Isolation and Characterization of Engineered Nucleoside Deoxyribosyltransferase with Enhanced Activity Toward 2'-Fluoro-2'-Deoxynucleoside.

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40°C) and 4.4-fold (at 50°C) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

Sympathetic Overactivity Based on Heart-Rate Variability in Patients with Obstructive Sleep Apnea and Cerebral Small-Vessel Disease.

BACKGROUND AND PURPOSE: Obstructive sleep apnea (OSA) is associated with cerebral white-matter changes (WMC), but the underlying mechanisms are not completely understood. Our aim was to identify the cardiovascular autonomic characteristics during sleep that are associated with cerebral WMC in OSA patients. METHODS: We recruited subjects from our sleep-center database who underwent both polysomnography and brain MRI within a 1-year period. Sixty patients who had OSA with WMC (OSA+WMC), 44 patients who had OSA without WMC (OSA-WMC), and 31 control subjects who had neither OSA nor WMC were analyzed. Linear and nonlinear indices of heart-rate variability (HRV) were analyzed in each group according to different sleep stages and also over the entire sleeping period. RESULTS: Among the nonlinear HRV indices, the Poincaré ratio (SD12) during the entire sleep period was significantly increased in the OSA+WMC group, even after age adjustment. Meanwhile, detrended fluctuation analysis 1 during non-rapid-eye-movement sleep tended to be lowest in the OSA+WMC group. These indices were altered regardless of the presence of hypertension or diabetes. In the subgroup analysis of middle-aged OSA patients, approximate entropy during rapid-eye-movement sleep was significantly lower in OSA+WMC patients than in OSA-WMC patients. Overall, the nonlinear HRV indices suggest that sympathetic activity was higher in the OSA+WMC group than in the OSA-WMC and control groups. CONCLUSIONS: Our findings suggest that dysregulation of HRV, especially overactivation of sympathetic tone, could be a pathophysiologic mechanism underlying the development of WMC in OSA patients.

Moderate-to-severe obstructive sleep apnea is associated with cerebral small vessel disease.

BACKGROUND: Cerebral small vessel disease (SVD) is associated with increased risk of cerebral infarction and hemorrhage. Obstructive sleep apnea (OSA) is known to increase the risk of cerebrovascular disease. This study aimed to investigate the association between cerebral SVD and severity of OSA. METHODS: A total of 170 patients were included from the patient registry at the present Sleep Center; these patients underwent both magnetic resonance imaging (MRI) of the brain and polysomnography (PSG) for suspected OSA. The presence and burden of white matter hyperintensities (WMHs), asymptomatic lacunar infarctions (ALIs), cerebral microbleeds (CMBs), and perivascular spaces (PVSs) were determined by MRI, and their relationships with the apnea-hypopnea index (AHI), as determined by PSG, were investigated. RESULTS: Among the 170 patients, 25 (14.7%) had high-grade WMHs, 21 (12.4%) had ALIs, 21 (12.4%) had CMBs, and 34 (20.0%) had high-grade PVSs. In the multivariable analysis, after adjusting for factors including age, sex, and other variables for which p <0.1 in univariable analysis (hypertension, diabetes mellitus, previous stroke, minimal SaO2 and arousal index), moderate-to-severe OSA was associated with high-grade WMHs (odds ratio [OR] 4.72; 95% confidence interval [CI] 1.14-19.47), CMBs (OR 3.47; 95% CI 0.89-15.18), or high-grade PVSs (OR 3.64; 95% CI 1.02-13.01), but not with ALIs. The total SVD score was independently associated with increased AHI (p = 0.017), particularly in patients with moderate-to-severe OSA (ß [standard error] = 0.448 (0.204), p = 0.030]. CONCLUSION: Moderate-to-severe OSA is positively associated with multiple indicators of cerebral SVD, including WMHs, CMBs, and PVSs.

Is obstructive sleep apnea associated with the presence of intracranial cerebral atherosclerosis?

