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High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/ßA20P/ßP38L/ßF118W/ßS130Y/ßC189N/ßC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.
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Sequência de Aminoácidos/genética , Estabilidade Enzimática/genética , Hidroliases/química , Catálise , Hidroliases/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Pseudonocardia/química , Pseudonocardia/genética , TemperaturaRESUMO
Gelsemium elegans Benth., a well-known toxic herbal plant, is widely used to treat rheumatic arthritis, inflammation and other diseases. Gelsemium contains humantenmine (HMT), which is an important bioactive and toxic alkaloid. Cytochrome P450 enzymes (CYPs) play important roles in the elimination and detoxification of exogenous substances. This study aimed to investigate the roles of CYPs in the metabolism and detoxification of HMT. First, metabolic studies were performed in vitro by using human liver microsomes, selective chemical inhibitors and recombinant human CYPs. Results indicated that four metabolites, including hydroxylation and oxidation metabolites, were found in human liver microsomes and identified based on their high-resolution mass spectrum. The isoform responsible for HMT metabolism was mainly CYP3A4/5. Second, the toxicity of HMT on L02 cells in the presence of the nicotinamide adenine dinucleotide phosphate system (NADPH) was significantly less than that without NADPH system. A CYP3A4/5 activity inhibition model was established by intraperitoneally injecting ketoconazole in mice and used to evaluate the role of CYP3A4/5 in HMT detoxification. In this model, the 14-day survival rate of the mice decreased to 17% after they were intragastrically treated with HMT, along with hepatic injury and increasing alanine aminotransferase (ALT) /aspartate aminotransferase (AST) levels. Overall, CYP3A4/5 mediated the metabolism and detoxification of HMT.
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Alcaloides/metabolismo , Alcaloides/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Gelsemium/química , Gelsemium/toxicidade , Inativação Metabólica , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Animais , Extratos Vegetais/metabolismo , Extratos Vegetais/toxicidade , Adulto JovemRESUMO
In this work, we reported an effective method for the synthesis of a multirecognition magnetic molecularly imprinted polymer (MMIP) with atom transfer radical polymerization (ATRP), using 2,4-diamino-6-methyl-1,3,5-triazine as pseudo-template. The resulting MMIP was characterized in detail by Fourier transform-infrared (FT-IR) spectra, scanning electron microscopy (SEM), thermogravimetic analysis (TGA), and vibrating sample magnetometry (VSM). These results indicated the successful synthesis of MMIP with sufficient thermal stability and magnetic properties. The adsorption experiments were carried out to evaluate the specific selectivity of MMIP related to the spatial structure of target molecules. The MMIP exhibited multirecognition ability and excellent binding capability for melamine (MEL), cyromazine (CYR), triamterene (TAT), diaveridine (DVD), and trimethoprim (TME), and the apparent maximum number of binding sites (Q max) was 77.5, 75.2, 72.5, 69.9, and 70.4 µmol g-1, respectively. The multirecognition MMIP not only possessed adequate magnetic responsiveness for fast separation but also avoided the risk of template leakage on trace component analysis. Therefore, it was suitable for serving as a magnetic solid-phase extraction (MSPE) adsorbent. MSPE coupled with high-performance liquid chromatography analysis was applied to enrich and separate five target molecules from three samples. Recoveries for all target molecules ranged from 81.6 to 91.5% with relative standard deviations of no more than 4.1% (n = 3). Graphical abstract Multirecognition property of magnetic molecularly imprinted polymer prepared with pseudo template.
