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BACKGROUND AND OBJECTIVE: The purpose of our study was to explore the immediate and long-term effects of socially assistive robots (SARs) on neuropsychiatric symptoms (NPSs), behavioral and psychological symptoms of dementia (BPSD), positive emotional experiences, and social interaction in older people living with dementia. METHODS: We set keywords and used Boolean operators to search the CINAHL, Cochrane Library, EMBASE, IEEE Digital Library, MEDLINE, PsycINFO, PubMed, Web of Science, Scopus, and Chinese Electronic Periodical Service from inception to February 2022 for randomized controlled trials. The Cochrane Collaboration bias assessment tool was used to assess article quality, and RevMan 5.4.1 software was used to conduct the meta-analysis. RESULTS: A total of 14 studies were included in the meta-analysis. SARs can help people living with dementia reduce their NPS of depression and anxiety, provide happiness from positive emotional experiences, and improve their social interaction through conversation. However, there was no significant improvement in agitation behavior, overall BPSD, or quality of life in people living with dementia. In follow-up, it was found that the effect of SRT was limited. CONCLUSION: SARs can reduce depression and increase positive emotions in people living with dementia. They may also reduce the burden on healthcare workers during the COVID-19 pandemic. This research was registered on PROSPERO CRD42020169340.
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COVID-19 , Demencia , Robótica , Humanos , Anciano , Demencia/terapia , Demencia/psicología , Calidad de Vida , Pandemias , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Antibiotic resistance has emerged as one of the most significant threats to global public health. Plasmids, which are highly efficient self-replicating genetic vehicles, play a critical role in the dissemination of drug-resistant genes. Previous studies have mainly focused on drug-resistant genes only, often neglecting the complete functional role of multidrug-resistant (MDR) plasmids in bacteria. In this study, we conducted a comprehensive investigation of the transcriptomes and proteomes of Escherichia coli J53 transconjugants harboring six major MDR plasmids of different incompatibility (Inc) groups, which were clinically isolated from patients. The RNA-seq analysis revealed that MDR plasmids influenced the gene expression in the bacterial host, in particular, the genes related to metabolic pathways. A proteomic analysis demonstrated the plasmid-induced regulation of several metabolic pathways including anaerobic respiration and the utilization of various carbon sources such as serine, threonine, sialic acid, and galactarate. These findings suggested that MDR plasmids confer a growth advantage to bacterial hosts in the gut, leading to the expansion of plasmid-carrying bacteria over competitors without plasmids. Moreover, this study provided insights into the versatility of prevalent MDR plasmids in moderating the cellular gene network of bacteria, which could potentially be utilized in therapeutics development for bacteria carrying MDR plasmids.
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Proteoma , Transcriptoma , Humanos , Proteoma/genética , Proteómica , Escherichia coli/genética , Plásmidos/genéticaRESUMEN
OBJECTIVES: Taxoid 10ß-O-acetyl transferase (DBAT) was redesigned to enhance its catalytic activity and substrate preference for baccatin III and taxol biosynthesis. RESULTS: Residues H162, D166 and R363 were determined as potential sites within the catalytic pocket of DBAT for molecular docking and site-directed mutagenesis to modify the activity of DBAT. Enzymatic activity assays revealed that the kcat/KM values of mutant H162A/R363H, D166H, R363H, D166H/R363H acting on 10-deacetylbaccatin III were about 3, 15, 26 and 60 times higher than that of the wild type of DBAT, respectively. Substrate preference assays indicated that these mutants (H162A/R363H, D166H, R363H, D166H/R363H) could transfer acetyl group from unnatural acetyl donor (e.g. vinyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate and isoamyl acetate) to 10-deacetylbaccatin III. CONCLUSION: Taxoid 10ß-O-acetyl transferase mutants with redesigned active sites displayed increased catalytic activities and modified substrate preferences, indicating their possible application in the enzymatic synthesis of baccatin III and taxol.