PURPOSE: Intracranial cerebral atherosclerosis (ICAS) is one of critical atherosclerosis which closely related with stroke. Obstructive sleep apnea (OSA) is associated with systemic atherosclerosis, but it is unclear whether OSA is related with the presence of ICAS. We aimed to investigate the association between the presence of ICAS and severity of OSA in patients with suspected OSA. METHODS: This retrospective, cross-sectional study included 283 patients who suspected OSA (presence of one or more OSA-related symptom and high-risk category in Berlin questionnaire) and underwent polysomnography and brain magnetic resonance angiography (MRA). The ICAS was defined as ≥50% decrease of luminal diameter in MRA. The severity of OSA was defined by apnea-hypopnea index (AHI). RESULTS: The mean age was 60.7 ± 13.5 years, and 55.8% (158/283) were male in all included patients. The 53 (18.7%) patients had ICAS and 117 (41.3%) patients had moderate to severe OSA (AHI ≥ 15). Higher AHI was noted in patients with ICAS compared to those without ICAS (31.7 ± 25.8 versus 15.2 ± 17.4, p = 0.001). In multivariable logistic analyses, after adjusting for age, sex, and variables with p < 0.1 in univariable analyses (hypertension, diabetes mellitus, atrial fibrillation, previous stroke history, body mass index, lipid-lowing agents, arousal index, and minimum oxygen saturation), moderate to severe OSA were independently related with the presence of ICAS (odds ratio 4.17, 95% confidence interval 1.40-12.40, p = 0.010). CONCLUSIONS: Our findings suggest that moderate to severe OSA is associated with the presence of ICAS in patients with suspected OSA.

Stage-Wise Identification and Analysis of miRNA from Root-Knot Nematode Meloidogyne incognita.

In this study, we investigated global changes in miRNAs of Meloidogyne incognita throughout its life cycle. Small RNA sequencing resulted in approximately 62, 38, 38, 35, and 39 Mb reads in the egg, J2, J3, J4, and female stages, respectively. Overall, we identified 2724 known and 383 novel miRNAs (read count > 10) from all stages, of which 169 known and 13 novel miRNA were common to all the five stages. Among the stage-specific miRNAs, miR-286 was highly expressed in eggs, miR-2401 in J2, miR-8 and miR-187 in J3, miR-6736 in J4, and miR-17 in the female stages. These miRNAs are reported to be involved in embryo and neural development, muscular function, and control of apoptosis. Cluster analysis indicated the presence of 91 miRNA clusters, of which 36 clusters were novel and identified in this study. Comparison of miRNA families with other nematodes showed 17 families to be commonly absent in animal parasitic nematodes and M. incognita. Validation of 43 predicted common and stage-specific miRNA by quantitative PCR (qPCR) indicated their expression in the nematode. Stage-wise exploration of M. incognita miRNAs has not been carried out before and this work presents information on common and stage-specific miRNAs of the root-knot nematode.

Quality characteristics and ginsenosides composition of ginseng-yakju according to the particle size of ginseng powder.

The aim of this study was to develop rice wine (Yakju) containing various amounts and particle sizes of ginseng powder and to analyze the physicochemical characteristics and content of ginsenosides in ginseng-Yakju. Soluble solid content, pH, ethanol concentration, acidity, amino acid content, and evaluation of preference showed no difference between four kinds of Yakju groups, regardless of ginseng supplementation and particle size of the ginseng powder. During fermentation of Yakju containing ginseng, the contents of ginsenosides Rb1, Rb2, Rb3, and Rc were decreased. Otherwise, the content of ginsenoside Rh1 was increased highly by brewing microorganisms in Yakju. Recovery ratios of ginsenosides in ginseng-Yakju were approximately 25.4% (coarse ginseng power) and 23.8% (fine ginseng powder), which were superior to the recovery ratio of ginsenosides in Yakju containing ginseng slices (5%).

Efficacy of levofloxacin and rifaximin based quadruple therapy in Helicobacter pylori associated gastroduodenal disease: a double-blind, randomized controlled trial.

The aim of this study was to evaluate the efficacy of levofloxacin and rifaximin based quadruple regimen as first-line treatment for Helicobacter pylori infection. A prospectively randomized, double-blinded, parallel group, comparative study was performed. Three hundred consecutive H. pylori positive patients were randomized to receive: omeprazole, amoxicillin, clarithromycin (OAC); omeprazole, amoxicillin, levofloxacin (OAL); and omeprazole, amoxicillin, levofloxacin, rifaximin (OAL-R). The eradication rates in the intention to treat (ITT) and per protocol (PP) analyses were: OAC, 77.8% and 85.6%; OAL, 65.3% and 73.6%; and OAL-R, 74.5% and 80.2%. The eradication rate achieved with OAC was higher than with OAL on the ITT (P = 0.05) and PP analysis (P = 0.04). OAL-R regimen was not inferior to OAC. The frequency of moderate to severe adverse effects was significantly higher in OAC treatment group. Especially, diarrhea was most common complaint, and there was a significantly low rate of moderate to severe diarrhea with the rifaximin containing regimen. In conclusion, the levofloxacin and rifaximin based regimen comes up to the standard triple therapy, but has a limited efficacy in a Korean cohort. The rifaximin containing regimen has a very high safety profile for H. pylori eradication therapy.

