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author:("Suzuki, makoo")
1.  Motor Cortex-Evoked Activity in Reciprocal Muscles Is Modulated by Reward Probability 
PLoS ONE  2014;9(3):e90773.
Horizontal intracortical projections for agonist and antagonist muscles exist in the primary motor cortex (M1), and reward may induce a reinforcement of transmission efficiency of intracortical circuits. We investigated reward-induced change in M1 excitability for agonist and antagonist muscles. Participants were 8 healthy volunteers. Probabilistic reward tasks comprised 3 conditions of 30 trials each: 30 trials contained 10% reward, 30 trials contained 50% reward, and 30 trials contained 90% reward. Each trial began with a cue (red fixation cross), followed by blue circle for 1 s. The subjects were instructed to perform wrist flexion and press a button with the dorsal aspect of middle finger phalanx as quickly as possible in response to disappearance of the blue circle without looking at their hand or the button. Two seconds after the button press, reward/non-reward stimulus was randomly presented for 2-s duration. The reward stimulus was a picture of Japanese 10-yen coin, and each subject received monetary reward at the end of experiment. Subjects were not informed of the reward probabilities. We delivered transcranial magnetic stimulation of the left M1 at the midpoint between center of gravities of agonist flexor carpi radialis (FCR) and antagonist extensor carpi radialis (ECR) muscles at 2 s after the red fixation cross and 1 s after the reward/non-reward stimuli. Relative motor evoked potential (MEP) amplitudes at 2 s after the red fixation cross were significantly higher for 10% reward probability than for 90% reward probability, whereas relative MEP amplitudes at 1 s after reward/non-reward stimuli were significantly higher for 90% reward probability than for 10% and 50% reward probabilities. These results implied that reward could affect the horizontal intracortical projections in M1 for agonist and antagonist muscles, and M1 excitability including the reward-related circuit before and after reward stimulus could be differently altered by reward probability.
doi:10.1371/journal.pone.0090773
PMCID: PMC3948372  PMID: 24603644
2.  A randomized trial of pregabalin in patients with neuropathic pain due to spinal cord injury 
Neurology  2013;80(6):533-539.
Objective:
To assess the efficacy and tolerability of pregabalin for the treatment of central neuropathic pain after spinal cord injury (SCI).
Methods:
Patients with chronic, below-level, neuropathic pain due to SCI were randomized to receive 150 to 600 mg/d pregabalin (n = 108) or matching placebo (n = 112) for 17 weeks. Pain was classified in relation to the neurologic level of injury, defined as the most caudal spinal cord segment with normal sensory and motor function, as above, at, or below level. The primary outcome measure was duration-adjusted average change in pain. Key secondary outcome measures included the change in mean pain score from baseline to end point, the percentage of patients with ≥30% reduction in mean pain score at end point, Patient Global Impression of Change scores at end point, and the change in mean pain-related sleep interference score from baseline to end point. Additional outcome measures included the Medical Outcomes Study–Sleep Scale and the Hospital Anxiety and Depression Scale.
Results:
Pregabalin treatment resulted in statistically significant improvements over placebo for all primary and key secondary outcome measures. Significant pain improvement was evident as early as week 1 and was sustained throughout the treatment period. Adverse events were consistent with the known safety profile of pregabalin and were mostly mild to moderate in severity. Somnolence and dizziness were most frequently reported.
Conclusions:
This study demonstrates that pregabalin is effective and well tolerated in patients with neuropathic pain due to SCI.
Classification of evidence:
This study provides Class I evidence that pregabalin, 150 to 600 mg/d, is effective in reducing duration-adjusted average change in pain compared with baseline in patients with SCI over a 16-week period (p = 0.003, 95% confidence interval = −0.98, −0.20).
doi:10.1212/WNL.0b013e318281546b
PMCID: PMC3589291  PMID: 23345639
3.  Characterization of blood pressure and endothelial function in TRPV4‐deficient mice with l‐NAME‐ and angiotensin II‐induced hypertension 
Physiological Reports  2014;2(1):e00199.
