RESUMEN
Background: Prescribing DOACs presents with challenges in the elderly and patients with renal and hepatic impairment. To mitigate safety risks, pharmacists have a role in detection, prevention, and resolution of DOAC-associated drug-related problems (DRPs). Objectives: To identify the types of DOAC-associated DRPs in patients on DOAC therapy and factors that predispose patients to DOAC-associated DRPs. Methods: An observational cross-sectional study was conducted in SGH from January 1, 2017, to May 31, 2019, on patients prescribed with a DOAC (rivaroxaban, dabigatran, and apixaban). Data were electronically extracted for patient demographics, clinical characteristics, and details of DOAC-related DRPs identified by pharmacists. Matching of DRP group to non-DRP group at a ratio of 1:2 based on gender, race, and DOAC was performed. The DRP group included patients with detected DRPs while non-DRP group included patients without them. Descriptive analysis was used to summarize patient characteristics and types of DOAC-associated DRPs. In the matched population, conditional logistic regression was used to calculate unadjusted (UOR) and adjusted odds (AOR) ratio to detect association of DOAC-associated DRPs with age, renal function, ≥2 comorbidities, and DOAC indication (atrial fibrillation [AF] vs venous thromboembolism). Results: A total of 8432 patients prescribed DOACs were analyzed, which consisted of 827 (9.8%) and 7602 (90.2%) patients with DRPs and no DRPs, respectively. The top DOAC-associated DRP was inappropriate drug regimen (n = 487, 60.1%). After matching, 2403 patients were analyzed, consisting of 801 patients from DRP group and 1602 from non-DRP group. Factors associated with DOAC-associated DRPs were statistically significant for renal function at creatinine clearance (CrCl) of >30 to 50 mL/min/1.73 m2 (AOR: 1.42; 95% CI: 1.14-1.76; P = .002), 15 to 30 mL/min/1.73 m2 (OR: 1.94; 95% CI: 1.42-2.66; P < .001), and <15 mL/min/1.73m2 (OR: 2.35; 95% CI: 1.13-4.88; P = .022), respectively, compared with a CrCl of >50 mL/min/1.73 m2 and DOAC indication for AF (AOR: 1.84; 95% CI: 1.47-2.30; P < .001) compared with venous thromboembolism. Conclusion: Inappropriate drug regimen was the most common DOAC-associated DRP. Impaired renal function and patients with AF increased the likelihood of DOAC-associated DRPs.
RESUMEN
AIMS: To validate host-specific Bacteroidales assays to identify faecal-source contamination of drinking water sources in the Kathmandu Valley, Nepal. METHODS AND RESULTS: A total of 54 composite faecal-source samples were collected from human sewage, ruminants, pigs, dogs, chickens and ducks, which were analysed by quantitative polymerase chain reaction using human-specific (BacHum, HF183 SYBR, gyrB and HF183 TaqMan), ruminant-specific (BacCow and BacR), pig-specific (Pig2Bac and PF163) and dog-specific assays (BacCan SYBR). The BacHum, BacR and Pig2Bac assays were judged the best performing human-specific, ruminant-specific and pig-specific assays respectively. The BacCan SYBR assay highly cross-reacted with other species, resulting in poor performance. Furthermore, these validated assays were applied to microbial source tracking (MST) of 74 drinking water samples. Out of these, 20, 12 and 4% samples were judged contaminated by human, ruminant and pig faeces respectively. Detection ratios of human and ruminant faecal markers were relatively higher in built-up and agricultural areas respectively. CONCLUSION: BacHum, BacR and Pig2Bac assays were found suitable for MST and both, human and animal faecal contaminations of drinking water sources were common in the valley. SIGNIFICANCE AND IMPACT OF THE STUDY: MST could be an effective tool for preparing the faecal pollution strategies as these are site specific.