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Acetilesterasa/metabolismo , Histidina , Mutagénesis Sitio-Dirigida/métodos , Proteínas Recombinantes/metabolismo , Taxoides/metabolismo , Acetilesterasa/genética , Escherichia coli/genética , Histidina/genética , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Simulación del Acoplamiento Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Especificidad por Sustrato , Taxus/enzimología , Taxus/genéticaRESUMEN
BACKGROUND: Mental health professionals experiencing work-related stress may experience burn out, leading to a negative impact on their organization and patients. AIM: The aim of this study was to examine the effects of yoga classes on work-related stress, stress adaptation, and autonomic nerve activity among mental health professionals. METHODS: A randomized controlled trial was used, which compared the outcomes between the experimental (e.g., yoga program) and the control groups (e.g., no yoga exercise) for 12 weeks. Work-related stress and stress adaptation were assessed before and after the program. Heart rate variability (HRV) was measured at baseline, midpoint through the weekly yoga classes (6 weeks), and postintervention (after 12 weeks of yoga classes). RESULTS: The results showed that the mental health professionals in the yoga group experienced a significant reduction in work-related stress (t = -6.225, p < .001), and a significant enhancement of stress adaptation (t = 2.128, p = .042). Participants in the control group revealed no significant changes. Comparing the mean differences in pre- and posttest scores between yoga and control groups, we found the yoga group significantly decreased work-related stress (t = -3.216, p = .002), but there was no significant change in stress adaptation (p = .084). While controlling for the pretest scores of work-related stress, participants in yoga, but not the control group, revealed a significant increase in autonomic nerve activity at midpoint (6 weeks) test (t = -2.799, p = .007), and at posttest (12 weeks; t = -2.099, p = .040). LINKING EVIDENCE TO ACTION: Because mental health professionals experienced a reduction in work-related stress and an increase in autonomic nerve activity in a weekly yoga program for 12 weeks, clinicians, administrators, and educators should offer yoga classes as a strategy to help health professionals reduce their work-related stress and balance autonomic nerve activities.
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Ejercicio Físico/psicología , Salud Mental , Estrés Psicológico/terapia , Lugar de Trabajo/psicología , Yoga/psicología , Adulto , Femenino , Humanos , Masculino , Estrés Psicológico/prevención & controlRESUMEN
Many microfluidic chip-based LC-MS systems have been utilized for high-throughput analysis in various fields of bioanalytical applications such as proteomic, glycomic, pharmaceutical, and clinical research. This review is an update of a previous review article (Electrophoresis 2012, 33, 635-643) to mainly cover the most recent advancements in chip-based LC-MS for determining small molecules in bioanalysis. First, the different types of microfluidic chip devices for chip-based LC-MS analysis will be discussed. Following the discussion of the recent developments in the chip-based instrumentation, the applications of chip-based LC-MS for determining small molecules, such as glycans, pharmaceutical drugs, drugs of abuse, drug metabolites, and biomarkers in various biological sample matrixes will also be included in this review.
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Biomarcadores/análisis , Cromatografía Liquida , Espectrometría de Masas , Técnicas Analíticas Microfluídicas , Línea Celular Tumoral , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Humanos , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Preparaciones Farmacéuticas/análisis , Polisacáridos/análisisRESUMEN
Ten new prostaglandin derivatives (PGs), sarcoehrendins A-J (1-10), together with five known analogues (11-15) were isolated from the soft coral Sarcophyton ehrenbergi. Compounds 4-8 represented the first examples of PGs featuring an 18-ketone group. The structures including the absolute configurations were elucidated on the basis of spectroscopic analysis and chemical evidence. All of the isolates and six synthetic analogues (3a, 3b, 4a, and 11a-11c) were screened for inhibitory activity against phosphodiesterase-4 (PDE4), which is a drug target for the treatment of asthma and chronic obstructive pulmonary disease. Compounds 2, 10, 11a, 11b, and 13-15 exhibited inhibition with IC50 values less than 10 µM, and compound 15 (IC50 = 1.4 µM) showed comparable activity to the positive control rolipram (IC50 = 0.60 µM). The active natural PGs (2, 10, and 13-15) represent the first examples of PDE4 inhibitors without an aromatic moiety, and a preliminary structure-activity relationship is also proposed.