Abstract
Transient receptor potential vanilloid type 4 (TRPV4) is an endothelial Ca2+ entry channel contributing to endothelium‐mediated dilation in conduit and resistance arteries. We investigated the role of TRPV4 in the regulation of blood pressure and endothelial function under hypertensive conditions. TRPV4‐deficient (TRPV4−/−) and wild‐type (WT) control mice were given l‐NAME (0.5 g/L) in drinking water for 7 days or subcutaneously infused with angiotensin (Ang) II (600 ng/kg per minute) for 14 days, and blood pressure measured by radiotelemetry. TRPV4−/− mice had a lower baseline mean arterial pressure (MAP) (12‐h daytime MAP, 94 ± 2 vs. 99 ± 2 mmHg in WT controls). l‐NAME treatment induced a slightly greater increase in MAP in TRPV4−/− mice (day 7, 13 ± 4%) compared to WT controls (6 ± 2%), but Ang II‐induced increases in MAP were similar in TRPV4−/− and WT mice (day 14, 53 ± 6% and 37 ± 11%, respectively, P < 0.05). Chronic infusion of WT mice with Ang II reduced both acetylcholine (ACh)‐induced dilation (dilation to 10−5 mol/L ACh, 71 ± 5% vs. 92 ± 2% of controls) and the TRPV4 agonist GSK1016790A‐induced dilation of small mesenteric arteries (10−8 mol/L GSK1016790A, 14 ± 5% vs. 77 ± 7% of controls). However, Ang II treatment did not affect ACh dilation in TRPV4−/− mice. Mechanistically, Ang II did not significantly alter either TRPV4 total protein expression in mesenteric arteries or TRPV4 agonist‐induced Ca2+ response in mesenteric endothelial cells in situ. These results suggest that TRPV4 channels play a minor role in blood pressure regulation in l‐NAME‐ but not Ang II‐induced hypertension, but may be importantly involved in Ang II‐induced endothelial dysfunction.
We investigated the role of transient receptor potential vanilloid type 4 (TRPV4) in regulating vascular tone in vivo under normal conditions and in response to hypertensive challenges. Our results indicate that TRPV4 channels may play a complex role in the control of vascular tone and endothelial function under stress.
doi:10.1002/phy2.199
PMCID: PMC3967682
Endothelium; endothelium‐derived hyperpolarizing factor; Hypertension; mesenteric arteries; nitric oxide; TRPV4
4.  Relationship between the Berg Balance Scale and Static Balance Test in Hemiplegic Patients with Stroke 
Journal of Physical Therapy Science  2013;25(8):1043-1049.
[Purpose] The purpose of this study was to analyze the relationship between results of the Berg Balance Scale (BBS) and Static Balance Test (SBT) in hemiplegic patients with stroke. [Subjects] The subjects were 39 hemiplegic patients (25 men, 14 women; mean age, 69.4 ± 11.0 years) with stroke that had occurred within the preceding 6 months and who had good understanding of verbal instructions. [Methods] The SBT consists of five posture-holding tasks (sitting, stride standing, close standing, one-foot standing on the unparalyzed leg, and one-foot standing on the paralyzed leg). Four grades, 1–4, are used to judge the ability of patients to hold these postures. The SBT and BBS were each implemented, and the relationship between test results was analyzed using correlation coefficients. [Results] The correlation coefficient for the BBS score and SBT score was 0.87. Thus, a strong correlation was seen between the BBS and SBT. [Conclusion] The SBT is thought to be an assessment index that can predict overall balance ability.
doi:10.1589/jpts.25.1043
PMCID: PMC3820219  PMID: 24259912
Stroke; Balance; Assessment
5.  Modulation of the cortical silent period elicited by single- and paired-pulse transcranial magnetic stimulation 
BMC Neuroscience  2013;14:43.
Background
The cortical silent period (CSP) elicited by transcranial magnetic stimulation (TMS) is affected by changes in TMS intensity. Some studies have shown that CSP is shortened or prolonged by short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF), Those studies, however, used different TMS intensities to adjust the amplitude of the motor evoked potential (MEP). Therefore, it is unclear whether changes in CSP duration are induced by changes in TMS intensities or by SICI and ICF. The purpose of this study was to confirm the effects of muscle contractions and stimulus intensities on MEP amplitude and the duration of CSP induced by single-pulse TMS and to clarify the effects of SICI and ICF on CSP duration.
