ABSTRACT
This paper reports a novel investigation of the voltage modulation efficiency (VME) in scanning capacitance microscopy (SCM). A signal intensity model was used to define the VME, which is dependent on the impedance components in an SCM setup. In SCM, the VME was found to play a key mediating role in the close relationship between the signal intensity and the modulation voltage, providing an indicator for the surface treatment and the back-contact process of an SCM specimen. We observed that, for silicon-based specimens, ultraviolet-assisted oxidation and microwave annealing improved the specimen surface and the back-contact, respectively, which increased the VME. It was also found that a high modulation voltage and a large back-contact area may induce a significant stray capacitance around the conductive tip and, hence, lower the VME. The VME degradation not only decreased the SCM signal intensity but also reduced the image contrast in the regions with high carrier concentrations. In addition, our experimental results further revealed that the signal intensity model also provided a promising opportunity to establish a precise and quantitative method for measuring the thickness of insulating layers.
ABSTRACT
We have previously identified prenylated Rab acceptor 1 (PRA1) as a novel cellular interacting partner for Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1). The intracellular trafficking and full signaling of LMP1 requires its interaction with PRA1. To further explore the role of PRA1 in Epstein-Barr virus-associated nasopharyngeal carcinoma (NPC) cells, we generated several PRA1-knockdown cell clones, which exhibited altered cell morphology and increased cell motility. We identified proteins differentially expressed in the knockdown clones by means of isobaric mass tags labeling coupled with multidimensional liquid chromatography-mass spectrometry. We validated a panel of proteins, which showed consistent up-regulation in PRA1-knockdown clones and participated in regulating lipid homeostasis and cell migration. Immunofluorescence staining further revealed altered localization of these proteins and accumulation of intracellular cholesterol in PRA1-knockdown clones. These effects were phenocopied by treatment with a cholesterol transport inhibitor, U18666A. Moreover, overexpressed PRA1 was able to alleviate the dysregulation of these affected proteins either from PRA1 knockdown or U18666A treatment, implying a role for PRA1 in regulating the levels of these affected proteins in response to altered cholesterol homeostasis. We further demonstrated that LMP1 expression caused PRA1 sequestration in NPC cells, leading to a consequence reminiscent of PRA1 knockdown. Finally, the immunohistochemistry showed a physiological relevance of the PRA1-associated proteome-wide changes in NPC biopsy tissues. In sum, our findings delineated novel roles of PRA1 in lipid transport and cell migration, and provided additional insights into the molecular basis of NPC morphogenesis, namely a consequence of LMP1-PRA1 interaction.
Subject(s)
Cell Movement , GTP-Binding Proteins/deficiency , GTP-Binding Proteins/metabolism , Lipid Metabolism , Proteome/metabolism , Vesicular Transport Proteins/deficiency , Vesicular Transport Proteins/metabolism , Androstenes/pharmacology , Biological Transport/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Shape/drug effects , Cholesterol/metabolism , Chromatography, Liquid , Gene Knockdown Techniques , Humans , Isotope Labeling , Lipid Metabolism/drug effects , Mass Spectrometry , Models, Biological , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/pathology , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Proteome/chemistry , Reproducibility of Results , Viral Matrix Proteins/metabolismABSTRACT
BACKGROUND: Spinal muscular atrophy (SMA) is a common inherited and fatal neuromuscular disease caused by deletions and/or mutations that lead to altered concentrations of proteins encoded by the survival motor neuron genes SMN1 and SMN2. Because of the high incidence (at least 1 in 10,000 live births and a carrier frequency of 1 in 35 to 1 in 50) and severity of the disease, precise quantification of SMN1 and SMN2 gene copy numbers is essential for diagnosis and genetic counseling. METHODS: We developed a genotyping platform combining capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to quantify absolute gene dosage. The absolute gene dosage can be determined by a multiplexed competitive PCR protocol followed by capillary electrophoresis analysis. The relative SMN1/SMN2 ratio can be analyzed by PinPoint assay followed by MALDI-TOF MS analysis. RESULTS: The complementary assays were evaluated in confirmed cases including 9 affected patients, 33 carriers, and 478 healthy individuals from the general population. We were able to determine all genotypes with different SMN1/SMN2 gene copy number ratios, which unambiguously diagnosed carrier status and the severity of SMA with 100% specificity. CONCLUSIONS: This quantitative genotyping platform is suitable for detection of SMA. The described approach may serve as a general quantitative genotyping method for molecular diagnosis of other inheritable diseases.
Subject(s)
Cyclic AMP Response Element-Binding Protein/genetics , Muscular Atrophy, Spinal/diagnosis , Nerve Tissue Proteins/genetics , RNA-Binding Proteins/genetics , Electrophoresis, Capillary , Gene Dosage , Genetic Testing , Genotype , Heterozygote , Humans , Muscular Atrophy, Spinal/genetics , Polymorphism, Single Nucleotide , Reproducibility of Results , SMN Complex Proteins , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spinal Muscular Atrophies of Childhood/diagnosis , Spinal Muscular Atrophies of Childhood/genetics , Survival of Motor Neuron 1 Protein , Survival of Motor Neuron 2 ProteinABSTRACT
Beta-thalassemia is a common monogenic disease caused by mutations in the human beta-globin gene (HBB), many of which are differentially represented in human subpopulations stratified by ethnicity. This study describes an efficient and highly accurate method to screen for the eight most-common disease-causing mutations, covering more than 98% of HBB alleles in the Taiwanese population, using parallel minisequencing and multiplex assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS was optimized for sensitivity and resolution by "mass tuning" the PinPoint assay for eight HBB SNPs. Because of the close proximity and clustering of mutations in HBB, primer extension reactions were conducted in parallel. Efficient sequential desalting using POROS and cationic exchange chromatography allowed for an unambiguous multiplex genotyping by MALDI-TOF MS. The embellishing SNP assay allowed for highly accurate identification of the eight most-common beta-thalassemia mutations in homozygous normal control, carrier, and eight heterozygous carrier mixtures, as well as the diagnosis of a high-risk family. The results demonstrated a flexible strategy for rapid identification of clustering SNPs in HBB with a high degree of accuracy and specificity. It can be adapted easily for high-throughput diagnosis of various hereditary diseases or to establish family heritage databases for clinical applications.