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Antozoos/química , Inhibidores de Fosfodiesterasa 4/aislamiento & purificación , Inhibidores de Fosfodiesterasa 4/farmacología , Prostaglandinas/aislamiento & purificación , Prostaglandinas/farmacología , Animales , China , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/efectos de los fármacos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Inhibidores de Fosfodiesterasa 4/química , Prostaglandinas/química , Rolipram/farmacología , Relación Estructura-ActividadRESUMEN
The development and integration of microfabricated liquid chromatography (LC) microchips have increased dramatically in the last decade due to the needs of enhanced sensitivity and rapid analysis as well as the rising concern on reducing environmental impacts of chemicals used in various types of chemical and biochemical analyses. Recent development of microfluidic chip-based LC mass spectrometry (chip-based LC-MS) has played an important role in proteomic research for high throughput analysis. To date, the use of chip-based LC-MS for determination of small molecules, such as biomarkers, active pharmaceutical ingredients (APIs), and drugs of abuse and their metabolites, in clinical and pharmaceutical applications has not been thoroughly investigated. This mini-review summarizes the utilization of commercial chip-based LC-MS systems for determination of small molecules in bioanalytical applications, including drug metabolites and disease/tumor-associated biomarkers in clinical samples as well as adsorption, distribution, metabolism, and excretion studies of APIs in drug discovery and development. The different types of commercial chip-based interfaces for LC-MS analysis are discussed first and followed by applications of chip-based LC-MS on biological samples as well as the comparison with other LC-MS techniques.
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Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Técnicas Analíticas Microfluídicas/métodos , Preparaciones Farmacéuticas/análisis , Animales , Cromatografía Liquida/instrumentación , Humanos , Espectrometría de Masas/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Nanotecnología/instrumentación , Preparaciones Farmacéuticas/sangre , Preparaciones Farmacéuticas/orina , Reproducibilidad de los ResultadosRESUMEN
Crystallinity and crystal orientation have a predominant impact on a materials' semiconducting properties, thus it is essential to manipulate the microstructure arrangements for desired semiconducting device performance. Here, ultra-uniform hole-transporting material (HTM) by self-assembling COOH-functionalized P3HT (P3HT-COOH) is fabricated, on which near single crystal quality perovskite thin film can be grown. In particular, the self-assembly approach facilitates the P3HT-COOH molecules to form an ordered and homogeneous monolayer on top of the indium tin oxide (ITO) electrode facilitate the perovskite crystalline film growth with high quality and preferred orientations. After detailed spectroscopy and device characterizations, it is found that the carboxylic acid anchoring groups can down-shift the work function and passivate the ITO surface, retarding the interface carrier recombination. As a result, the device made with the self-assembled HTM show high open-circuit voltage over 1.10 V and extend the lifetime over 4,300 h when storing at 30% relative humidity. Moreover, the cell works efficiently under much reduced light power, making it useful as power source under dim-light conditions. The demonstration suggests a new facile way of fabricating monolayer HTM for high efficiency perovskite devices, as well as the interconnecting layer needed for tandem cell.
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Chemokines orchestrate leukocyte migration toward sites of inflammation and infection and target leukocytes via chemokine receptors such as CCR6, a subfamily of the seven-transmembrane G-protein-coupled receptors. Lipid rafts are cholesterol and sphingolipid-enriched liquid-ordered membrane microdomains thought to serve as scaffolding platforms for signal transduction. To globally understand the dynamic changes of proteins within lipid rafts upon CCR6 activation in T cells, we quantitatively analyzed the time-dependent changes of lipid raft proteome using our recently reported membrane proteomics strategy combining gel-assisted digestion, iTRAQ labeling and LC-MS/MS. To our knowledge, the error-free identification of 852 proteins represents the first data set of the raft proteome in T cells upon chemokine receptor activation, including 354 previously annotated raft proteins and 85 dynamically recruited proteins that are potential raft-associated proteins. The temporal profiles revealed that many proteins involved in the actin cytoskeleton rearrangement are actively recruited into lipid rafts upon CCR6 activation. We further confirmed the proteomics results by Western blotting and used small interfering RNA-mediated knockdown to evaluate their roles upon CCR6 activation. In sum, we employed quantitative proteomic strategy to analyze raft proteome and identified many molecules actively involved in the control of actin assembly and disassembly regulating CCR6 activation-induced cell migration.
