ABSTRACT
OBJECTIVE@#To establish a quantitative fluorescent detection method using DAPI for detecting inorganic polyphosphate (polyP) in enterohemorrhagic Escherichia coli (EHEC) O157:H7.@*METHODS@#The DNA of wild-type strain of EHEC O157:H7 was extracted and purified. DAPI was combined with the extracted DNA and polyP45 standards for measurement of the emission spectra at 360 nm and 415 nm fluorescence spectrophotometry. The fluorescence of DAPI-DNA and DAPI-polyP complexes was detected by fluorescence confocal microscopy to verify the feasibility of DAPI for detecting polyP. To determine the optimal pretreatment protocol for improving the cell membrane permeability, the effects of 6 pretreatments of the cells (namely snap-freezing in liquid nitrogen, freezing at -80 ℃, and freezing at -20 ℃, all followed by thawing at room temperature; heating at 60 ℃ for 10 min; treatment with Triton x-100; and placement at room temperature) were tested on the survival of EHEC O157:H7. The fluorescence values of the treated bacteria were then measured after DAPI staining. A standard calibration curve of polyP standard was established for calculation of the content of polyP in the live cells of wildtype EHEC strain and two mutant strains.@*RESULTS@#At the excitation wavelength of 360 nm, the maximum emission wavelength of DAPI-DNA was 460 nm, and the maximum emission wavelength of DAPI-polyP was 550 nm at the excitation wavelength of 415 nm. The results of confocal microscopy showed that 405 nm excitation elicited blue fluorescence from DAPIDNA complex with the emission wavelength of 425-475 nm; excitation at 488 nm elicited green fluorescence from the DAPIpolyP complex with the emission wavelength of 500-560 nm of. Snap-freezing of cells at -80 ℃ followed by thawing at room temperature was the optimal pretreatment to promote DAPI penetration into the live cells. The standard calibration curve was =1849+127.5 (R=0.991) was used for determining polyP content in the EHEC strains. The experimental results showed that wild-type strain had significantly higher polyP content than the mutant strains with deletion.@*CONCLUSIONS@#We established a convenient quantitative method for direct and reliable detection polyP content to facilitate further study of polyP and its catalytic enzymes in EHEC O157:H7.
Subject(s)
Escherichia coli O157 , Escherichia coli Proteins , PolyphosphatesABSTRACT
Objective To investigate the clinical features of moyamoya disease complicated with Graves′disease and the efficacy of extra-and intra-cranial revascularization. Methods The clinical data of 4 patients with moyamoya disease complicated with Graves′disease were analyzed retrospectively. Among them,three were females and one was a male. Their mean age was 32 ± 7 years. After medical treatment, their thyroid function was normal. The patients were treated with superficial temporal artery-middle cerebral artery bypass grafting. Results (1) Three patients showed cerebral infarction and one showed frequent transient ischemic attack. DSA confirmed that 2 patients had unilateral moyamoya disease and 2 had bilateral moyamoya disease. Head MRI revealed brain infarcts. (2) The thyroid function was normal after drug treat-ment,the symptoms of moyamoya disease were stable in 3 cases. One patient had high metabolic symptoms, such as high fever and accelerated heart rate within one week after procedure. The patients were followedup for 6 to 18 months,one was good,3 were excellent,and there was no recurrence of Graves′disease. Postoperative head MRI revealed that the 4 patients did not have new brain infarcts. MRA showed that the arterial filling in cerebral sulci in the ischemic lesion areas was obviously improved compared with that before procedure. Retrograde filling of the ipsilateral middle cerebral artery M2-M3 segment was observed in 2 patients. Postoperative single photon emission computed tomography perfusion imaging revealed that the ischemic perfusion lesions on the operated sides were obviously improved compared with those before procedure. Conclusion When complicated with Graves′ disease,the symptoms of moyamoya disease will aggravate. It manifests as acute and chronic cerebral ischemia. After controlling the symptoms of hyperthyroidism,most cerebral ischemic symptoms can be alleviated. Superficial temporal artery-middle cerebral artery bypass grafting may establish an effective collateral circulation and improve the clinical symptoms.