Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis

<p>Abstract</p> <p>Background</p> <p>Wheat (<it>Triticum aestivum </it>L.) <it>O</it>-methyltransferase (TaOMT2) catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin), 3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous <it>Medicago sativa </it>caffeic acid/5-hydroxyferulic acid <it>O</it>-methyltransferase (MsCOMT) as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat.</p> <p>Results</p> <p>This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate.</p> <p>Conclusions</p> <p>We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.</p

Serum markers and development of delayed neuropsychological sequelae after acute carbon monoxide poisoning: anion gap, lactate, osmolarity, S100B protein, and interleukin-6

Objective Reliable biomarkers of delayed neuropsychological sequelae (DNS) after acute carbon monoxide (CO) poisoning are lacking. This study investigated the associations between potential serum markers and the development of DNS after acute CO poisoning. Methods Retrospective chart reviews were conducted for patients diagnosed with acute CO poisoning during a 28-month period. The patients were divided into two groups according to the presence or absence of having developed DNS. Multivariate analysis was performed to identify predictors of DNS after CO poisoning. Results Of a total of 102 patients, 10 (9.8%) developed DNS. The levels of serum osmolarity, S100B protein, and serum lactate, as well as serum anion gap, were statistically significant in univariate analysis. Multiple logistic regression analysis showed that anion gap (adjusted odds ratio [AOR], 1.36; 95% confidence interval [CI], 1.11 to 1.88), serum lactate level (AOR, 1.74; 95% CI, 1.26 to 2.75), and serum S100B protein level ([AOR, 7.02×105; 95% CI, 4.56×102 to 9.00×1010] in model 1, [AOR, 3.69×105; 95% CI, 2.49×102 to 2.71×1011] in model 2) were independently associated with DNS development. Conclusion Based on our preliminary results, serum lactate level, serum anion gap, and serum S100B protein level in the emergency department could be informative predictors of DNS development in patients with acute CO poisoning. These markers might have the potential to improve early recognition of DNS in patients with acute CO poisoning

Gene Expression Profile of the Hypothalamus in DNP-KLH Immunized Mice Following Electroacupuncture Stimulation

Clinical evidence indicates that electroacupuncture (EA) is effective for allergic disorder. Recent animal studies have shown that EA treatment reduces levels of IgE and Th2 cytokines in BALB/c mice immunized with 2,4-dinitrophenylated keyhole limpet protein (DNP-KLH). The hypothalamus, a brain center of the neural-immune system, is known to be activated by EA stimulation. This study was performed to identify and characterize the differentially expressed genes in the hypothalamus of DNP-KLH immunized mice that were stimulated with EA or only restrained. To this aim, we conducted a microarray analysis to evaluate the global gene expression profiles, using the hypothalamic RNA samples taken from three groups of mice: (i) normal control group (no treatments); (ii) IMH group (DNP-KLH immunization + restraint); and (iii) IMEA group (immunization + EA stimulation). The microarray analysis revealed that total 39 genes were altered in their expression levels by EA treatment. Ten genes, including T-cell receptor alpha variable region family 13 subfamily 1 (Tcra-V13.1), heat shock protein 1B (Hspa1b) and 2′–5′ oligoadenylate synthetase 1F (Oas1f), were up-regulated in the IMEA group when compared with the IMH group. In contrast, 29 genes, including decay accelerating factor 2 (Daf2), NAD(P)H dehydrogenase, quinone 1 (Nqo1) and programmed cell death 1 ligand 2 (Pdcd1lg2) were down-regulated in the IMEA group as compared with the IMH group. These results suggest that EA treatment can modulate immune response in DNP-KLH immunized mice by regulating expression levels of genes that are associated with innate immune, cellular defense and/or other kinds of immune system in the hypothalamus

Brain computed tomography angiography in postcardiac arrest patients and neurologic outcome