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Actinas/metabolismo , Citoesqueleto/metabolismo , Microdominios de Membrana/metabolismo , Proteómica/métodos , Receptores CCR6/metabolismo , Linfocitos T/metabolismo , Western Blotting , Quimiocina CCL20 , Quimiotaxis , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Células Jurkat , Microdominios de Membrana/química , Proteínas de Microfilamentos/metabolismo , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Microfluidic chip-based high-performance-liquid-chromatography coupled to mass spectrometry (chip-HPLC-MS) has been widely used in proteomic research due to its enhanced sensitivity. We employed a chip-HPLC-MS system for determining small molecules such as drug metabolites in biological fluids. This chip-HPLC-MS system integrates a microfluidic switch, a 2-dimensional column design including an enrichment column (160 nL) for sample pre-concentration and an analytical column for chromatographic separation, as well as a nanospray emitter on a single polyimide chip. In this study, a relatively large sample volume (500 nL) was injected into the enrichment column for pre-concentration and an additional 4 µL of the initial mobile phase was applied to remove un-retained components from the sample matrix prior to chromatographic separation. The 2-dimensional column design provides the advantages of online sample concentration and reducing matrix influence on MS detection. 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples using the integrated chip-HPLC-MS system. The linear range was 0.1-10 ng mL(-1) and the method detection limit (signal-to-noise ratio of 3) was 0.05 ng mL(-1) for 7-aminoFM2. After consecutive liquid-liquid extraction (LLE) and solid-phase extraction (SPE), the chip-HPLC-MS exhibited high correlation between 7-aminoFM2 spiked Milli-Q water and 7-aminoFM2 spiked urine samples. This system also showed good precision (n = 5) and recovery for spiked urine samples at the levels of 0.1, 1.0, and 10 ng mL(-1). Intra-day and inter-day precision were 2.0-7.1% and 4.3-6.0%, respectively. Clinical urine samples were also analyzed by this chip-HPLC-MS system and acceptable relative differences (-1.3 to -13.0%) compared with the results using a GC-MC method were determined. Due to its high sensitivity and ease of operation, the chip-HPLC-MS system can be utilized for the determination of small molecules such as drug metabolites and neurotransmitters in biological fluids for clinical diagnosis.
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Ansiolíticos/orina , Cromatografía Líquida de Alta Presión/métodos , Flunitrazepam/análogos & derivados , Técnicas Analíticas Microfluídicas/métodos , Espectrometría de Masas en Tándem/métodos , Ansiolíticos/aislamiento & purificación , Ansiolíticos/metabolismo , Flunitrazepam/aislamiento & purificación , Flunitrazepam/metabolismo , Flunitrazepam/orina , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Extracción en Fase SólidaRESUMEN
OBJECTIVE: To demonstrate laparoscopic surgery for a patient with unicornuate uterus and a large hematometra in the noncommunicating uterine horn. DESIGN: Narrated video featuring the diagnostic tests and surgical management. SETTING: Academic tertiary hospital. PATIENT(S): A 13-year-old woman, gravida 0 with menarche at 9 years old, had severe dysmenorrhea during her recent menstrual cycles. The results of transrectal three-dimensional sonography, hysteroscopy, and magnetic resonance imaging (MRI) were consistent for a uterine didelphys composed of a right unicornuate uterus and a left hemiuterus with hematometra and no connection to the cervix. Left adnexal endometrioma and ipsilateral renal agenesis were also noted. INTERVENTION(S): Laparoscopic adhesiolysis and enucleation of the left ovarian endometrioma were first performed. Then, implementing the concepts of Strassman unification method, a horizontal incision was made on the medial side of each hemicorpus, starting from the left rudimentary horn and ending at the fundal area of the right hemiuterus. The incision was carried deep enough to reach the endometrium. The opposing, reverted myometrium was then reapproximated from the lower end of the uterus to the upper fundal part in two layers, using absorbable bidirectional monofilament barbed suture. The outer serosal layer was closed with 1-0 Monocryl via a running continuous suture. On postoperative day 2, the patient was discharged uneventfully. MAIN OUTCOME MEASURE(S): Symptomatic relief and restoration of normal and functional anatomy. RESULT(S): The patient has regular menstrual cycles with no recurrence of dysmenorrhea or ovarian endometrioma. At a 2-year postoperative follow-up evaluation, hysteroscopic examination revealed a single uterine cavity with a small fundal septum. The MRI scans showed an anteverted, symmetrical uterus. CONCLUSION(S): In comparison with the widely applied hemihysterectomy, reconstructive laparoscopic metroplasty not only effectively achieves symptomatic relief but also offers better uterine symmetry and a larger intrauterine cavity via unification of the uterus.
