenin in canine cutaneous melanotic tumors, semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed. The canine ctnnb1 sequence showed a high degree of similarity to those of human and mouse. Semiquantitative RT-PCR showed a substantial increase in expression of ctnnb1 mRNA in canine cutaneous melanotic tumors compared to normal canine melanocytes, regardless of whether the tumor was benign or malignant. Immunohistochemistry revealed cytoplasmic accumulation of beta-catenin in melanotic tumors. In melanoma tissues, nuclear translocation of beta-catenin was also observed. The present study demonstrated that abnormal intracellular accumulation and substantially increased expression of beta-catenin are involved in canine cutaneous melanotic tumor.

The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath.

Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.

Anti-atherogenic effect of citrus flavonoids, naringin and naringenin, associated with hepatic ACAT and aortic VCAM-1 and MCP-1 in high cholesterol-fed rabbits.

The anti-atherogenic effects of the citrus flavonoids, naringin and naringenin, were evaluated in high cholesterol-fed rabbits. At 3 months of age, 30 male New Zealand White (NZW) rabbits were divided into three groups (n = 10 per group). The rabbits were fed a 1% cholesterol diet alone (control group) or a diet supplemented with either 0.1% naringin or 0.05% naringenin for 8 weeks. The plasma lipoprotein levels, total cholesterol, triglyceride, and high-density lipoprotein showed no significant differences in the control and experimental groups. Hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity was slightly low in naringin (5.0%)- and naringenin (15.0%)-fed rabbits, compared to control group. The aortic fatty streak areas were significantly lower in both the naringin (19.2 +/- 5.6%)- and naringenin (18.1 +/- 6.5%)-supplemented groups than in the control group (60.4 +/- 14.0%). The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), by semiquantitative RT-PCR analysis of the thoracic aorta, were significantly lower in the flavonoids supplemented groups than in the control group. These results suggest that the anti-atherogenic effect of the citrus flavonoids, naringin and naringenin, is involved with a decreased hepatic ACAT activity and with the downregulation of VCAM-1 and MCP-1 gene expression.

Arthrobacter siderocapsulatus Dubinina and Zhdanov 1975AL is a later subjective synonym of Pseudomonas putida (Trevisan 1889) Migula 1895AL.

The taxonomic position of Arthrobacter siderocapsulatus Dubinina and Zhdanov 1975AL was investigated using 16S rDNA, fatty acid and phenotypic analyses. The type strain (NCIMB 11286T) showed 99.85% 16S rDNA similarity to the type strain of Pseudomonas putida. Phenotypic properties of the two strains were compared using API 20NE and BIOLOG kits. Identical reactions were recorded for all tests, except for assimilation of malonic acid. The two strains also showed almost identical cellular fatty acid profiles. On the basis of evidence presented in this and earlier studies, it is proposed that Arthrobacter siderocapsulatus is a later subjective synonym of Pseudomonas putida (Trevisan 1889) Migula 1895AL.

Production of hydrogen peroxide by serum and its involvement in cell proliferation in ROS 17/2.8 osteoblasts.

The effects of hydrogen peroxide, which fetal bovine serum (FBS) releases, on proliferation have been studied in ROS 17/2.8 osteoblasts. Cell proliferation, when activated by FBS, was inhibited by catalase in ROS 17/2.8 osteoblasts, but did not in primary osteoblast-like cells. Serum-induced extracellular signal-regulated kinase(ERK) activity was reduced by the pretreated catalase in ROS 17/2.8 osteoblasts. In addition, the present studies demonstrate that addition of FBS led to an increase of fluorescence of dihydrorhodamine 123, indicating formation of free radicals including hydrogen peroxide in ROS 17/2.8 osteoblasts, but not in primary osteoblast-like cells. These phenomena may account for the generation of reactive oxygen species during cellular proliferation in ROS 17/2.8 osteoblasts.

Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol.

Studies were performed to determine if the growth of Methylomicrobium album BG8 on methanol could be enhanced through the provision of chloromethane. M. album BG8 was found to be able to oxidize chloromethane via the particulate methane monooxygenase with an apparent K(s) of 11+/-3 microM and V(max) of 15+/-0.6 nmol (min mg protein)(-1). When up to 2.6 mM chloromethane was provided with 5 mM methanol, methanotrophic growth was significantly enhanced in comparison to the absence of chloromethane, indicating that methanotrophs can utilize chloromethane to support growth, although it could not serve as a sole growth substrate. [(14)C]chloromethane was found to be oxidized to [(14)C]CO(2) as well as incorporated into biomass. These results indicate that reactions previously thought to be cometabolic may actually provide some benefit to methanotrophs and that these cells can use multiple compounds to enhance growth.

Degradation of chlorinated and brominated hydrocarbons by Methylomicrobium album BG8.

The degradation kinetics of ten halogenated hydrocarbons by Methylomicrobium album BG8 expressing particulate methane monooxygenase (pMMO) and the inhibitory effects of these compounds on microbial growth and whole-cell pMMO activity were measured. When M. album BG8 was grown with methane, growth was completely inhibited by dichloromethane (DCM), bromoform (BF), chloroform (CF), vinyl chloride (VC), 1,1-dichloroethylene (1,1-DCE), and cis-dichloroethylene (cis-DCE). Trichloroethylene (TCE) partially inhibited growth on methane, while dibromomethane (DBM), trans-dichloroethylene (trans-DCE), and 1,1,1-trichloroethane (1,1, 1-TCA) had no effect. If the cells were grown with methanol, DCM, BF, CF, and 1,1-DCE completely inhibited growth, while VC, trans-DCE, TCE, and 1,1,1-TCA partially inhibited growth. Both DBM and cis-DCE had no effect on growth with methanol. Whole-cell pMMO activity was also affected by these compounds, with all but 1,1,1-TCA, DCM, and DBM reducing activity by more than 25%. DCM, DBM, VC, trans-DCE, cis-DCE, 1,1-DCE, and TCE were degraded and followed Michaelis-Menten kinetics. CF, BF, and 1,1,1-TCA were not measurably degraded. These results suggested that the products of DCM, TCE, VC, and 1,1-DCE inactivated multiple enzymatic processes, while trans-DCE oxidation products were also toxic but to a lesser extent. cis-DCE toxicity, however, appeared to be localized to pMMO. Finally, DBM and 1,1,1-TCA were not inhibitory, and CF and BF were themselves toxic to M. album BG8. Based on these results, the compounds could be separated into four general categories, namely (1) biodegradable with minimal inactivation, (2) biodegradable with substantial inactivation, (3) not biodegradable with minimal inactivation, and (4) not biodegradable but substantial inactivation of cell activity.

Effect of ionizing radiation on the differentiation of ROS 17/2.8 osteoblasts through free radicals.

Although the acceleration of bone regeneration by radiation has been reported, the mechanisms of action of radiation on bone are unclear. The present results indicate that ionizing radiation-stimulated differentiation could result from the generation of reactive oxygen species during radiation exposure. The free radical release is considered as the most important mechanism of bone effect by radiation treatment. In addition, we report that radiation induced transient activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation and the transcription factor, AP-1. The JNK and AP-1 activation is mediated with radiation-released free radicals in ROS 17/2.8 osteoblasts. These results indicate that ionizing radiation at a single dose of up to 5 Gray stimulates differentiation of ROS 17/2.8 osteoblasts via free radial release which may affect JNK/SAPK and AP-1 activities.