Objective This study aimed to analyze intracranial vessels using brain computed tomography angiography (CTA) and scoring systems to diagnose brain death and predict poor neurologic outcomes of postcardiac arrest patients. Methods Initial brain CTA images of postcardiac arrest patients were analyzed using scoring systems to determine a lack of opacification and diagnose brain death. The primary outcome was poor neurologic outcome, which was defined as cerebral performance category score 3 to 5. The frequency, sensitivity, specificity, positive predictive value, negative predictive value, and area under receiver operating characteristic curve for the lack of opacification of each vessel and for each scoring system used to predict poor neurologic outcomes were determined. Results Patients with poor neurologic outcomes lacked opacification of the intracranial vessels, most commonly in the vein of Galen, both internal cerebral veins, and the mid cerebral artery (M4). The 7-score results (P=0.04) and 10-score results were significantly different (P=0.04) between outcome groups, with an area under receiver operating characteristic of 0.61 (range, 0.48 to 0.72). The lack of opacification of each intracranial vessel and all scoring systems exhibited high specificity (100%) and positive predictive values (100%) for predicting poor neurologic outcomes. Conclusion Lack of opacification of vessels on brain CTA exhibited high specificity for predicting poor neurologic outcomes of patients after cardiac arrest

Design of a dye-doped liquid crystal cell with a constant transmittance-difference contour map

Thus far, the trial-and-error method has been used to find the condition for a dye-doped liquid crystal cell with desired performances. In this paper, we report a systematic design process to find the condition for the desired performances without trial-and-error process

Pilot study on a rewarming rate of 0.15°C/hr versus 0.25°C/hr and outcomes in post cardiac arrest patients

Objective Cerebral hemodynamic and metabolic changes may occur during the rewarming phase of targeted temperature management in post cardiac arrest patients. Yet, studies on different rewarming rates and patient outcomes are limited. This study aimed to investigate post cardiac arrest patients who were rewarmed with different rewarming rates after 24 hours of hypothermia and the association of these rates to the neurologic outcomes. Methods This study retrospectively investigated post cardiac arrest patients treated with targeted temperature management and rewarmed with rewarming rates of 0.15°C/hr and 0.25°C/hr. The association of the rewarming rate with poor neurologic outcomes (cerebral performance category score, 3 to 5) was investigated. Results A total of 71 patients were analyzed (0.15°C/hr, n=36; 0.25°C/hr, n=35). In the comparison between 0.15°C/hr and 0.25°C/hr, the poor neurologic outcome did not significantly differ (24 [66.7%] vs. 25 [71.4%], respectively; P=0.66). In the multivariate analysis, the rewarming rate of 0.15°C/hr was not associated with the 1-month neurologic outcome improvement (odds ratio, 0.54; 95% confidence interval, 0.16 to 1.69; P=0.28). Conclusion The rewarming rates of 0.15°C/hr and 0.25°C/hr were not associated with the neurologic outcome difference in post cardiac arrest patients

Preoperative Lymph Node Staging by FDG PET/CT With Contrast Enhancement for Thyroid Cancer: A Multicenter Study and Comparison With Neck CT

Objectives The purpose of this study was to compare lymph node (LN) staging using 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) with contrast-enhancement (CE) PET/CT and contrast-enhanced neck CT (neck CT) in patients with thyroid cancer with level-by-level comparison with various factors. Methods This was a retrospective multicenter study. A total of 85 patients were enrolled. Patients who underwent a preoperative evaluation by CE PET/CT and neck CT for thyroid cancer were enrolled. The gold standard for LN was the combination of surgical pathology and clinical follow-up. We compared CE PET/CT with neck CT using a level-by-level method. Factors, including age, sex, camera, arm position, tumor size, extra-thyroidal extension, tumor location, number of primary tumors, primary tumor maximum standardized uptake value, and the interval from scan to operation were also analyzed. Results Overall accuracy was 81.2% for CE PET/CT and 68.2% for neck CT. CE PET/CT was more sensitive than neck CT (65.8% vs. 44.7%). Also, CE PET/CT showed higher negative predictive value (77.2% vs. 66.1%). CE PET/CT showed good agreement with the gold standard (weighted kappa [κ], 0.7) for differentiating N0, N1a, and N1b, whereas neck CT showed moderate agreement (weighted κ, 0.5). CE PET/CT showed better agreement for the number of levels involved with the gold standard (weighted κ, 0.7) than that of neck CT with the gold standard (weighted κ, 0.5). The accuracies for differentiating N0, N1a, and N1b were 81.2% for CE PET/CT and 68.2% for neck CT. Level-by-level analysis showed that CE PET/CT was more sensitive and has higher negative predictive value for detecting ipsilateral level IV and level VI LNs than neck CT. Other analyzed factors were not related to accuracies of both modalities. Conclusion CE PET/CT was more sensitive and reliable than neck CT for preoperative LN staging in patients with thyroid cancer