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Acetaminophen (APAP) is one of the most commonly used oral analgesics and antipyretics, but hepatotoxicity including liver failure may occur after overdose. The therapeutic options for treating APAP hepatotoxicity are limited. Eriodictyol, a dietary flavonoid with anti-inflammatory and antioxidant properties, was used here to determine its protective effects against APAP-induced hepatotoxicity in mice. Various administration routes and pharmacokinetics-pharmacodynamics (PK-PD) analyses were used to determine these effects. Protective effects were observed in intravenously and intraperitoneally but not in intragastrically administered eriodictyol. LC-MS/MS analysis revealed two monoglucuronide metabolites of eriodictyol in liver and intestine microsomes. Recombinant human uridine-5'-diphospho -glucuronosyltransferase (UGT) isoforms and chemical inhibition studies demonstrated that UGT1As (mainly UGT1A1, UGT1A9, UGT1A10) and UGT2B7 were likely the main contributors to eriodictyol glucuronidation. Intragastric administration of eriodictyol, which displayed lower parent and higher metabolite concentrations in the plasma, did not elicit protective effects against APAP hepatotoxicity, when compared to the intraperitoneal injection of eriodictyol. The relative bioavailability of eriodictyol was increased to 216.84% with the coadministration of glycyrrhetinic acid (GA), an inhibitor of UGT1As. Intragastric administration of eriodictyol in combination with GA also induced protective effects against APAP hepatotoxicity. Furthermore, intragastric administration of eriodictyol attenuated APAP hepatotoxicity in heterozygous Ugt1 (Ugt1+/-) mice but not in its wild-type littermates. Thus, UGT1A-mediated metabolic inactivation reduced the protective effect of eriodictyol. Eriodictyol attenuated APAP hepatotoxicity via inhibition of hepatic cytochrome P450 (cyp) 2e1 and cyp3a11 activities; reserve of glutathione (GSH) by improvement of glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione S-transferase (GST) activities; elevation of superoxide dismutase (SOD) activity; and reduction of malondialdehyde (MDA) level. Our findings indicate that parenterally administered eriodictyol may be used to treat APAP-induced hepatotoxicity, and its efficacy can be enhanced by UGT1As down-regulation.
Assuntos
Acetaminofen/toxicidade , Flavanonas/farmacologia , Glucuronídeos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Flavanonas/metabolismo , Glucuronídeos/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Ácido Glicirretínico/farmacologia , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Superóxido Dismutase/metabolismoRESUMO
In this study, a new molecularly imprinted polymer chiral stationary phase (MIP-CSP) was prepared utilizing molecular crowding agent for improvement the selective separation ability. S-amlodipine (S-AML), methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA), and polymethyl methacrylate (PMMA) were selected as template, functional monomer, cross-linker, and molecular crowding agent, respectively. The composition of formulas for MIP-CSP was optimized, and the permeability and structural feature of resultant MIP-CSP were characterized. The effect of mobile-phase composition, including ionic strength, pH, and organic modifier content, was investigated for achieving the selective separation of rac-amlodipine (rac-AML) on MIP-CSP. The baseline separation of rac-AML was achieved with resolution of 1.58, whereas no selective separation was observed on the imprinted monolith without molecular crowding agent. The perturbation chromatography method was successfully applied to evaluate the recognition mechanism of templates on MIP-CSP. The retention time of S-AML detected in typical analytical conditions was obviously greater than the time of negative peak derived from perturbation, which indicated the retention of template may be due to the imprinted cavities on MIP-CSP. Additionally, the result of Van't Hoff analysis indicated that the chiral separation of rac-AML on MIP-CSP was an entropy-driven process, which supported the molecular imprinting theory. These results reveal that molecular crowding is a potential strategy for preparation of MIP-CSP with excellent selective separation ability. Graphical Abstract Improvement of chiral separation on molecularly imprinted monolith by molecular crowding condition.
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Humantenmine (HMT), the most toxic compound isolated from Gelsemium elegans Benth, is a well-known active herbal compound. A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to estimate the absolute oral bioavailability of HMT in rats. Quantification was performed by multiple reaction monitoring using electrospray ionization operated in positive ion mode with transitions of m/z 327.14 â m/z 296.19 for HMT and m/z 323.20 â m/z 236.23 for gelsemine (internal standard, IS). The linear range of the calibration curve was 1-256 nmol/L, with a lower limit of quantification at 1 nmol/L. The accuracy of HMT ranged from 89.39 to 107.5%, and the precision was within 12.24% (RSD). Excellent recovery and negligible matrix effect were observed. HMT remained stable during storage, preparation and analytical procedures. The pharmacokinetics of HMT in rats showed that HMT reached the concentration peak at 12.50 ± 2.74 min with a peak concentration of 28.49 ± 6.65 nmol/L, and the corresponding area under the concentration-time curve (AUC0-t ) was 1142.42 ± 202.92 nmol/L min after 200 µg/kg HMT was orally administered to rats. The AUC0-t of HMT given at 20 µg/kg by tail vein administration was 1518.46 ± 192.24 nmol/L min. The calculated absolute bioavailability of HMT was 7.66%.