Asunto(s)
Bacteroidetes/genética , ADN Bacteriano , Agua Potable/microbiología , Reacción en Cadena de la Polimerasa/métodos , Animales , ADN Bacteriano/análisis , ADN Bacteriano/genética , Perros , Patos , Heces/microbiología , Humanos , Nepal , Reproducibilidad de los Resultados , PorcinosRESUMEN
Retinoic acid (RA) receptor (RAR) agonists are potential teratogens to various vertebrates. Their contamination has been detected in municipal wastewater in different countries. This study involved field investigations and laboratory batch treatment experiments to elucidate the removal characteristics by activated sludge treatment of RAs (all-trans RA and 13-cis RA) and 4-oxo-RAs (4-oxo-all-trans RA and 4-oxo-13-cis RA), which were identified as major RAR agonists in municipal wastewater. Results obtained in this study show that currently employed activated sludge treatments can remove RAs, 4-oxo-RAs and overall RAR agonist contamination effectively from municipal wastewater in general, although high RAR agonistic activity might sometimes remain in the effluent. Laboratory experiments revealed that RAs were removed rapidly from the aqueous phase by adsorption to the sludge, after which they were removed further by biological and/or chemical degradation. Aside from adsorption to the sludge, 4-oxo-RAs were also apparently removed by biological and chemical degradation. Biodegradation contributed greatly to the removal. Results of additional experiments indicated that novel non-identifiable RAR agonists can occur through the biodegradation of 4-oxo-RAs by activated sludge and that they can persist for a long period.
Asunto(s)
Aguas del Alcantarillado/química , Tretinoina/análogos & derivados , Eliminación de Residuos Líquidos/métodos , Factores de Tiempo , Tretinoina/química , Contaminantes Químicos del Agua/químicaAsunto(s)
Receptores de Ácido Retinoico/agonistas , Tretinoina/análogos & derivados , Eliminación de Residuos Líquidos , Contaminantes Químicos del Agua/toxicidad , Animales , Humanos , Japón , Aguas del Alcantarillado , Tretinoina/análisis , Tretinoina/toxicidad , Técnicas del Sistema de Dos Híbridos , Contaminantes Químicos del Agua/análisisRESUMEN
OBJECTIVES: Various forms of adrenocortical insufficiency can cause musculoskeletal symptoms such as muscle pain, tautness of the limbs, arthralgia, and flexion contractures. However, the findings of neurological investigations are inconclusive and have not been well summarized. METHODS: We report the case of a 61-year-old man with isolated adrenocorticotropic hormone deficiency who presented with musculoskeletal symptoms, including flexion contractures. We performed three neurological investigations: nerve conduction studies, electromyography, and muscle biopsy analysis. Further, we reviewed reports of 16 patients with various forms of adrenocortical insufficiency and musculoskeletal symptoms by considering the findings of these three investigations. RESULTS: From the literature review, we found that (a) analysis of muscle biopsy is the most sensitive technique, followed by electromyography and then nerve conduction studies; and (b) the longer the duration of the musculoskeletal symptoms, the greater the incidence of abnormal findings with all three techniques. CONCLUSIONS: Physicians may prioritize neurological investigations, depending on these findings.
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Enfermedad de Addison/complicaciones , Enfermedades Musculoesqueléticas/diagnóstico , Enfermedades Musculoesqueléticas/etiología , Enfermedad de Addison/diagnóstico , Enfermedad de Addison/fisiopatología , Adulto , Anciano , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Enfermedades Musculoesqueléticas/fisiopatologíaRESUMEN
In this study, the microbial community structure and carbon source utilisation profile of activated sludge samples collected from full-scale municipal wastewater treatment plants (WWTPs) operated under different conditions were characterised and compared, respectively, using terminal-restriction fragment length polymorphism (T-RFLP) analysis and Biolog assay. Samples were collected from each biological treatment tank of six conventional activated sludge, two anaerobic-oxic, two anaerobic-anoxic-oxic, and one step-aeration processes in eight full-scale WWTPs in Osaka, Japan. Results of the T-RFLP analysis of eubacterial 16S rDNA showed that microbial communities of activated sludge differed greatly among samples, and that they were affected by process-based operational conditions. In contrast, the carbon source utilisation profiles of activated sludge samples were mutually similar, but appeared to be influenced slightly by aerated conditions at each reaction tank. Similar carbon source utilisation profiles among all samples suggest that the activated sludge community possesses functions that are necessary for wastewater treatment even if the phylogenetic composition is different. Different results from the T-RFLP analysis and Biolog assay suggest that the phylogenetic composition of microbial community might not necessarily reflect the wastewater treatment functions of the activated sludge.