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Laparoscopía/métodos , Procedimientos de Cirugía Plástica/métodos , Anomalías Urogenitales/cirugía , Útero/anomalías , Útero/cirugía , Adolescente , Dismenorrea/diagnóstico por imagen , Dismenorrea/cirugía , Femenino , Humanos , Anomalías Urogenitales/diagnóstico por imagen , Útero/diagnóstico por imagenRESUMEN
Poly(1-vinylimidazole-co-ethylene dimethacrylate) (VIM-EDMA) monolithic stationary phase was applied for separation of inorganic anions by capillary ion chromatography (IC). The retention of inorganic anions on the VIM-EDMA column was investigated using various salt solutions as the eluent. Good separation of inorganic anions was obtained on VIM-EDMA monolithic column using NH4Cl as the eluent. Good mechanical stability and low swelling propensity values (0.12 and -0.02 for ACN and MeOH, respectively) were obtained. The column repeatability was also examined by determining the relative standard deviations (RSDs) of retention time and peak area of target anions. Relatively low RSDs (nâ¯=â¯7) of retention time (<2.3%) and peak area (<8.8%) were obtained on the VIM-EDMA column. The utilization of VIM-EDMA column for potential environmental application was also demonstrated by determining the occurrence of inorganic anions in various environmental water samples without sample preparation process.
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Simultaneous determination of nitrate (NO3â¾) and nitrite (NO2â¾) in vegetables was performed on a poly(1-vinylimidazole-co-ethylene dimethacrylate) (VIM-EDMA) monolithic column by capillary liquid chromatography (LC) with UV detection. Good linearity (0.5-100⯵gâ¯mL-1 i.e. 12.5 -2500⯵gâ¯g-1 in vegetables) was obtained with coefficient of determination > 0.996. Limits of detection (signal-to-noise ratio: S/N= 3) were estimated at 0.06 and 0.05⯵gâ¯mL-1, which corresponded to 1.50 and 1.25⯵gâ¯g-1 for NO2â¾ and NO3â¾, respectively, in vegetables. The limits of quantification (S/N= 10) were estimated at 0.17 and 0.16⯵gâ¯mL-1 (4.25 and 4.00⯵gâ¯g-1 in vegetables) for NO2â¾ and NO3â¾, respectively. Although the detection limits were relatively higher than other LC-UV techniques, this proposed method offered satisfactory sensitivity for complying the Acceptable Daily Intake (ADI) levels set by EU to monitor the occurrence of NO2â¾ and NO3â¾in vegetables. The intra-day/inter-day precision (0.14-3.35%/0.06-6.93%) and accuracy (90.33-103.32%/96.00-101.26%) were also examined for method validation. No obvious carry-over and decline of separation efficiency were observed for more than 200 analyses of real samples. The occurrence of NO2â¾ and NO3â¾in various vegetable samples was investigated to demonstrate the potential of utilizing the developed polymeric monolith-based capillary LC-UV method for food safety application.
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Cromatografía Liquida/métodos , Análisis de los Alimentos/métodos , Imidazoles/química , Metacrilatos/química , Nitratos/análisis , Nitritos/análisis , Polietilenglicoles/química , Polivinilos/química , Verduras/química , Cloruro de Amonio/química , Inocuidad de los Alimentos , Límite de Detección , Reproducibilidad de los Resultados , Rayos UltravioletaRESUMEN
BACKGROUND AND AIM: Infection or bleeding after transrectal prostate biopsy remains a concern of both patients and urologists. We explored the risk of association of certain co-morbidities with both complications. PATIENTS AND METHODS: Using the Taiwan National Health Insurance Research Database, we identified patients undergoing prostate biopsy from 2000 to 2013. We used logistic multivariable regression to search for associations between post-biopsy hospitalization and the two co-morbidities within a year after biopsy. RESULTS: Among 3,601 prostate biopsies, 100 infections (3.77%) and 52 (1.44%) bleeding-related emergency room visits and hospitalizations were recorded within 30 days after biopsy. The group having the biopsy as an inpatient exhibited older age (p < 0.0001) and a higher percentage of having diabetes mellitus (p = 0.015) than patients without either complication. The logistic multivariable regression analysis showed that urinary retention, freedom from diabetes, and performance as an outpatient procedure were independent risk factors for infection-related hospitalization (odds ratios 1.81, 1.96, and 1.72; p values 0.031, 0.037, and 0.010, respectively). CONCLUSION: Patients with a recent history of urinary retention have a higher probability of infection-related hospitalization after prostate biopsy.