MEP evoked by TMS was detected from the right first dorsal interosseous muscle in 15 healthy subjects. First, MEP and CSP were induced by single-pulse TMS with an intensity of 100% active motor threshold (AMT) at four muscle contraction levels [10%, 30%, 50%, and 70% electromyogram (EMG)]. Next, MEP and CSP were induced by seven TMS intensities (100%, 110%, 120%, 130%, 140%, 150%, and 160% AMT) during muscle contraction of 10% EMG. Finally, SICI and ICF were recorded at the four muscle contraction levels (0%, 10%, 30%, and 50% EMG).
Results
MEP amplitudes increased with increases in muscle contraction and stimulus intensity. However, CSP duration did not differ at different muscle contraction levels and was prolonged with increases in stimulus intensity. CSP was shortened with SICI compared with CSP induced by single-pulse TMS and with ICF at all muscle contraction levels, whereas CSP duration was not significantly changed with ICF.
Conclusions
We confirmed that CSP duration is affected by TMS intensity but not by the muscle contraction level. This study demonstrated that CSP is shortened with SICI, but it is not altered with ICF. These results indicate that after SICI, CSP duration is affected by the activity of inhibitory intermediate neurons that are activated by the conditioning SICI stimulus.
doi:10.1186/1471-2202-14-43
PMCID: PMC3621611  PMID: 23547559
Transcranial magnetic stimulation; Motor evoked potential; Cortical silent period; Short-interval intracortical inhibition; Intracortical facilitation
6.  Neuromagnetic activation following active and passive finger movements 
Brain and Behavior  2013;3(2):178-192.
The detailed time courses of cortical activities and source localizations following passive finger movement were studied using whole-head magnetoencephalography (MEG). We recorded motor-related cortical magnetic fields following voluntary movement and somatosensory-evoked magnetic fields following passive movement (PM) in 13 volunteers. The most prominent movement-evoked magnetic field (MEF1) following active movement was obtained approximately 35.3 ± 8.4 msec after movement onset, and the equivalent current dipole (ECD) was estimated to be in the primary motor cortex (Brodmann area 4). Two peaks of MEG response associated with PM were recorded from 30 to 100 msec after movement onset. The earliest component (PM1) peaked at 36.2 ± 8.2 msec, and the second component (PM2) peaked at 86.1 ± 12.1 msec after movement onset. The peak latency and ECD localization of PM1, estimated to be in area 4, were the same as those of the most prominent MEF following active movement. ECDs of PM2 were estimated to be not only in area 4 but also in the supplementary motor area (SMA) and the posterior parietal cortex (PPC) over the hemisphere contralateral to the movement, and in the secondary somatosensory cortex (S2) of both hemispheres. The peak latency of each source activity was obtained at 54–109 msec in SMA, 64–114 msec in PPC, and 84–184 msec in the S2. Our results suggest that the magnetic waveforms at middle latency (50–100 msec) after PM are different from those after active movement and that these waveforms are generated by the activities of several cortical areas, that is, area 4 and SMA, PPC, and S2. In this study, the time courses of the activities in SMA, PPC, and S2 accompanying PM in humans were successfully recorded using MEG with a multiple dipole analysis system.
doi:10.1002/brb3.126
PMCID: PMC3607158  PMID: 23531918
Magnetoencephalography; MEF1; MEG; MRCF; PPC; S2; SEF; SMA
8.  Aberrant methylation of LINE-1, SLIT2, MAL and IGFBP7 in non-small cell lung cancer 
Oncology Reports  2013;29(4):1308-1314.