Asunto(s)
Aguas del Alcantarillado/microbiología , Eliminación de Residuos Líquidos/métodos , Aerobiosis , Anaerobiosis , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción/genética , ARN Ribosómico 16S/genética , Microbiología del AguaRESUMEN
An energy consumption model was developed for evaluating sewage sludge treatment plants (SSTPs) incorporating various treatment processes such as thickening, anaerobic digestion, dewatering, incineration, and melting. Based on data analyses from SSTPs in Osaka, Japan, electricity consumption intensities for thickening, anaerobic digestion, dewatering, incineration, and melting and heat consumption intensities for anaerobic digestion, incineration, and melting were expressed as functions of sludge-loading on each unit process. The model was applied for predicting the energy consumption and greenhouse gas (GHG) emissions of SSTPs using various treatment processes and power and heat generation processes using digestion gas. Results showed that SSTPs lacking incineration and melting processes but having power generation processes showed excess energy production at the high sludge-loading rate. Energy consumption of the SSTPs without incineration and melting processes were low, but their GHG emissions were high because of CH(4) and N(2)O emissions from sludge cake at the landfill site. Incineration and melting processes consume much energy, but have lower CH(4) and N(2)O emissions.
Asunto(s)
Conservación de los Recursos Energéticos , Efecto Invernadero , Modelos Químicos , Aguas del Alcantarillado/química , Eliminación de Residuos Líquidos/métodos , Modelos Teóricos , Eliminación de Residuos Líquidos/instrumentaciónRESUMEN
Retinoic acid receptor (RAR) is a nuclear receptor involved in vertebrate morphogenesis, growth, cellular differentiation, and tissue homeostasis. Excess expression of the retinoid signaling can cause various developmental toxicities in animals and humans. We previously found that influents from sewage treatment plants (STPs) in Japan had a RAR agonistic activity and the activity cannot be removed completely by conventional biological treatments. In this study, we assessed the performance of chemical and physical sewage treatment technologies-ozonation, ultraviolet treatment, chlorination, coagulation using polyaluminium chloride (PAC) and ferric sulfate, and filtration with ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO) membranes-in removal of RAR agonistic activity of STP effluent. All water treatment experiments were conducted in laboratory-scale reactors. The RAR agonistic activity of samples was measured using a yeast two-hybrid assay. Results showed that the effectiveness of tested technologies on the removal of RAR agonistic activity can be ranked as RO or NF > chlorination > ozonation > MF > UV > coagulation with ferric sulfate>>coagulation with PAC. Furthermore, the effectiveness of chlorination might rank lower because excess reaction might bring a side effect by producing some RAR agonistic by-product(s).
Asunto(s)
Receptores de Ácido Retinoico/agonistas , Aguas del Alcantarillado , Eliminación de Residuos Líquidos/métodos , Contaminantes Químicos del Agua/análisis , Purificación del Agua/métodos , Filtración , Halogenación , Aguas del Alcantarillado/análisis , Rayos Ultravioleta , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/aislamiento & purificaciónRESUMEN
The aim of this study was to assess the availability of the biodegradation potential of aniline and phenol as the indicator for evaluating pollutant impact on a river environment. Biodegradation tests employing river water microorganisms were carried out by a modified TOC-Handai method using aniline and phenol as substrates. Complete degradation time and half-life were determined as indicators expressing the biodegradation potential of aniline and phenol, respectively. Investigations in Lake Biwa-Yodo River basin for more than two years showed that the biodegradation potential of both compounds varied seasonally. In addition, aniline biodegradation potential seemed to be influenced by the hydraulic retention time at each sampling station, while downstream stations with large input of wastewater from the surrounding cities were divided from upstream stations by phenol biodegradation potential. Comparison of the biodegradation potential in rivers at different pollution levels also showed that polluted and less polluted rivers were clearly divided by phenol biodegradation potential. These results indicated that phenol biodegradation potential can be applied as an indicator for evaluating the soundness of river environment from the view point of ecological function.
Asunto(s)
Biodegradación Ambiental , Ríos , Microbiología del Agua , Contaminantes Químicos del Agua/metabolismo , Compuestos de Anilina/metabolismo , Geografía , Japón , Fenol/metabolismoRESUMEN
DNA microarray mounted 85 functional gene sequences related to carbon, nitrogen and sulphur cycles, chemical degradation, metal metabolisms, and energy flows was developed to evaluate the function and status of the environment. Total of 24 river water samples from 6 sampling stations in 2 rivers in 4 seasons were analyzed using constructed DNA microarray. The numbers and constitution of the functional genes were much affected by the seasonal change. Some of the functional genes related to methane oxidation, nitrite reduction, nitrogen fixation, aromatic compounds degradation (catechol 2,3-dioxygenase), alkane degradation (group I and III) and iron reduction were detected in most of all the samples, suggesting that these could be the general functions of the river environment. Some other functional genes related to ammonium oxidation, aromatic compounds degradation (catechol 1,2-dioxygenase) and alkane degradation (group II) can be a certain indicator for the evaluation of the environmental condition.