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Alcaloides/sangue , Alcaloides/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Alcaloides/química , Animais , Disponibilidade Biológica , Estabilidade de Medicamentos , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Background: The problem of maternal mental health is a priority issue of global concern. Dyadic coping refers to the co-managing and making decisions between two parties in response to a joint stressful event. At present, china has limited focus on dyadic coping for pregnant women during pregnancy. This study aimed to investigate different categories and characteristics of dyadic coping in pregnant women throughout pregnancy and to analyze the factors that influence these categories. Methods: This study was a cross-sectional, and 376 pregnant women who visited the obstetric clinic at a tertiary hospital in Sichuan province from June to September 2023 were interviewed face-to-face using convenience sampling. Data were collected using a sociodemographic questionnaire, dyadic coping scale, and family adaptability and cohesion evaluation scale. The data were imported into excel and exported to spss 27.0 to analyze the potential characteristics of pregnant women's dyadic coping during pregnancy and to explore the effects of this using univariate analysis and multifactorial logistic regression. Results: A total of 376 valid questionnaires were collected. The results of the potential profile analysis showed that the dyadic coping of pregnant women during pregnancy could be categorized into three different groups: the "low coping group" (21.3%), the "general coping group" (67.5%), and the "high coping group" (11.2%). Multiple logistic regression analyses revealed that low monthly family income, early pregnancy, primipara, family adaptability and cohesion were the factors influencing the dyadic coping of pregnant women during pregnancy. Conclusion: During pregnancy, pregnant women exhibit moderate levels of dyadic coping. Three different categories of dyadic coping patterns were exhibited: low coping group, general coping group, and high coping group, with significant heterogeneity. Therefore, there is a need to focus on the dyadic coping status of various categories of pregnant women and implement targeted couple and family-wide interventions.
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Glycans mediate various biological processes through carbohydrate-protein interactions, and glycan microarrays have become indispensable tools for understanding these mechanisms. However, advances in functional glycomics are hindered by the absence of convenient and universal methods for obtaining natural glycan libraries with diverse structures from glycoconjugates. To address this challenge, we have developed an integrative approach that enables one-pot release and simultaneously capture, separation, structural characterization, and functional analysis of N/O-glycans. Using this approach, glycoconjugates are incubated with a pyrazolone-type heterobifunctional tag-ANPMP to obtain glycan-2ANPMP conjugates, which are then converted to glycan-AEPMP conjugates. We prepared a tagged glycan library from porcine gastric mucin, soy protein, human milk oligosaccharides, etc. Following derivatization by N-acetylation and permethylation, glycans were subjected to detailed structural characterization by ESI-MSn analysis, which revealed >83 highly pure glycan-AEPMPs containing various natural glycan epitopes. A shotgun microarray is constructed to study the fine details of glycan-bindings by proteins and antisera.
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Proteínas , Pirazolonas , Animais , Humanos , Suínos , Glicoconjugados , Polissacarídeos/química , Glicômica/métodosRESUMO
Glycans have been proven to play special roles in keeping human health as a class of nutritional and bioactive ingredients in many food materials. However, their broad use in the food industry is hindered by the lack of comprehensive analytical methods for high-quality food glycomics studies and large-quantity raw materials for their production. This study focuses on structural identification and quantitative comparison of bioactive N-glycans in seven species of livestock and poultry plasma as potential natural glycan resources by a novel comprehensive relative quantification strategy based on stable isotope labeling with nondeuterated and deuterated 4-methyl-1-(2-hydrazino-2-oxoethyl)-pyridinium bromide (d0/d7-HMP) in combination with linkage-specific derivatization of sialic acid residues. Methodological validation of the method in terms of detection sensitivity, signal resolution, quantification linearity, precision, and accuracy on model neutral and complicated sialylated glycans demonstrated its advantages over the existing methods. Based on this method, a series of bioactive N-glycans were found in seven species of livestock and poultry plasma, and their differences in structure, abundance percentages, and relative contents of N-glycans were revealed, demonstrating their excellent applicability for comprehensive food glycomics analysis and great exploitation potential of these plasma samples as large-quantity raw materials in producing bioactive N-glycans for application in food and pharmaceutical industries.
