Identification of mild cognitive impairment subtypes predicting conversion to Alzheimer's disease using multimodal data.

Mild cognitive impairment (MCI) is a high-risk condition for conversion to Alzheimer's disease (AD) dementia. However, individuals with MCI show heterogeneous patterns of pathology and conversion to AD dementia. Thus, detailed subtyping of MCI subjects and accurate prediction of the patients in whom MCI will convert to AD dementia is critical for identifying at-risk populations and the underlying biological features. To this end, we developed a model that simultaneously subtypes MCI subjects and predicts conversion to AD and performed an analysis of the underlying biological characteristics of each subtype. In particular, a heterogeneous mixture learning (HML) method was used to build a decision tree-based model based on multimodal data, including cerebrospinal fluid (CSF) biomarker data, structural magnetic resonance imaging (MRI) data, APOE genotype data, and age at examination. The HML model showed an average F1 score of 0.721, which was comparable to the random forest method and had significantly more predictive accuracy than the CART method. The HML-generated decision tree was also used to classify-five subtypes of MCI. Each MCI subtype was characterized in terms of the degree of abnormality in CSF biomarkers, brain atrophy, and cognitive decline. The five subtypes of MCI were further categorized into three groups: one subtype with low conversion rates (similar to cognitively normal subjects); three subtypes with moderate conversion rates; and one subtype with high conversion rates (similar to AD dementia patients). The subtypes with moderate conversion rates were subsequently separated into a group with CSF biomarker abnormalities and a group with brain atrophy. The subtypes identified in this study exhibited varying MCI-to-AD conversion rates and differing biological profiles.

Role of a mutated residue at the entrance of the substrate access channel in cytochrome p450 engineered for vitamin D(3) hydroxylation activity.

The cytochrome P450 enzyme engineered for enhancement of vitamin D(3) (VD(3)) hydroxylation activity, Vdh-K1, includes four mutations (T70R, V156L, E216M, and E384R) compared to the wild-type enzyme. Plausible roles for V156L, E216M, and E384R have been suggested by crystal structure analysis (Protein Data Bank 3A50 ), but the role of T70R, which is located at the entrance of the substrate access channel, remained unclear. In this study, the role of the T70R mutation was investigated by using computational approaches. Molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations were performed, and differences between R70 and T70 were compared in terms of structural change, binding free energy change (PMF), and interaction force between the enzyme and substrate. MD simulations revealed that R70 forms a salt bridge with D42 and the salt bridge affects the locations and the conformations of VD(3) in the bound state. SMD simulations revealed that the salt bridge tends to be formed strongly when VD(3) passes through the binding pocket. PMFs showed that the T70R mutation leads to energetic stabilization of enzyme-VD(3) binding in the region near the heme active site. Interestingly, these results concluded that the D42-R70 salt bridge at the entrance of the substrate access channel affects the region near the heme active site where the hydroxylation of VD(3) occurs; i.e., it is thought that the T70R mutation plays an important role in enhancing VD(3) hydroxylation activity. A significant future challenge is to compare the hydroxylation activities of R70 and T70 directly by a quantum chemical calculation, and three-dimensional coordinates of the enzyme and VD(3) obtained from MD and SMD simulations will be available for the future challenge.

Comparative analysis of proteome and transcriptome in human hepatocellular carcinoma using 2D-DIGE and SAGE.

Proteome analysis of human hepatocellular carcinoma was conducted using two-dimensional difference gel electrophoresis, and the protein expression profiles were compared to the mRNA expression profiles made from serial analysis of gene expression (SAGE) in identical samples from a single patient. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the protein expression profiles. A total of 188 proteins were identified, and the expression profiles of 164 proteins which had the corresponding SAGE data were compared to the mRNA expression profiles. Among them, 40 proteins showed significant differences in the mRNA expression levels between non HCC and HCC. We compared expression changes of proteins with those of mRNAs. We found that the expression tendency of 24 proteins were similar to that of mRNA, whereas 16 proteins showed different or opposite tendency to the mRNA expression.