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Biopsia/efectos adversos , Complicaciones Posoperatorias/epidemiología , Enfermedades de la Próstata/diagnóstico , Infección de la Herida Quirúrgica/epidemiología , Retención Urinaria/complicaciones , Anciano , Anciano de 80 o más Años , Biopsia/métodos , Estudios de Cohortes , Hemorragia/epidemiología , Hospitalización/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Taiwán/epidemiologíaRESUMEN
In this study, we applied 1-vinylimidazole (VIM) as the functional monomer to prepare a series of VIM-based monolithic stationary phases for both reversed-phase and hydrophilic interaction capillary liquid chromatography (LC) using various dimethacrylates (EDMA: ethylene dimethacrylate; HDDMA: 1,6-hexanediol dimethacrylate; DDDMA: 1,10-decanediol dimethacrylate) as cross-linkers. With a simple thermally initiated free-radical cross-linking polymerization process, VIM-based monolithic stationary phases have been successfully prepared. The porosity, permeability, and column efficiency of synthesized VIM-based monolithic stationary phases were characterized. With similar total porosity (85-90%), the VIM-HDDMA monoliths showed the lowest permeability among the three sets of VIM-based stationary phases. Various sets of non-polar (alkyl benzenes and polycyclic aromatic hydrocarbons) and polar analytes (phenol derivatives and amphenicol antibiotics) were applied as model compounds to further investigate the retention behavior of the VIM-based monolithic stationary phases for reversed-phase capillary LC analysis using selected VIM-based monolithic columns. While a mixture of organic acids was employed to perform HILIC analysis using the selected VIM-based monolithic columns. The separation selectivity and retention behavior of the VIM-based monolithic stationary phases were compared to those obtained using three previously prepared alkyl methacrylate-based monolithic columns. Strong retention and good resolution of polar analytes (such as phenol derivatives, amphenicol antibiotics, and organic acids) were observed using the selected VIM-based monolithic columns. The strong retention and good resolution might be attributed to the additional hydrogen-bonding between the hydrogen-donating analytes and the hydrogen-accepting imidazolium functionality on the VIM-based stationary phase. The applicability for both reversed-phase and HILIC capillary LC analysis has also been demonstrated using the selected VIM-based monolithic columns.
RESUMEN
Using a simple liquid-liquid extraction (LLE) procedure for sample pretreatment, 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The linearity was found in the range of 0.1-50ngmL-1 with a method detection limit (signal-to-noise ratio of 3) estimated at 0.05ngmL-1. Using the proposed method, good precision and recovery were also found in spiked urine samples at the levels of 0.5, 5.0, and 50ngmL-1 (intra-day/inter-day precision: 0.6-1.8% / 0.1-0.8%; post-spiked/pre-spiked recovery: 95.4-102.9% / 96.3-102.5%). In addition, acceptable relative differences (-24.2 - 0.8%) were observed by analyzing clinical urine samples using this monolith-based capillary LC-MS/MS method compared with the results obtained by the routine GC-MC method. Using the monolithic column, no noticeable deterioration of separation efficiency or carry-over was observed for more than 200 injections of urine samples. The applicability of the developed monolith-based capillary LC-MS/MS method was demonstrated by quantifying 7-aminoFM2 in various clinical urine samples. Based on these experimental results, the proposed LLE-monolith-based capillary LC-MS/MS method shows the potential for routine determination of drug metabolites in human urine for clinical and forensic applications.
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Cromatografía Liquida/métodos , Flunitrazepam/análogos & derivados , Espectrometría de Masas en Tándem/métodos , Flunitrazepam/química , Flunitrazepam/orina , Humanos , Extracción Líquido-Líquido , Metacrilatos/química , Polímeros/químicaRESUMEN
Natural production of anti-cancer drug taxol from Taxus has proved to be environmentally unsustainable and economically unfeasible. Currently, bioengineering the biosynthetic pathway of taxol is an attractive alternative production approach. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) was previously characterized as an acyltransferase, using 10-deacetylbaccatin III (10-DAB) and acetyl CoA as natural substrates, to form baccatin III in the taxol biosynthesis. Here, we report that other than the natural acetyl CoA (Ac-CoA) substrate, DBAT can also utilize vinyl acetate (VA), which is commercially available at very low cost, acylate quickly and irreversibly, as acetyl donor in the acyl transfer reaction to produce baccatin III. Furthermore, mutants were prepared via a semi-rational design in this work. A double mutant, I43S/D390R was constructed to combine the positive effects of the different single mutations on catalytic activity, and its catalytic efficiency towards 10-DAB and VA was successfully improved by 3.30-fold, compared to that of wild-type DBAT, while 2.99-fold higher than the catalytic efficiency of WT DBAT towards 10-DAB and Ac-CoA. These findings can provide a promising economically and environmentally friendly method for exploring novel acyl donors to engineer natural product pathways.