[Effect of acid pump antagonist (Revaprazan, Revanex(R)) on result of 13C urea breath test in patients with Helicobacter pylori associated peptic ulcer disease].

BACKGROUND/AIMS: Revaprazan (Revanex(R)) is a novel proton pump inhibitor (PPI) that has a somewhat different effect on proton pump compared with the other PPI's, also (called as 'acid pump antagonist'). We aimed to examine the false negative rate of 13C-urea breath test (UBT) in the patients with Helicobacter pylori (H. pylori) associated peptic ulcer disease who were treated with revaprazan and evaluate the anti-urease activity of revaprazan. METHODS: Total 55 patients were enrolled in this study. They received EGD examination between January 2009 and December 2009 and diagnosed histologically as H. pylori associated peptic ulcer disease. All patients took revaprazan only. Three patients were excluded because of underlying chronic disease and inappropriate breath sampling. The remaining 52 patients had UBT at 0, 2 and 4 weeks of revaprazan use. After 2 weeks of the cessation of revaprazan, they had the fourth UBT. RESULTS: At 2 and 4 weeks, the false negative rates of UBT were 5.8% and 23.1%, respectively (p=0.05). After 2 weeks of the cessation, the cases of the false negative result were five. Four out of five patients had prolonged negative results on two or three successive tests, and baseline 13C difference value did not predict the false negative results. CONCLUSIONS: False negative results of UBT were common and increased with prolonged use of acid pump antagonist. As PPI, it had also anti-urease activity and most patients (47/52, 90.4%) reverted to positive results by 2 weeks after the cessation of taking the medication.

Intracellular expression of the T-cell factor-1 RNA aptamer as an intramer.

T-cell factor (TCF)-1 protein forms the transcriptional complex with beta-catenin and regulates the expression of diverse target genes during early development and carcinogenesis. We have selected previously an RNA aptamer that binds to the DNA-binding domain of TCF-1 and have shown that it interfered with binding of TCF-1 to its specific DNA recognition sequences in vitro. As an approach to modulate the transcription by TCF/beta-catenin complex in the cells, we have developed the RNA expression vector for stable expression of RNA aptamer inside of the mammalian cells. High level of RNA was expressed as an intramer in the fusion with the stable RNA transcript. The RNA intramer inhibited TCF/beta-catenin transcription activity as shown by luciferase assay. It also modulated the expression of TCF/beta-catenin target genes, such as cyclin D1 and matrix metalloproteinase-7, as predicted to be as an effective inhibitor of the TCF function. In addition, it efficiently reduced the growth rate and tumorigenic potential of HCT116 colon cancer cells. Such RNA intramer could lead to valuable gene therapeutics for TCF/beta-catenin-mediated carcinogenesis.

Inhibition of the DNA binding by the TCF-1 binding RNA aptamer.

The DNA binding architectural protein, TCF, and the transcriptional activator, beta-catenin, form a complex that regulates the expression of diverse target genes during early development and carcinogenesis. As an approach to modulating transcription by this complex, we selected an RNA aptamer that binds to the DNA binding domain of TCF-1. The aptamer interfered with the binding of TCF-1 to its specific DNA recognition sequences in vitro and also inhibited DNA binding of cellular TCF-1. We also developed the truncated version of the aptamer for efficient delivery to the cells. Structural analysis of the truncated aptamer revealed that a stem-loop with an internal loop was responsible for the binding to TCF-1. Similar approach may well be applicable to other proteins, especially DNA binding transcription factors, in order to modulate their DNA binding and transcriptional activity in the cells.

Specific modulation of the anti-DNA autoantibody-nucleic acids interaction by the high affinity RNA aptamer.

Anti-DNA autoantibodies are one of the frequently found autoantibodies in systemic lupus erythematosus patient sera. RNA aptamers for the monoclonal G6-9 anti-DNA autoantibody were selected from a random pool of RNA library. Binding affinity of the best aptamer is around 2nM, which is at least 100-fold higher than that of cognate DNA antigen to the autoantibody. Aptamer binds specifically to the G6-9 autoantibody but not to other similar autoantibodies. Minimal binding motif of the aptamer was mapped, providing a hint for a natural epitope of the autoantibody. DNA binding to the G6-9 autoantibody is shown to be efficiently inhibited by the aptamer. Such binding property of the RNA aptamer could be used not only as a modulator for the pathogenic anti-DNA autoantibody, but also as a useful biochemical reagent for elucidating a fine specificity of the autoantibody-nucleic acid interaction.