RESUMO
We investigated the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pan-drug-resistant Acinetobacter baumannii (PDR-AB) with carbapenem resistance. Eight strains were randomly selected from 84 clinical isolates of PDR-AB strains obtained by the Kirby-Bauer and agar dilution methods. An efflux pump inhibition test was used to screen for the efflux pump phenotype. An ethylenediaminetetraacetic acid (EDTA) synergy test was used to screen for the ß-lactamase phenotype, and a three-dimensional test was used to detect extended spectrum ß-lactamase (ESBL) and ampicillin C, KPC, and carbapenemase. ESBL genes were amplified by polymerase chain reaction and sequenced. Outer membrane proteins were extracted from a sensitive strain and the PDR-AB strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected to LC-MS/MS. Peptide mass fingerprinting data were retrieved, and proteins with differential expression were identified. Results of the efflux pump inhibition tests showed that the minimum inhibitory concentrations for meropenem were decreased in 4 of the 8 strains by at least 25% of the original value. The results of the EDTA synergy test were negative, and the modified Hodge's tests were positive for all strains. PCR and sequencing confirmed that seven, five, and all eight of the PDR-AB strains contained blaOXA-23, blaTEM-1, and KPC-2, respectively. OXA-23 and CsuC proteins were differentially expressed between the drug-resistant and -sensitive strains. Production of blaOXA-23 enzyme and pilus molecular chaperone to guide synthesis of CsuC protein may be involved in the resistance of A. baumannii to carbapenems. LC-MS/ MS provides a quick and easy method for carbapenemase detection.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , beta-Lactamases/isolamento & purificação , Acinetobacter baumannii/química , Acinetobacter baumannii/efeitos dos fármacos , Proteínas de Bactérias/química , Farmacorresistência Bacteriana/genética , Humanos , beta-Lactamases/químicaRESUMO
Regulatory factors governing the formation of bone in the glenoid fossa in response to functional appliance therapy have not been identified. Therefore, the purpose of this study was to investigate the temporal pattern of expression of two key chondrogenesis markers-SOX9 and its target gene, type II collagen-in the glenoid fossa by immunostaining in a 35-day-old Sprague Dawley rat model during both natural growth and forward mandibular positioning. The expression of both factors was up-regulated when the mandible was positioned forward, indicating an enhancement of chondrocyte differentiation and chondroid matrix formation. Our results indicate that chondroid bone formation in the glenoid fossa in response to forward mandibular positioning is regulated by molecular markers indicative of endochondral ossification.
Assuntos
Colágeno Tipo II/análise , Proteínas de Grupo de Alta Mobilidade/análise , Mandíbula/anatomia & histologia , Aparelhos Ortodônticos Funcionais , Osso Temporal/metabolismo , Fatores de Transcrição/análise , Regulação para Cima , Análise de Variância , Animais , Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/genética , Matriz Extracelular/efeitos dos fármacos , Feminino , Proteínas de Grupo de Alta Mobilidade/genética , Mandíbula/crescimento & desenvolvimento , Modelos Animais , Osteogênese/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOX9 , Osso Temporal/crescimento & desenvolvimento , Fatores de Transcrição/genética , Regulação para Cima/genéticaRESUMO
The present study was designed to quantitatively assess the temporal pattern of expression of Sox 9, the regulator of chondrocyte differentiation and type II collagen, the major component of the cartilage matrix during forward mandibular positioning, and compare it with the expression during natural growth. Female Sprague-Dawley rats, 5 weeks old, were used. Results showed that the expression of Sox 9 and type II collagen are accelerated and enhanced when the mandible is positioned forward. Furthermore, we monitored the amount of new bone formation during mandibular advancement and after the removal of bite-jumping appliances. A substantial increase was observed in the amount of newly formed bone when the mandible was positioned forward. No significant difference in new bone formation could be found after the appliance was removed when compared with natural growth. Thus, functional appliance therapy accelerates and enhances condylar growth by accelerating the differentiation of mesenchymal cells into chondrocytes, leading to an earlier formation and increase in amount of cartilage matrix. This enhancement of growth did not result in a subsequent pattern of subnormal growth for most of the growth period; this indicates that functional appliance therapy can truly enhance condylar growth.