Genome-wide DNA hypomethylation and gene hypermethylation play important roles in instability and carcinogenesis. Methylation in long interspersed nucleotide element 1 (LINE-1) is a good indicator of the global DNA methylation level within a cell. Slit homolog 2 (SLIT2), myelin and lymphocyte protein gene (MAL) and insulin-like growth factor binding protein 7 (IGFBP7) are known to be hypermethylated in various malignancies. The aim of the present study was to assess the precise methylation levels of LINE-1, SLIT2, MAL and IGFBP7 in non-small cell lung cancer (NSCLC) using a pyrosequencing assay. Methylation of all regions was examined in 56 primary NSCLCs using a pyrosequencing assay. Changes in mRNA expression levels of SLIT2, MAL and IGFBP7 were measured before and after treatment with a demethylating agent. Methylation of these genes was also examined in 9 lung cancer cell lines using RT-PCR and a pyrosequencing assay. Frequencies of hypomethylation of LINE-1 and hypermethylation of SLIT2, MAL and IGFBP7, defined by predetermined cut off values, were 55, 64, 46 and 54% in NSCLCs, respectively and exhibited tumor-specific features. The hypermethylation of all genes was well correlated with changes in expression. The methylation level and frequency of MAL were significantly higher in smokers and in patients without EGFR mutations. Through accurate measurement of methylation levels using pyrosequencing, hypomethylation of LINE-1 and hypermethylation of SLIT2, MAL and IGFBP7 were frequently detected in NSCLCs and associated with various clinical features. Analysis of the methylation profiles of these genes may, therefore, provide novel opportunities for the therapy of NSCLCs.
doi:10.3892/or.2013.2266
PMCID: PMC3621652  PMID: 23381221
LINE-1; SLIT2; MAL; IGFBP7; pyrosequencing; methylation
9.  Predicting Recovery of Cognitive Function Soon after Stroke: Differential Modeling of Logarithmic and Linear Regression 
PLoS ONE  2013;8(1):e53488.
Cognitive disorders in the acute stage of stroke are common and are important independent predictors of adverse outcome in the long term. Despite the impact of cognitive disorders on both patients and their families, it is still difficult to predict the extent or duration of cognitive impairments. The objective of the present study was, therefore, to provide data on predicting the recovery of cognitive function soon after stroke by differential modeling with logarithmic and linear regression. This study included two rounds of data collection comprising 57 stroke patients enrolled in the first round for the purpose of identifying the time course of cognitive recovery in the early-phase group data, and 43 stroke patients in the second round for the purpose of ensuring that the correlation of the early-phase group data applied to the prediction of each individual's degree of cognitive recovery. In the first round, Mini-Mental State Examination (MMSE) scores were assessed 3 times during hospitalization, and the scores were regressed on the logarithm and linear of time. In the second round, calculations of MMSE scores were made for the first two scoring times after admission to tailor the structures of logarithmic and linear regression formulae to fit an individual's degree of functional recovery. The time course of early-phase recovery for cognitive functions resembled both logarithmic and linear functions. However, MMSE scores sampled at two baseline points based on logarithmic regression modeling could estimate prediction of cognitive recovery more accurately than could linear regression modeling (logarithmic modeling, R2 = 0.676, P<0.0001; linear regression modeling, R2 = 0.598, P<0.0001). Logarithmic modeling based on MMSE scores could accurately predict the recovery of cognitive function soon after the occurrence of stroke. This logarithmic modeling with mathematical procedures is simple enough to be adopted in daily clinical practice.
doi:10.1371/journal.pone.0053488
PMCID: PMC3543398  PMID: 23326439
10.  Midwall ejection fraction for assessing systolic performance of the hypertrophic left ventricle 
Background
In patients with left ventricular hypertrophy (LVH), LV midwall fractional shortening (FS) is used as a measure of LV systolic performance that is more physiologically appropriate than conventional FS. For evaluation of LV volume and ejection fraction (EF), 2-dimensional (2D) echocardiography is more accurate than M-mode echocardiography. The purpose of this study was to assess systolic performance by midwall EF using 2D speckle tracking echocardiography (STE).
Methods
Sixty patients were enrolled in the study. Patients were divided into two groups with LVH (n = 30) and without LVH (control group, n = 30). LV systolic function was compared between the two groups and the relationships of left ventricular mass index (LVMI) with LV systolic parameters, including midwall EF, were investigated.
