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1.  The Prediction of Response to Galantamine Treatment in Patients with Mild to Moderate Alzheimer’s Disease 
Current Alzheimer Research  2014;11(2):110-118.
The prediction of efficacy in long-term treatment of acetylcholinesterase inhibitors (AChEIs) is a major clinical issue, although no consistently strong predictive factors have emerged thus far. The present analyses aimed to identify factors for predicting long-term outcome of galantamine treatment. Analyses were conducted with data from a 24 weeks randomized, double-blind, placebo controlled trial to evaluate the efficacy and the safety of galantamine in the treatment of 303 patients with mild to moderate AD. Patients were divided into responders (4 or more point improvement of ADAS-cog scores at 24 weeks of treatment) and non-responders. We explored whether patients’ background (e.g. sex, age, and duration of disease) and scores of cognitive scales at early stage, are relevant to the long-term response to AChEIs. Predictive values were estimated by the logistic regression model. The responder rate was 31.7 %. We found that changes in scores of ADAS-J cog subscales between week 4 and baseline, especially word recognition, can be a good variable to predict subsequent response to galantamine, with approximately 75% of predictive performance. Characteristics of patients, including demographic characteristics, severity of disease and neuropsychological features before treatment were poorly predictive. The present study indicate that initial response to galantamine administration in patients with mild to moderate AD seems to be a reliable predictor of response of consequent galantamine treatment. Patients who show improvement of episodic memory function during the first 4 weeks of galantamine administration may be likely to particularly benefit from galantamine treatment.
PMCID: PMC3979115  PMID: 24156269
Acetylcholinesterase inhibitor; Alzheimer's disease; Alzheimer’s disease assessment scale-cognitive subscale (ADAS-cog); galantamine, pharmacotherapy; responders.
2.  Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids 
A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neor), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neor marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neor mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C20:3n-6) and eicosatetraenoic acid (C20:4n-3), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C20:4n-6) and eicosapentaenoic acid (C20:5n-3), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.
PMCID: PMC3346472  PMID: 22344656
3.  Increase of Eicosapentaenoic Acid in Thraustochytrids through Thraustochytrid Ubiquitin Promoter-Driven Expression of a Fatty Acid Δ5 Desaturase Gene▿† 
Applied and Environmental Microbiology  2011;77(11):3870-3876.
Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C16:0), n − 6 docosapentaenoic acid (DPA) (C22:5n − 6), and docosahexaenoic acid (DHA) (C22:6n − 3), with eicosapentaenoic acid (EPA) (C20:5n − 3) and arachidonic acid (AA) (C20:4n − 6) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C20:4n − 3) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6- and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C20:3n − 6) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs.
PMCID: PMC3127612  PMID: 21478316
4.  Clinical Omics Analysis of Colorectal Cancer Incorporating Copy Number Aberrations and Gene Expression Data 
Cancer Informatics  2010;9:147-161.
Colorectal cancer (CRC) is one of the most frequently occurring cancers in Japan, and thus a wide range of methods have been deployed to study the molecular mechanisms of CRC. In this study, we performed a comprehensive analysis of CRC, incorporating copy number aberration (CRC) and gene expression data. For the last four years, we have been collecting data from CRC cases and organizing the information as an “omics” study by integrating many kinds of analysis into a single comprehensive investigation.
In our previous studies, we had experienced difficulty in finding genes related to CRC, as we observed higher noise levels in the expression data than in the data for other cancers.
Because chromosomal aberrations are often observed in CRC, here, we have performed a combination of CNA analysis and expression analysis in order to identify some new genes responsible for CRC.
This study was performed as part of the Clinical Omics Database Project at Tokyo Medical and Dental University. The purpose of this study was to investigate the mechanism of genetic instability in CRC by this combination of expression analysis and CNA, and to establish a new method for the diagnosis and treatment of CRC.
Materials and methods:
Comprehensive gene expression analysis was performed on 79 CRC cases using an Affymetrix Gene Chip, and comprehensive CNA analysis was performed using an Affymetrix DNA Sty array. To avoid the contamination of cancer tissue with normal cells, laser micro-dissection was performed before DNA/RNA extraction. Data analysis was performed using original software written in the R language.
We observed a high percentage of CNA in colorectal cancer, including copy number gains at 7, 8q, 13 and 20q, and copy number losses at 8p, 17p and 18. Gene expression analysis provided many candidates for CRC-related genes, but their association with CRC did not reach the level of statistical significance. The combination of CNA and gene expression analysis, together with the clinical information, suggested UGT2B28, LOC440995, CXCL6, SULT1B1, RALBP1, TYMS, RAB12, RNMT, ARHGDIB, S1000A2, ABHD2, OIT3 and ABHD12 as genes that are possibly associated with CRC. Some of these genes have already been reported as being related to CRC. TYMS has been reported as being associated with resistance to the anti-cancer drug 5-fluorouracil, and we observed a copy number increase for this gene. RALBP1, ARHGDIB and S100A2 have been reported as oncogenes, and we observed copy number increases in each. ARHGDIB has been reported as a metastasis-related gene, and our data also showed copy number increases of this gene in cases with metastasis.
The combination of CNA analysis and gene expression analysis was a more effective method for finding genes associated with the clinicopathological classification of CRC than either analysis alone. Using this combination of methods, we were able to detect genes that have already been associated with CRC. We also identified additional candidate genes that may be new markers or targets for this form of cancer.
PMCID: PMC2918356  PMID: 20706620
colorectal cancer; clinical omics; microarray; copy number aberration
5.  Identification of dendritic cells, B cell and T cell subsets in Tasmanian devil lymphoid tissue; evidence for poor immune cell infiltration into devil facial tumors 
The Tasmanian devil is under threat of extinction due to the transmissible devil facial tumor disease (DFTD). This fatal tumor is an allograft that does not induce an immune response, raising questions about the activity of Tasmanian devil immune cells. T and B cell analysis has been limited by a lack of antibodies, hence the need to produce such reagents. Amino acid sequence analysis revealed that CD4, CD8, IgM, and IgG were closely related to other marsupials. Monoclonal antibodies were produced against CD4, CD8, IgM, and IgG by generating bacterial fusion proteins. These, and commercial antibodies against CD1a and CD83, identified T cells, B cells and dendritic cells by immunohistochemistry. CD4+ and CD8+ T cells were identified in pouch young thymus, adult lymph nodes, spleen, bronchus‐ and gut‐associated lymphoid tissue. Their anatomical distribution was characteristic of mammalian lymphoid tissues with more CD4+ than CD8+ cells in lymph nodes and splenic white pulp. IgM+ and IgG+ B cells were identified in adult lymph nodes, spleen, bronchus‐associated lymphoid tissue and gut‐associated lymphoid tissue, with more IgM+ than IgG+ cells. Dendritic cells were identified in lymph node, spleen and skin. This distribution is consistent with eutherian mammals and other marsupials, indicating they have the immune cell subsets for an anti‐tumor immunity. Devil facial tumor disease tumors contained more CD8+ than CD4+ cells, but in low numbers. There were also low numbers of CD1a+ and MHC class II+ cells, but no CD83+ IgM+ or IgG+ B cells, consistent with poor immune cell infiltration. Anat Rec, 297:925–938, 2014. © 2014 The Authors. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology Published by Wiley Periodicals, Inc.
PMCID: PMC4112814  PMID: 24664954
devil facial tumour disease; immunohistochemistry; lymphocyte subsets; dendritic cells; marsupial immunology; monoclonal antibody development

Results 1-5 (5)