Protein expression profile characteristic to hepatocellular carcinoma revealed by 2D-DIGE with supervised learning.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Although several major risks related to HCC, e.g., hepatitis B and/or hepatitis C virus infection, aflatoxin B1 exposure, alcohol drinking and genetic defects have been revealed, the molecular mechanisms leading to the initiation and progression of HCC have not been clarified. To reduce the mortality and improve the effectiveness of therapy, it is important to detect the proteins which are associated with tumor progression and may be useful as potential therapeutic or diagnosis targets. However, previous studies have not yet revealed the associations among HCC cells, histological grade and AFP. Here, we performed two-dimensional difference gel electrophoresis (2D-DIGE) combined with MS for 18 HCC patients. To focus not on individual proteins but on multiple proteins associated with pathogenesis, we introduce the supervised feature selection based on stochastic gradient boosting (SGB) for identifying protein spots that discriminate HCC/non HCC, histological grade of moderate/well and high alpha-fetoprotein (AFP)/low AFP level without arbitrariness. We detected 18, 25 and 27 protein spots associated with HCC, histological grade and AFP level, respectively. We confirmed that SGB is able to identify the known HCC-related proteins, e.g., heat shock proteins, carbonic anhydrase 2. Moreover, we identified the differentially expressed proteins associated with histological grade of HCC and AFP level and found that aldo-keto reductase 1B10 (AKR1B10) is related to well differentiated HCC, keratin 8 (KRT8) is related to both histological grade and AFP level and protein disulfide isomerase-associated 3 (PDIA3) is associated with both HCC and AFP level. Our pilot study provides new insights on understanding the pathogenesis of HCC, histological grade and AFP level.

Comparative proteomic and transcriptomic profiling of the human hepatocellular carcinoma.

Proteome analysis of human hepatocellular carcinoma (HCC) was done using two-dimensional difference gel electrophoresis. To gain an understanding of the molecular events accompanying HCC development, we compared the protein expression profiles of HCC and non-HCC tissue from 14 patients to the mRNA expression profiles of the same samples made from a cDNA microarray. A total of 125 proteins were identified, and the expression profiles of 93 proteins (149 spots) were compared to the mRNA expression profiles. The overall protein expression ratios correlated well with the mRNA ratios between HCC and non-HCC (Pearson's correlation coefficient: r=0.73). Particularly, the HCC/non-HCC expression ratios of proteins involved in metabolic processes showed significant correlation to those of mRNA (r=0.9). A considerable number of proteins were expressed as multiple spots. Among them, several proteins showed spot-to-spot differences in expression level and their expression ratios between HCC and non-HCC poorly correlated to mRNA ratios. Such multi-spotted proteins might arise as a consequence of post-translational modifications.

Proteomic analysis of rice leaf, stem and root tissues during growth course.

Rice proteins were isolated from leaf, stem and root tissues, harvesting at 1, 2, 4, 8 and 10 weeks after budding. Each tissue of each age was separately pulverized in liquid nitrogen, and the resulted tissue powders were suspended in 10% TCA-acetone and followed by acetone suspension to precipitate at low temperature, which resulted in the tissue-specific and age-specific protein mixture. The protein mixtures were separated by 2-DE using polyacrylamide gels (26 x 20 cm). The protein spots were identified by N-terminal sequence analysis and by MALDI and LC-MS/MS analyses after in-gel tryptic digestion. From a total of 4532 spots, 676 unique proteins were identified, of which 80 proteins (12%) were observed in all three tissues: leaf, stem and root. In addition, 45 (7%) were common in leaf and stem, 57 (8%) in stem and root, and 10 (2%) proteins in root and leaf. Also 141 unique proteins (21%) were observed only for leaf, 96 (14%) for stem, and 247 (36%) for root tissue. Proteins playing a role for photosynthesis and energy production were most abundant in leaf and stem, and those for cell defense were rich in roots.

High-throughput and high-resolution two dimensional mapping of pI and m/z using a microchip in a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer.

We have developed a high-throughput, two-dimensional-mapping (isoelectric point [pI], mass-to-charge ratio [m/z]) method by combining a capillary isoelectric focusing chip sealed with removable resin tape and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Sample proteins are separated in a meandering channel on the chip and immediately frozen. The tape is then removed and the proteins are freeze-dried. The freeze-drying maintains the separation state of the proteins and prevents movement of the sample solution, which can reduce pI resolution. A matrix solution is then applied and mass spectrometry is carried out by laser irradiation. The whole process takes less than 70 min, more than 10 times faster than with two-dimensional, polyacrylamide gel electrophoresis.

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database.

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.

Characteristics of dipoles in clustered individual spikes and averaged spikes.