Results
Midwall EF in the LVH group was significantly lower than that in the control group (42.8±4.4% vs. 48.1±4.1%, p <0.0001). Midwall FS was also significantly lower in the LVH group (13.4±2.8% vs. 16.1±1.5%, p <0.0001), but EF did not differ significantly between the two groups. There were significant correlations between midwall EF and LVMI (r=0.731, p <0.0001) and between midwall FS and LVMI (r=0.693, p <0.0001), with midwall EF having the higher correlation.
Conclusions
These results show that midwall EF can be determined using 2D STE. Midwall EF can be used to monitor LV systolic dysfunction, which is not possible with conventional EF. Evaluation of midwall EF may allow assessment of new parameters of LV systolic function in patients with LV geometric variability.
doi:10.1186/1476-7120-10-45
PMCID: PMC3552820  PMID: 23167789
Midwall ejection fraction; Left ventricular systolic function; Left ventricular hypertrophy; Speckle tracking echocardiography
11.  A randomized, double-blind, multicenter, placebo-controlled phase III trial to evaluate the efficacy and safety of pregabalin in Japanese patients with fibromyalgia 
Arthritis Research & Therapy  2012;14(5):R217.
Introduction
Fibromyalgia is a chronic disorder characterized by widespread pain and tenderness. Prior trials have demonstrated the efficacy of pregabalin for the relief of fibromyalgia symptoms, and it is approved for the treatment of fibromyalgia in the United States. However, prior to this study, there has not been a large-scale efficacy trial in patients with fibromyalgia in Japan.
Methods
This randomized, double-blind, multicenter, placebo-controlled trial was conducted at 44 centers in Japan to assess the efficacy and safety of pregabalin for the symptomatic relief of pain in fibromyalgia patients. Patients aged ≥18 years who had met the criteria for fibromyalgia were randomized to receive either pregabalin, starting at 150 mg/day and increasing to a maintenance dose of 300 or 450 mg/day, or placebo, for 15 weeks. The primary efficacy endpoint was mean pain score at final assessment. Secondary endpoints included Patient Global Impression of Change (PGIC) together with measures of sleep, physical functioning and quality of life.
Results
A total of 498 patients (89% female) were randomized to receive either pregabalin (n = 250) or placebo (n = 248). Pregabalin significantly reduced mean pain score at final assessment (difference in mean change from baseline, compared with placebo -0.44; P = 0.0046) and at every week during the study (P <0.025). Key secondary endpoints were also significantly improved with pregabalin treatment compared with placebo, including PGIC (percentage reporting symptoms "very much improved" or "much improved", 38.6% vs 26.7% with placebo; P = 0.0078); pain visual analog scale (difference in mean change from baseline, compared with placebo -6.19; P = 0.0013); Fibromyalgia Impact Questionnaire total score (-3.33; P = 0.0144); and quality of sleep score (-0.73; P <0.0001). Treatment was generally well tolerated, with somnolence and dizziness the most frequently reported adverse events.
Conclusions
This trial demonstrated that pregabalin, at doses of up to 450 mg/day, was effective for the symptomatic relief of pain in Japanese patients with fibromyalgia. Pregabalin also improved measures of sleep and functioning and was well tolerated. These data indicate that pregabalin is an effective treatment option for the relief of pain and sleep problems in Japanese patients with fibromyalgia.
Trial Registration
ClinicalTrials.gov: NCT00830167
doi:10.1186/ar4056
PMCID: PMC3580529  PMID: 23062189
12.  Application of a methylation gene panel by quantitative PCR for lung cancers 
Cancer Letters  2006;247(1):56-71.