Association of carotid and intracranial stenosis with Alzheimers disease biomarkers

Background To clarify whether atherosclerosis of the carotid and intracranial arteries is related to Alzheimers disease (AD) pathology in vivo, we investigated the associations of carotid and intracranial artery stenosis with cerebral beta-amyloid (Aβ) deposition and neurodegeneration in middle- and old-aged individuals. Given different variations of the pathologies between cognitive groups, we focused separately on cognitively normal (CN) and cognitively impaired (CI) groups. Methods A total of 281 CN and 199 CI (mild cognitive impairment and AD dementia) subjects underwent comprehensive clinical assessment, [11C] Pittsburgh compound B-positron emission tomography, and magnetic resonance (MR) imaging including MR angiography. We evaluated extracranial carotid and intracranial arteries for the overall presence, severity (i.e., number and degree of narrowing), and location of stenosis. Results We found no associations between carotid and intracranial artery stenosis and cerebral Aβ burden in either the CN or the CI group. In terms of neurodegeneration, exploratory univariable analyses showed associations between the presence and severity of stenosis and regional neurodegeneration biomarkers (i.e., reduced hippocampal volume [HV] and cortical thickness in the AD-signature regions) in both the CN and CI groups. In confirmatory multivariable analyses controlling for demographic covariates and diagnosis, the association between number of stenotic intracranial arteries ≥ 2 and reduced HV in the CI group remained significant. Conclusions Neither carotid nor intracranial artery stenosis appears to be associated with brain Aβ burden, while intracranial artery stenosis is related to amyloid-independent neurodegeneration, particularly hippocampal atrophy.This study was supported by a grant from the Ministry of Science and ICT, Republic of Korea (grant nos. NRF-2014M3C7A1046042, 2017R1A2B2008412, and 2018M3C7A1056888); by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant nos. HI18C0630 and HI19C0149); by a grant no. 04-20190500 from the SNUH Research Fund; and by a grant no. 06-20191860 from the Scientific Research Fund of the Korean Society of Magnetic Resonance in Medicine (2019). The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication

A questionnaire survey on the diagnosis and treatment of Fabry nephropathy in clinical practice

Background Fabry nephropathy is characterized by a deficiency of lysosomal alpha-galactosidase A, which results in proteinuria and kidney disease. The ineffectiveness of enzyme replacement therapy (ERT) for severe kidney failure highlights the need for early detection and meaningful markers. However, because the diagnosis and treatment of Fabry disease can vary according to the expertise of physicians, we evaluated the opinions of Korean specialists. Methods A questionnaire regarding the management of Fabry nephropathy was emailed to healthcare providers with the experience or ability to treat individuals with Fabry nephropathy. Results Of the 70 experts who responded to the survey, 43 were nephrologists, and 64.3% of the respondents reported having treated patients with Fabry disease. Pediatricians are treating primarily patients with classic types of the disease, while nephrologists and cardiologists are treating more patients with variant types. Only 40.7% of non-nephrologists agreed that a kidney biopsy was required at the time of diagnosis, compared with 81.4% of nephrologists. Thirty-eight of 70 respondents (54.3%) reported measuring globotriaosylsphingosine (lyso-Gb3) as a biomarker. The most common period to measure lyso-Gb3 was at the time of diagnosis, followed by after ERT, before ERT, and at screening. For the stage at which ERT should begin, microalbuminuria and proteinuria were chosen by 51.8% and 28.6% of respondents, respectively. Conclusion Nephrologists are more likely to treat variant Fabry disease rather than classic cases, and they agree that ERT should be initiated early in Fabry nephropathy, using lyso-Gb3 as a biomarker