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发布于:2020-11-27 15:58:15  访问:1 次 回复:0 篇
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Exclusion criteria also included heavy cigarette smoking, alcohol use other than socially, and any psychotherapeutic use.Finally, it should be emphasized that although no pathological conrmation had been obtained in any of these subjects, all of them had been followed for approximately three years without demonstrating any symptoms or signs of neurological disorders, including mild cognitive impairment. Nonetheless, all patients included in this study had sustained response to DA drugs and there was no need to revise clinical diagnosis on any of these patients after followup evaluation when this manuscript was written.Only patients with postmortem conrmation of DLB were included in this study.Individuals remained at bed rest for one hour following LP.All CSF for proteomic analysis was taken from the th to th ml collected to limit variations arising from rostralcaudal gradient.In addition, all LP was performed in the morning to limit potential circadian uctuation of CSF proteins and metabolites.The protein concentration in CSF is relatively low Targetmol‘s Sarcosine compared to plasma, and in addition, the protein proles in CSF are similar to those in plasma, suggesting that even a minor contamination of CSF with blood could signicantly confound the interpretation of quantitative proteomic analysis of CSF.To circumvent this difculty, in the last few years we have adopted the following strategy: discovering potential biomarkers with pooled samples with extensive chromatographic separation of peptides and multiple injections to reach the bottom of the iceberg.After potential biomarkers are identi ed, we conrm andor validate them in individual samples to achieve information related to the sensitivity and specicity of each marker. Hence, in the current study, in discovery phase we pooled CSF samples from AD, PD, DLB, and controls before proteomic analysis.Samples were spotted at seconds intervals using a array pattern for a total of spots per plate.A maximum of peaks were selected per spot, with a minimum signalnoise ratio of and cluster area of. More than precursors were selected and submitted for MSMS, where a positive ion mode with collision induceddissociation cell and kV collision energy were used, and shots were accumulated per spectrum.A total of MS and more than MSMS spectra were acquired for each spotted plate.The secondary antibodies were added, and detected by enhanced chemiluminescence or by ECL plus western blotting detection system. Relative levels of each protein were quanti ed by measuring optical densities of the corresponding bands compared to a pooled sample containing all cases.The sensitivity of each marker was also computed from the ROC curve at specicity.To aid interpretation of our data when comparing markers, raw data was transformed with the natural log so its behavior among healthy subjects more accurately reected a normal distribution with a mean of and unit standard deviation. Standardization of the markers, which leaves the ROC curves unchanged, also facilitates the comparison of two different markers because the units of measurement are now F.Logistic regression has several theoretical properties that make it convenient for applied biomarker research. For instance, the P values for each marker in the composite evaluate whether it signi cantly increases the distance between cases and controls.
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