Summary
Detection of lung cancer at early stages could potentially increase survival rates. One promising approach is the application of suitable lung cancer-specific biomarkers to specimens obtained by non-invasive methods. Thus far, clinically useful biomarkers that have high sensitivity have proven elusive. Certain genes, which are involved in cellular pathways such as signal transduction, apoptosis, cell to cell communication, cell cycles and cytokine signaling are down-regulated in cancers and may be considered as potential tumor suppressor genes. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. Using quantitative real time PCR, we tested 11 genes (3-OST-2, RASSF1A, DcR1, DcR2, P16, DAPK, APC, ECAD, HCAD, SOCS1, SOCS3) for levels of methylation within their promoter sequences in non-small cell lung cancers (NSCLC), adjacent non-malignant lung tissues, in peripheral blood mononuclear cells (PBMC) from cancer free patients, in sputum of cancer patients and controls. Of all the 11 genes tested 3-OST-2 showed the highest levels of promoter methylation in tumors combined with lowest levels of promoter methylation in control tissues. 3-OST-2 followed by, RASSF1A showed increased levels of methylation with advanced tumor stage (P<0.05). Thus, quantitative analysis of 3-OST-2 and RASSF1A methylation appears to be a promising biomarker assay for NSCLC and should be further explored in a clinical study. Our preliminary data on the analysis of sputum DNA specimens from cancer patients further support these observations.
doi:10.1016/j.canlet.2006.03.020
PMCID: PMC3379713  PMID: 16644104
Real time PCR; Tumor suppressor gene; Non-small cell lung cancer
13.  Aberrant methylation of N-methyl-D-aspartate receptor type 2B (NMDAR2B) in non-small cell carcinoma 
BMC Cancer  2011;11:220.
Background
N-methyl-D-aspartate receptors (NMDAR) act as tumor suppressors of digestive malignancies. The expression and genetic methylation patterns of NMDAR2B in non-small cell lung cancer (NSCLC) are unknown.
Methods
The relationship between gene methylation and expression of NMDAR2B was analyzed in NSCLC cell lines (N = 9) and clinical tissues (N = 216). The cell lines were studied using RT-PCR and 5-aza-2'-deoxycytidine treatment, while the clinical tissues were examined by methylation specific real-time quantitative PCR and immunohistochemistry. Retrospective investigation of patient records was used to determine the clinical significance of NMDAR2B methylation.
Results
NMDAR2B was silenced in five of the nine cell lines; 5-aza-2'-deoxycytidine treatment restored expression, and was inversely correlated with methylation. Aberrant methylation of NMDAR2B, detected in 61% (131/216) of clinical NSCLC tissues, was inversely correlated with the status of protein expression in 20 randomly examined tumors. Aberrant methylation was not associated with clinical factors such as gender, age, histological type, or TNM stage. However, aberrant methylation was an independent prognostic factor in squamous cell carcinoma cases.
Conclusions
Aberrant methylation of the NMDAR2B gene is a common event in NSCLC. The prognosis was significantly better for cases of squamous cell carcinoma in which NMDAR2B was methylated. It may have different roles in different histological types.
doi:10.1186/1471-2407-11-220
PMCID: PMC3149028  PMID: 21639937
14.  Oxidative Stress and Longevity in Okinawa: An Investigation of Blood Lipid Peroxidation and Tocopherol in Okinawan Centenarians 
Background. The Free Radical Theory of Aging mechanistically links oxidative stress to aging. Okinawa has among the world's longest-lived populations but oxidative stress in this population has not been well characterized. Methods. We compared plasma lipid peroxide (LPO) and vitamin E—plasma and intracellular tocopherol levels (total α, β, and γ), in centenarians with younger controls. Results. Both LPO and vitamin E tocopherols were lower in centenarians, with the exception of intracellular β-tocopherol, which was significantly higher in centenarians versus younger controls. There were no significant differences between age groups for tocopherol: cholesterol and tocopherol: LPO ratios. Correlations were found between α-Tocopherol and LPO in septuagenarians but not in centenarians. Conclusions. The low plasma level of LPO in Okinawan centenarians, compared to younger controls, argues for protection against oxidative stress in the centenarian population and is consistent with the predictions of the Free Radical Theory of Aging. However, the present work does not strongly support a role for vitamin E in this phenomenon. The role of intracellular β-tocopherol deserves additional study. More research is needed on the contribution of oxidative stress and antioxidants to human longevity.