Asunto(s)
Monitoreo del Ambiente/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Ríos , Alcanos/metabolismo , Secuencia de Bases , Biodegradación Ambiental , Carbono/metabolismo , Catecol 1,2-Dioxigenasa/metabolismo , Hidrocarburos Aromáticos/metabolismo , Metales/metabolismo , Nitritos , Nitrógeno/metabolismo , Oxidación-Reducción , Compuestos de Amonio Cuaternario/metabolismo , Estaciones del Año , Azufre/metabolismo , Microbiología del Agua , Contaminantes Químicos del AguaRESUMEN
The presence of arsenic in groundwater has been of great public concern because of its high toxicity. For purification of arsenic-contaminated groundwater, bacterial oxidation of arsenite, As(III), with a chemical adsorption process was examined in this study. After As(III) oxidation to arsenate, As(V), arsenic is easily removable from contaminated groundwater because As(V) is more adsorptive to absorbents than As(III). By acclimation to As(III) of high concentrations, a mixed culture of heterotrophic bacteria with high As(III)-oxidizing activity was obtained from a soil sample that was free from contamination. With initial concentration up to 1,500 mg l(-1) As(III), the mixed culture showed high As(III)-oxidizing activity at pH values of 7-10 and at temperatures of 25-35 degrees C. The mixed culture contained several genera of heterotrophic As(III)-oxidizing and arsenic-tolerant bacteria: Haemophilus, Micrococcus, and Bacillus. Activated alumina was added to the basal salt medium containing 75 mg l(-1) As(III) before and after bacterial oxidation. Arsenic removal by activated alumina was greatly enhanced by bacterial oxidation of As(III) to As(V). The isotherms of As(III) and As(V) onto activated alumina verified that bacterial As(III) oxidation is a helpful pretreatment process for the conventional adsorption process for arsenic removal.
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Arsénico/metabolismo , Bacterias Anaerobias/metabolismo , Microbiología del Agua , Contaminantes Químicos del Agua/metabolismo , Arsénico/aislamiento & purificación , Bacterias Anaerobias/crecimiento & desarrollo , Biodegradación Ambiental , Agua Dulce/análisis , Agua Dulce/microbiología , Oxidación-Reducción , Contaminantes Químicos del Agua/aislamiento & purificaciónRESUMEN
Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25 microg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.
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Azidas/química , ADN Bacteriano/genética , Viabilidad Microbiana/genética , Reacción en Cadena de la Polimerasa/métodos , Técnicas Bacteriológicas/métodos , ADN Bacteriano/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Modelos TeóricosRESUMEN
Fermentation modes and microorganisms related to two typical free-floating aquatic plants, water hyacinth and water lettuce, were investigated for their use in ethanol production. Except for arabinose, sugar contents in water lettuce resembled those in water hyacinth leaves. Water lettuce had slightly higher starch contents and lower contents of cellulose and hemicellulose. A traditional strain, Saccharomyces cerevisiae NBRC 2346, produced 14.4 and 14.9 g l(-1) ethanol, respectively, from water hyacinth and water lettuce. Moreover, a recombinant strain, Escherichia coli KO11, produced 16.9 and 16.2 g l(-1) ethanol in the simultaneous saccharification and fermentation mode (SSF), which was more effective than the separated hydrolysis and fermentation mode (SHF). The ethanol yield per unit biomass was comparable to those reported for other agricultural biomasses: 0.14-0.17 g g-dry(-1) for water hyacinth and 0.15-0.16 g g-dry(-1) for water lettuce.