The aim of this study is to analyze the characteristics of dipoles in clustered individual spikes and averaged spikes, we compared electroencephalography (EEG) dipole localizations from patients with intractable extratemporal lobe epilepsy (IETLE) and from patients with benign epilepsy with centrotemporal spikes (BECTS). We studied 10 patients; five with IETLE who underwent epilepsy surgery after subdural EEG and five with BECTS. We recorded 19-channel digital scalp EEGs and used clustering analysis for individual spikes to characterize interictal spikes. We selected and averaged one representative spike group at the maximum negative peak electrode. We used a single dipole method with three-shell spherical head model. We compared dipole localizations of both averaged and individual spikes.IETLE data had more identifiable spike clusters and fewer spikes in each cluster than BECTS (P<0.05). Dipole sources with goodness-of-fit >or=95% in averaged spikes were less frequent in IETLE than in BECTS (P<0.05). For IETLE, averaged spikes showed no dipoles (two patients), while individual spikes gave dipole sources reliably in the epileptic region. For BECTS, individual and averaged spike sources were clustered. More than 80% of dipoles in averaged spikes were stable, in close proximity, for prolonged periods in BECTS. More spike groups after clustering and fewer acceptable dipoles from averaged spikes in IETLE reflect variable spike activity over extensive epileptic regions. Fewer spike groups producing more acceptable dipoles in BECTS correlate with stable spike sources within the isolated epileptic central region. Characteristics of clustered interictal spikes need careful examination before the use of dipole analysis of averaged spikes for epilepsy evaluation.

Visual event-related potentials during movement imagery and the dipole analysis.

Visual event-related potentials during an oddball paradigm with movement imagery tasks were recorded in 10 right-handed subjects from 32 scalp electrodes. Rare targets and non-targets elicited early (P3e) and late (P31) P300 components. In the P3e there was no difference between the rare target and non-target. In the right-imagery task the rare target P31 amplitude was larger than the rare non-target one, whereas the rare non-target P31 amplitude was larger than the rare target one in the left-imagery task. Some of the 4 equivalent current dipole (ECD) sources were located at the subcortical regions, the cerebellum and the cingulate cortex, common to the P3e and the P31. Moreover, another P3e dipole was localized to the parietal regions, while that of the P31 dipoles to the contralateral premotor cortex. This difference between the P3e and P31 dipoles might reflect two different neural networks related with the transformation of coordinates from visual to motor space.

Suppressive efficacies by adaptive temporal filtering system on photoparoxysmal response elicited by flickering pattern stimulation.

PURPOSE: Based on our previous study that validated efficacies of an adaptive temporal filtering system (ATFS) suppressing a photoparoxysmal response (PPR) elicited by a chromatic flicker stimulation, we further studied ATFS efficacies on PPRs elicited by pattern-flicker stimulation in 13 photosensitive epilepsy patients. METHODS: Subjects were 13 photosensitive epilepsy patients (two male and 11 female patients; mean age +/- SD, 20.9 +/- 8.9 years) who were all sensitive to a flickering geometric-pattern scene. We used a scene consisting of 15-Hz flickering 4 c/deg stripe images lasting for 4 s. With a 14-inch television set 2 m before a subject, we displayed the following video scenes: nonfiltered and filtered flickering-stripe scenes; for the latter, two kinds of ATFSs with mild efficacy and strong efficacy were used. Three flickering-stripe scenes altogether, each of which lasted for 4 s, were given at random with a 10-s interval. RESULTS: A nonfiltered flickering-stripe scene elicited generalized PPRs in all patients; a filtered scene by use of an ATFS with mild efficacy elicited generalized PPRs in six patients (46%), whereas that by an ATFS with strong efficacy exhibited no PPRs. CONCLUSIONS: This study, using an ATFS, again shows suppressive efficacy on PPRs elicited by flickering-pattern stimulation. Therefore a series of our studies suggested that ATFS may be useful as a preventive measure for photosensitive seizures triggered by stimulative flickering images from televisions or other displays.

Electroencephalographic dipoles of spikes with and without myoclonic jerks caused by epilepsia partialis continua.

We used electroencephalographic (EEG) dipole analysis to investigate the generators of spikes with and without myoclonic jerks in a 12-year-old patient with epilepsia partialis continua secondary to left parietal cortical dysplasia. We recorded EEG and right wrist extensor electromyography (EMG) and collected 42 spikes with jerks (jerking spikes) and 42 spikes without jerks (nonjerking spikes). We applied a single moving dipole model to the individual and averaged spikes. Dipoles at the negative peak of individual jerking and nonjerking spikes were localized in the dysplastic area. At the onset of the averaged jerking spike that preceded the EMG discharges by 20 ms, the dipole was in the motor cortex, whereas for the averaged nonjerking spike, the dipole was in the sensory cortex. The dipole moment at averaged jerking spike onset was twice that of the averaged nonjerking spike. Electroencephalographic dipole analysis of averaged spikes differentiated the generator of jerking and nonjerking spikes in epilepsia partialis continua. Individual dipoles demonstrated the area of epileptogenic cortical dysplasia.