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Gado , Aves Domésticas , Animais , Humanos , Polissacarídeos/química , Ácido N-Acetilneuramínico , Glicômica/métodosRESUMO
Glycomics analysis has been undermined by the lack of structurally defined individual glycans as model compounds. However, it is challenging to prepare individual glycans from natural resources, mainly due to separation difficulties caused by highly diverse structure, complicated mixture form and chromophore-free property of naturally-existing glycans. In this study, we report a simple, universal and low-cost glycan separation strategy, glycoselection, which allows preparation of individual reducing glycans from their mixtures through reversible chromogenic derivatization by hydrazide chemistry in combination with two-dimensional high-performance liquid chromatography (2D-HPLC). Investigations on reaction conditions using lactose and maltodextrin as model glycans showed the feasibility of reversible hydrazide labeling and one-pot hydrazone conversion under mild conditions, the good stability of hydrazone-form derivatives of glycans in solution and the difference among seven selected hydrazine-carrying chromogenic reagents in product yields during glycan labeling and post-column detagging. The 2D-HPLC separation conditions were established on maltodextrin, from which fourteen highly-purified individual reducing oligo-glucans were ultimately obtained. Using this strategy, we also successfully prepared and identified eleven individual neutral reducing N-glycans from chicken ovalbumin and thirteen individual neutral reducing oligosaccharides and eight individual sialylated ones from human milk, demonstrating its good applicability to different types of reducing glycans as well as biological samples. Given the compatibility of individual reducing glycans with almost all of glycan derivatization protocols and analytical techniques of glycans and the potential of the method for larger scale application, this work provides a universal approach to compound-specific analysis of natural glycans and has great significance for glycomics studies.
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Glicômica , Hidrazonas , Humanos , Cromatografia Líquida de Alta Pressão , Glicômica/métodos , Polissacarídeos/química , HidrazinasRESUMO
Metamaterials are artificial multifunctional materials that acquire their material properties from their structure, rather than inheriting them directly from the materials they are composed of, and they may provide novel tools to significantly enhance the sensitivity and resolution of sensors. In this paper, we derive the dispersion relation of a cylindrical dielectric waveguide loaded on a negative permeability metamaterial (NPM) layer, and compute the resonant frequencies and electric field distribution of the corresponding Whispering-Gallery-Modes (WGMs). The theoretical resonant frequency and electric field distribution results are in good agreement with the full wave simulation results. We show that the NPM sensor based on a microring resonator possesses higher sensitivity than the traditional microring sensor since with the evanescent wave amplification and the increase of NPM layer thickness, the sensitivity will be greatly increased. This may open a door for designing sensors with specified sensitivity.
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Técnicas Biossensoriais , Teste de Materiais , Algoritmos , Simulação por Computador , Computadores , Radiação Eletromagnética , Análise de Elementos Finitos , Modelos Estatísticos , Permeabilidade , Espalhamento de Radiação , Software , Propriedades de SuperfícieRESUMO
Boronate affinity materials are usually used for selective enrichment of cis-diol-containing compounds, mainly based on formation of pH-dependent cyclic ester between cis-diol and boronic acid. Recently, B-N coordination, or combined with hydrogen-bonding interaction, was employed as primary interaction for the extraction of nitrogen-containing compounds. However, there are no reports about the combination of hydrophobic (or π-π) interaction and B-N coordination for the extraction. Here, we prepared a novel hydrophobic phenyl-boronic acid polymer (PBAP) through initiator-free ring-opening polymerization. The adsorption experiment indicated that the PBAP could combine hydrophobic (or π-π) interaction and B-N coordination to enhance their adsorption capacity toward hydrophobic and nitrogen-containing compounds, for example sulfamethoxazole (SMX) and trimethoprim (TMP). In addition, the PBAP monolith synthesized in pipette tip was used as solid phase microextraction (SPME) sorbent with combination of ultra high performance liquid chromatography to extract and monitor SMX and TMP from animal-originated foodstuffs. The proposed method exhibited low limit of quantitation as 5.0 and 1.0 ng mL-1 for SMX and TMP, respectively. The recoveries at three spiked levels were between 92.4% to 100.5% for SMX, and 92.7% to 102.6% for TMP, with intra-day and inter-day relative standard deviations no more than 5.3% and 8.6%, respectively. These results well demonstrated that the combination of hydrophobic (or π-π) interaction and B-N coordination played an important role in the extraction of hydrophobic and nitrogen-containing compounds.