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Araceae/metabolismo , Productos Agrícolas/metabolismo , Eichhornia/metabolismo , Etanol/metabolismo , Biomasa , FermentaciónRESUMEN
There is a group of compounds structurally similar to bisphenol-A (BPA), namely bisphenols (BPs), and some of them are considered to be able to partially replace BPA. In order to assess their biodegradability in the aquatic environment, a variety of BPs; BPA, bis(4-hydroxyphenyl)methane (BPF), bis(4-hydroxyphenyl)ethane (BPE), 2,2-bis(4-hydroxy-phenyl)butane (BPB), 2,2-bis(4- hydroxy-3-methylphenyl)propane (BPP), bis(4-hydroxyphenyl)sulfone (BPS), thiodiphenol (TDP) and 4,4'-dihydroxybenzophenone (HBP); were subjected to biodegradation tests under both aerobic and anaerobic conditions. For the aerobic degradation test, a kind of river-die-away method using several river water samples was used, while pond sediments were used for the anaerobic degradation tests in sealed anoxic bottles. As a whole, the examined BPs could be ranked by their biodegradability under aerobic conditions; BPF, HBP > > BPA > BPP > BPE > BPB > TDP > > BPS. On the other hand, the tendency for the anaerobic biodegradability was; BPF > HBP > BPS, BPA, TDP > BPE > BPB. From the viewpoint of biodegradability, BPF seems to be more environmentally-friendly than BPA and, therefore, may be a candidate to replace BPA for reducing the environmental risks.
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Bacterias Aerobias/metabolismo , Bacterias Anaerobias/metabolismo , Biodegradación Ambiental , Fenoles/química , Purificación del Agua/métodos , Compuestos de Bencidrilo , Monitoreo del Ambiente , Contaminantes Ambientales , Modelos Químicos , Ríos/química , Factores de Tiempo , Microbiología del Agua , Contaminantes Químicos del AguaRESUMEN
Two different types of hyaluronic acid (HA) hydrogels were synthesized by crosslinking HA with divinyl sulfone (DVS) and poly(ethylene glycol)-divinyl sulfone (VS-PEG-VS). Vitamin E succinate (VES), an anti-inflammatory drug, and bovine serum albumin (BSA), a model of anti-inflammatory protein drugs, were loaded into the gels and their release kinetics were measured in vitro. VES and BSA released with a burst from both HA hydrogels during the first few hours, and release continued gradually for several days. The rate of release from HA-VS-PEG-VS-HA hydrogels was faster than that from HA-DVS-HA hydrogels, presumably due to the lower crosslink density in the former. The anti-inflammatory action of released VES was tested by incubating peripheral blood mononuclear cells (PBMC) on HA hydrogels with and without VES in the gel. The number of cells adhering on HA hydrogels was very low compared to that on tissue culture polystyrene (TCPS), which might be one of the important advantages of using HA hydrogels for implant coatings or tissue engineering applications. ELISA test results showed that the tumor necrosis factor-alpha (TNF-alpha) concentration was very low in the supernatant of the wells containing the HA hydrogel with VES in contact with the activated macrophages compared to that without VES. This is probably the effect of the released VES reducing the production of anti-inflammatory cytokine, TNF-alpha. HA hydrogels containing anti-inflammatory drugs may have potential for use in tissue engineering and also as biocompatible coatings of implants.
Asunto(s)
Antiinflamatorios no Esteroideos/administración & dosificación , Materiales Biocompatibles/síntesis química , Hidrogeles/síntesis química , Vitamina E/análogos & derivados , Animales , Materiales Biocompatibles/química , Bovinos , Reactivos de Enlaces Cruzados , Sistemas de Liberación de Medicamentos , Humanos , Ácido Hialurónico/química , Hidrogeles/química , Técnicas In Vitro , Activación de Macrófagos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Estructura Molecular , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Albúmina Sérica Bovina/administración & dosificación , Sulfonas/química , Tocoferoles , Vitamina E/administración & dosificaciónRESUMEN
Pathologic calcification is the leading cause of the clinical failure of glutaraldehyde-fixed bovine pericardium used in bioprosthetic valves. A novel surface modification of glutaraldehyde fixed bovine pericardium was carried out with high molecular weight hyaluronic acid (HA). HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional groups onto the HA backbone. Glutaraldehyde-fixed bovine pericardium (GFBP) was modified by grafting this HA to the free aldehyde groups on the tissue via the hydrazide groups. Following a 2-week subcutaneous implantation in osteopontin (OPN)-null mice, the calcification of HA-modified bovine pericardium was drastically reduced (by 84.5%) compared to positive controls (tissue without HA-modification) (p = 0.005). The calcification-mitigating effect of HA surface modification was also confirmed by microscopic analysis of explanted tissue stained with Alizarin Red S for calcium.