doi:10.1155/2010/380460
PMCID: PMC3068305  PMID: 21490698
15.  Mass Spectra-Based Framework for Automated Structural Elucidation of Metabolome Data to Explore Phytochemical Diversity 
A novel framework for automated elucidation of metabolite structures in liquid chromatography–mass spectrometer metabolome data was constructed by integrating databases. High-resolution tandem mass spectra data automatically acquired from each metabolite signal were used for database searches. Three distinct databases, KNApSAcK, ReSpect, and the PRIMe standard compound database, were employed for the structural elucidation. The outputs were retrieved using the CAS metabolite identifier for identification and putative annotation. A simple metabolite ontology system was also introduced to attain putative characterization of the metabolite signals. The automated method was applied for the metabolome data sets obtained from the rosette leaves of 20 Arabidopsis accessions. Phenotypic variations in novel Arabidopsis metabolites among these accessions could be investigated using this method.
doi:10.3389/fpls.2011.00040
PMCID: PMC3355805  PMID: 22645535
metabolome analysis; liquid chromatography-mass spectrometry; structural elucidation; database searching; natural variations in secondary metabolite
16.  RiceFOX: A Database of Arabidopsis Mutant Lines Overexpressing Rice Full-Length cDNA that Contains a Wide Range of Trait Information to Facilitate Analysis of Gene Function 
Plant and Cell Physiology  2010;52(2):265-273.
Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named ‘RiceFOX’. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.
doi:10.1093/pcp/pcq190
PMCID: PMC3037076  PMID: 21186176
Arabidopsis thaliana; Full-length cDNA; Gene function; Oryza sativa; Trait; Transgenic plant
17.  Tissue-specific transcript annotation and expression profiling with complementary next-generation sequencing technologies 
Nucleic Acids Research  2010;38(16):e165.
Next-generation sequencing is excellently suited to evaluate the abundance of mRNAs to study gene expression. Here we compare two alternative technologies, cap analysis of gene expression (CAGE) and serial analysis of gene expression (SAGE), for the same RNA samples. Along with quantifying gene expression levels, CAGE can be used to identify tissue-specific transcription start sites, while SAGE monitors 3′-end usage. We used both methods to get more insight into the transcriptional control of myogenesis, studying differential gene expression in differentiated and proliferating C2C12 myoblast cells with statistical evaluation of reproducibility and differential gene expression. Both CAGE and SAGE provided highly reproducible data (Pearson's correlations >0.92 among biological triplicates). With both methods we found around 10 000 genes expressed at levels 2 transcripts per million (0.3 copies per cell), with an overlap of 86%. We identified 4304 and 3846 genes differentially expressed between proliferating and differentiated C2C12 cells by CAGE and SAGE, respectively, with an overlap of 2144. We identified 196 novel regulatory regions with preferential use in proliferating or differentiated cells. Next-generation sequencing of CAGE and SAGE libraries provides consistent expression levels and can enrich current genome annotations with tissue-specific promoters and alternative 3′-UTR usage.
doi:10.1093/nar/gkq602
PMCID: PMC2938216  PMID: 20615900
18.  PIK3CA Mutations and Copy Number Gains in Human Lung Cancers 
Cancer research  2008;68(17):6913-6921.
We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non–small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference–mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.
doi:10.1158/0008-5472.CAN-07-5084
PMCID: PMC2874836  PMID: 18757405
19.  TRPV4-DEFICIENT MICE EXHIBIT IMPAIRED ENDOTHELIUM-DEPENDENT RELAXATION INDUCED BY ACETYLCHOLINE IN VITRO AND IN VIVO 
Hypertension  2009;53(3):532-538.