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Cromatografia Líquida de Alta Pressão/métodos , Resíduos de Drogas , Produtos da Carne/análise , Microextração em Fase Sólida/métodos , Sulfametoxazol , Trimetoprima , Ácidos Borônicos/química , Resíduos de Drogas/análise , Resíduos de Drogas/química , Resíduos de Drogas/isolamento & purificação , Polímeros/química , Sulfametoxazol/análise , Sulfametoxazol/química , Sulfametoxazol/isolamento & purificação , Trimetoprima/análise , Trimetoprima/química , Trimetoprima/isolamento & purificaçãoRESUMO
This article analyzes the external environments facing community health services (CHS) facilities that belong to state-owned enterprises (SOEs) and public services units (PSUs) in urban China. Semistructured and open-ended key informant interviews were conducted in six such CHS facilities in Wuhan City. A total of 45 chief directors and various other classifications of health professionals from CHS facilities participated in the interviews. Behavioral responses of local government authorities, immediate managerial agencies (SOEs and PSUs), other medical facilities, and the general public were revealed as the external influences impacting these kinds of CHS facilities. Conclusions arrived at were that these CHS facilities need sound external environments to fulfill their roles, including financial support from government and immediate agencies, professional guidance and support, appropriate referrals from other medical facilities, and the cooperation and confidence of the general public in the work of the facilities.
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Atitude do Pessoal de Saúde , Serviços de Saúde Comunitária/organização & administração , Governo Local , Programas Nacionais de Saúde/organização & administração , China , Política de Saúde , Humanos , Relações Interinstitucionais , Entrevistas como Assunto , Estudos de Casos OrganizacionaisRESUMO
Nitrile hydratase (NHase) from Rhodococcus rhodochrous J1 is widely used for industrial production of acrylamide and nicotinamide. However, the two types of NHases (L-NHase and H-NHase) from R. rhodochrous J1 were only slightly expressed in E. coli by routine methods, which limits the comprehensive and systematic characterization of the enzyme properties. We successfully expressed the two types of recombinant NHases in E. coli by codon-optimization, engineering of Ribosome Binding Site (RBS) and spacer sequences. The specific activity of the purified L-NHase and H-NHase were 400 U/mg and 234 U/mg, respectively. The molecular mass of L-NHase and H-NHase was identified to be 94 kDa and 504 kDa, respectively, indicating that the quaternary structure of the two types of NHases was the same as those in R. rhodochrous J1. H-NHase exhibited higher substrate and product tolerance than L-NHase. Moreover, higher activity and shorter culture time were achieved in recombinant E. coli, and the whole cell catalyst of recombinant E. coli harboring H-NHase has equivalent efficiency in tolerance to the high-concentration product relative to that in R. rhodochrous J1. These results indicate that biotransformation of nitrile by R. rhodochrous J1 represents a potential alternative to NHase-producing E. coli.
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Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidroliases/genética , Hidroliases/metabolismo , Rhodococcus/enzimologia , Rhodococcus/genética , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação/genética , Biotransformação , Códon , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/enzimologia , Expressão Gênica , Engenharia Genética , Hidroliases/síntese química , Hidroliases/isolamento & purificação , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Several randomized controlled trials (RCTs) have compared endoscopic and symptomatic relapses in patients with erosive gastroesophageal reflux disease (GERD). We have summarized current evidence for rabeprazole 10 or 20 mg once daily for GERD maintenance treatment over 1 or 5 years. METHODS: MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials were searched, through August 2012, for eligible RCTs of adults with erosive GERD. The efficacies of rabeprazole 10 and 20 mg/d were compared. RESULTS: The search identified 288 citations, and five RCTs containing 1480 patients were considered eligible. Heartburn relapse rates did not differ significantly between patients treated with rabeprazole 10 and 20 mg/d for 1 year (relative risk (RR) = 1.29; 95% confidence interval (CI): 0.97-1.72), but differed in patients treated for 5 years (RR = 1.274; 95% CI: 1.005-1.615). Endoscopic relapse rates differed significantly between rabeprazole 10 and 20 mg/d for 1 year (RR = 1.92; 95% CI: 1.21-3.06), for 5 years (RR = 1.667; 95% CI: 1.073-2.589), and in combined 1- and 5-year maintenance trials (RR = 1.785; 95% CI: 1.298-2.456). CONCLUSION: Rabeprazole 20 mg/d was superior to rabeprazole 10 mg/d in preventing endoscopic relapse of erosive GERD, but that the two dosages were equivalent in symptomatic relief over 1 year.