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Bioprótesis , Calcinosis/prevención & control , Prótesis Valvulares Cardíacas , Pericardio/trasplante , Animales , Materiales Biocompatibles/química , Bioprótesis/efectos adversos , Bovinos , Femenino , Fijadores , Glutaral , Prótesis Valvulares Cardíacas/efectos adversos , Humanos , Ácido Hialurónico/química , Ensayo de Materiales , Ratones , Ratones Noqueados , Osteopontina , Sialoglicoproteínas/deficiencia , Sialoglicoproteínas/genéticaRESUMEN
Five orange pigmented filamentous strains of Eikelboom's type 1851 were isolated from bulking activated sludge, and were examined for their phylogenetic lineage, morphology, and physiology. The analysis of 16S rDNA sequence revealed that the isolates belong to Chloroflexus subdivision in green non sulfur bacteria. They shared most common features with type 1851, but the result of gram stain was negative. Furthermore, they showed polymorphic nature and gliding motility, which could not be seen in activated sludge samples. General physiological tests were conducted under aerobic conditions although they could also grow by fermentation. Good growth was generally seen on sugars. The extremely slow growth rate (0.48-0.93 day(-1)) suggests the stimulation of this type exclusively in long SRT systems fed with sugars.
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Chlorobi/aislamiento & purificación , Chlorobi/fisiología , Aguas del Alcantarillado/microbiología , Biomasa , Metabolismo de los Hidratos de Carbono , Fermentación , Dinámica Poblacional , Eliminación de Residuos LíquidosRESUMEN
For rapid and sensitive detection of poly(3-hydroxybutyrate) (PHB)-degrading bacteria, a PCR primer set (PHB primers) and a gene probe (PHB probe) were designed, based on the homologous regions of six fibronectin type III linker domain-encoding sequences laid on a variety of PHB depolymerase genes listed in the GenBank. PCR using PHB primers amplified DNA fragments with the expected sizes from all the tested bacterial strains used for primer design; and all of the amplified fragments gave positive signals by Southern hybridization with the PHB probe. No amplified fragments were observed from negative controls. To evaluate the availability of the PHB primers and PHB probe, they were applied to 57 wild-type, PHB-degrading bacteria newly isolated from a variety of environments. The PHB primers amplified DNA fragments with expected sizes from 50 of the 57 wild-type strains, while the PHB probe showed positive signals against the amplified fragments from 47 strains. These results suggest that the primer and probe system established in this study can detect a considerable proportion of the potential PHB-degrading bacteria and can be applied to evaluate PHB-degradation potential in a natural environment, in combination with direct DNA extraction methods.
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Bacterias/aislamiento & purificación , Bacterias/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Reacción en Cadena de la Polimerasa , Bacterias/genética , Biodegradación Ambiental , Southern Blotting , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Cartilla de ADN , Sondas de ADN , Fibronectinas/química , Genes Bacterianos , Estructura Terciaria de ProteínaRESUMEN
alpha1-Acid glycoprotein (AGP) showed multiple peaks on separation using capillary electrophoresis in a chemically modified capillary with dimethylpolysiloxane at slightly acidic conditions. We analyzed glycoforms of AGP species after separation by ion-exchange chromatography, Con A affinity chromatography, and Cu(II)-chelating affinity chromatography. The AGP species thus obtained were digested with N-glycosidase F, and the released carbohydrate chains were analyzed by high-performance liquid chromatography after labeling with 3-aminobenzoic acid. The results afforded basic information on the contribution of carbohydrate chains to the separation mechanism of glycoforms of AGP by capillary electrophoresis. In addition, we describe an easy method for AGP analysis in serum samples using the electrokinetic injection.
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Carbohidratos/química , Electroforesis Capilar/métodos , Glicoproteínas/sangre , Ácido N-Acetilneuramínico/química , Orosomucoide/análisis , Isoformas de Proteínas/sangre , Secuencia de Carbohidratos , Cromatografía Liquida/métodos , Glicoproteínas/química , Humanos , Modelos Químicos , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. Various authentic bacterial strains carrying neither C12O nor C23O genes were used as negative controls. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 amplified fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C23O-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/or benzoate-degrading bacteria newly isolated from a variety of environments. The C12O and/or C23O primers amplified DNA fragments of the expected sizes from 69 of the 106 wild-type strains tested, while the C12Op and/or C23Op detected positive signals in the amplified fragments from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways.