ABSTRACT
Mass spectrometry has become an important analytical tool for protein research studies to identify, characterise and quantify proteins with unmatched sensitivity in a highly parallel manner. When transferred into clinical routine, the cumbersome and error-prone sample preparation workflows present a major bottleneck. In this work, we demonstrate tryptic digestion of human serum that is fully automated by centrifugal microfluidics. The automated workflow comprises denaturation, digestion and acidification. The input sample volume is 1.3 µl only. A triplicate of human serum was digested with the developed microfluidic chip as well as with a manual reference workflow on three consecutive days to assess the performance of our system. After desalting and liquid chromatography tandem mass spectrometry, a total of 604 proteins were identified in the samples digested with the microfluidic chip and 602 proteins with the reference workflow. Protein quantitation was performed using the Hi3 method, yielding a 7.6% lower median intensity CV for automatically digested samples compared to samples digested with the reference workflow. Additionally, 17% more proteins were quantitated with less than 30% CV in the samples from the microfluidic chip, compared to the manual control samples. This improvement can be attributed to the accurate liquid metering with all volume CVs below 1.5% on the microfluidic chip. The presented automation solution is attractive for laboratories in need of robust automation of sample preparation from small volumes as well as for labs with a low or medium throughput that does not allow for large investments in robotic systems.
Subject(s)
Microfluidics , Proteomics , Automation , Chromatography, Liquid , Digestion , Humans , Mass SpectrometryABSTRACT
The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Krüppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.
Subject(s)
Apoptosis/physiology , DNA-Binding Proteins/antagonists & inhibitors , Leukemia, T-Cell/metabolism , Lymphoma/metabolism , Repressor Proteins/antagonists & inhibitors , T-Lymphocytes/metabolism , Tumor Suppressor Proteins/antagonists & inhibitors , Blotting, Western , Cell Line, Tumor , Flow Cytometry , Humans , Jurkat Cells , Leukemia, T-Cell/genetics , Lymphoma/genetics , RNA Interference , RNA, Messenger/analysis , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/pathology , TNF-Related Apoptosis-Inducing Ligand/metabolism , Transcription, Genetic , bcl-X Protein/metabolismABSTRACT
The Memory and Aging Project Satellite (MAPS) of the Washington University Alzheimer's Disease Research Center was developed to meet the medical, social, and housing needs of minority and medically underserved elders with cognitive impairments. MAPS is located in the offices of the St. Louis Area Agency on Aging (AAA). This program provides multidisciplinary outreach, as well as home-based diagnosis, treatment, and case management. It differs from most other satellite programs in that it seeks to provide service to individuals who do not voluntarily seek help for dementia. Cognitively impaired clients had numerous, unmanaged medical conditions and social problems. Few clients were adequately served by health and social service systems. Despite recent contact, only 10% of clients received a formal diagnosis of dementia from a physician. Treatment was hampered by the absence or limitations of caregivers. Despite the complexities of these cases, the MAPS staff have been generally successful in addressing client problems.
