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Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry
Elisa Legnini,Joe J. Orsini,Adolf Mühl,Britt Johnson,Angela Dajnoki,Olaf A Bodamer 대한진단검사의학회 2012 Annals of Laboratory Medicine Vol.32 No.5
Background: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS)method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. Methods: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6- sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). Results: ASM activities were stable for up to 2 months at or below 4˚C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intraand inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 μmol/h/L (mean 10.6) with a SD of 5.06 μmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 μmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 μmol/h/L). Conclusions: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP. Background: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS)method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. Methods: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6- sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). Results: ASM activities were stable for up to 2 months at or below 4˚C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intraand inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 μmol/h/L (mean 10.6) with a SD of 5.06 μmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 μmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 μmol/h/L). Conclusions: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.
Joe, M.M.,Benson, A.,Saravanan, V.S.,Sa, T. Academic Press 2015 Physiological and molecular plant pathology Vol.91 No.-
In the present investigation the in vitro activity of nanoemulsion (AUSN1) was evaluated against the plant pathogen Pectobacterium carotovorum sub sp. carotovorum causing the soft rot disease in numerous horticultural crops. With AUSN1 nanoemulsion treatment, minimum inhibitory concentration (MIC) of 0.09 mg ml<SUP>-1</SUP>, minimum bacteriostatic concentration (MBC) of 1.30 mg ml<SUP>-1</SUP> and zone of inhibition of 2.90 cm was observed against P. carotovorum. This formulation was able to reduce the bacterial population by 32.2-51.6%, under biofilm formation condition and a complete elimination of population under normal cultivation conditions. When subjected AUSN1 treatment, reduction in biofilm formation, swarming and swimming motility was observed in P. carotovorum strain and also inhibited the N-acyl homoserine lactones (AHL) activity by 13.27-62.3% and AHL production by 32.4-76.13%. Reduction in exo polysaccharide (EPS) synthesis and hydrolytic enzymes production in P. carotovorum strain was observed due to AUSN1 treatment. Reduced hydrophobicity (36.9-56.4%) and adhesion to polystyrene (22.9-47.5%) and potato tuber surfaces (19.5-46.8%) was observed with AUSN1 treatment. All these collective evidences clearly show that, under laboratory conditions, AUSN1 treatment was able to inhibit the soft root incidence in potato tubers.
Nanopatterning by multiple-ion-beam sputtering
Joe, M,Kim, J-H,Choi, C,Kahng, B,Kim, J-S IOP Pub 2009 Journal of Physics, Condensed Matter Vol.21 No.22
<P>We conducted a systematic study on nanopatterning by multiple-ion-beam sputtering, focusing on the superposition of the simple patterns formed by individual ion beams. When Au(001) is simultaneously sputtered by two ion beams at grazing incidence, both nanodot and nanohole patterns are obtained. If a rippled surface is subsequently sputtered at normal incidence, a nanobead pattern is obtained. All of the obtained patterns consist of the nanopatterns formed by individual ion beams; however, the superposition of nanopatterns is not realized in its ideal form. We also discuss the microscopic mechanism of pattern formation by multiple-ion-beam sputtering, and consider the questions and possibilities remaining to be explored. </P>