Agonist-induced Ca2+ entry is important for the synthesis and release of vasoactive factors in endothelial cells. The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+-permeant cation channel, is expressed in endothelial cells and involved in the regulation of vascular tone. Here we investigated the role of TRPV4 channels in acetylcholine-induced vasodilation in vitro and in vivo using the TRPV4 knockout mouse model. The expression of TRPV4 mRNA and protein was detected in both conduit and resistance arteries from wild-type mice. In small mesenteric arteries from wild-type mice, the TRPV4 activator 4α-phorbol-12,13-didecanoate increased endothelial [Ca2+]i in situ, which was reversed by the TRPV4 blocker ruthenium red. In wild-type animals, acetylcholine dilated small mesenteric arteries that involved both nitric oxide (NO) and endothelium-derived hyperpolarizing factor(s) (EDHF). In TRPV4-deficient mice, the NO component of the relaxation was attenuated and the EDHF component was largely eliminated. Compared to their wild-type littermates, TRPV4-deficient mice demonstrated a blunted endothelial Ca2+ response to acetylcholine in mesenteric arteries, and reduced NO release in carotid arteries. Acetylcholine (5 mg/kg, iv) decreased blood pressure by 37.0±6.2 mmHg in wild-type animals but only 16.6±2.7 mmHg in knockout mice. We conclude that acetylcholine-induced endothelium-dependent vasodilation is reduced both in vitro and in vivo in TRPV4 knockout mice. These findings may provide novel insight into mechanisms of Ca2+ entry evoked by chemical agonists in endothelial cells.
doi:10.1161/HYPERTENSIONAHA.108.127100
PMCID: PMC2694062  PMID: 19188524
Transient receptor potential; endothelium; endothelium-derived factors; nitric oxide; calcium
20.  TRPP2 and TRPV4 form a polymodal sensory channel complex 
The Journal of Cell Biology  2008;182(3):437-447.
The primary cilium has evolved as a multifunctional cellular compartment that decorates most vertebrate cells. Cilia sense mechanical stimuli in various organs, but the molecular mechanisms that convert the deflection of cilia into intracellular calcium transients have remained elusive. Polycystin-2 (TRPP2), an ion channel mutated in polycystic kidney disease, is required for cilia-mediated calcium transients but lacks mechanosensitive properties. We find here that TRPP2 utilizes TRPV4 to form a mechano- and thermosensitive molecular sensor in the cilium. Depletion of TRPV4 in renal epithelial cells abolishes flow-induced calcium transients, demonstrating that TRPV4, like TRPP2, is an essential component of the ciliary mechanosensor. Because TRPV4-deficient zebrafish and mice lack renal cysts, our findings challenge the concept that defective ciliary flow sensing constitutes the fundamental mechanism of cystogenesis.
doi:10.1083/jcb.200805124
PMCID: PMC2500130  PMID: 18695040
21.  Distribution of ultrasonic radiofrequency signal amplitude detects lipids in atherosclerotic plaque of coronary arteries: an ex-vivo study 
Background
Accumulation of lipids within coronary plaques is an important process in disease progression. However, gray-scale intravascular ultrasound images cannot detect plaque lipids effectively. Radiofrequency signal analysis could provide more accurate information on preclinical coronary plaques.
Methods
We analyzed 29 zones of mild atheroma in human coronary arteries acquired at autopsy. Two histologic groups, i.e., plaques with a lipid core (group L) and plaques without a lipid core (group N), were analyzed by automatic calculation of integrated backscatter. One hundred regions of interest were set on the target zone. Radiofrequency signals from a 50 MHz transducer were digitized at 240 MHz with 12-bit resolution. The intensity of integrated backscatter and its distribution within each plaque were compared between the two groups.
Results
Although the mean backscatter was similar between the groups, intraplaque variation of backscatter and backscatter in the axial direction were larger in group L than in group N (p = 0.02). Conventional intravascular ultrasound showed extremely low sensitivity for lipid detection, despite a high specificity. In contrast, a cut-off value>32 for the total variance of integrated backscatter identified lipid-containing plaque with a high sensitivity (85%) and specificity (75%).
Conclusion
Compared with conventional imaging, assessment of the intraplaque distribution of integrated backscatter is more effective for detecting lipid. As coronary atheroma progresses, its composition becomes heterogeneous and multi-layered. This radiofrequency technique can portray complex plaque histology and can detect the early stage of plaque progression.
doi:10.1186/1476-7120-6-18
PMCID: PMC2391144  PMID: 18471302
22.  Phase II Study of S-1 Monotherapy as a First-line, Combination Therapy of S-1 plus Cisplatin as a Second-line, and Weekly Paclitaxel Monotherapy as a Third-line Therapy in Patients with Advanced Gastric Carcinoma 
Clinical Medicine. Oncology  2008;2:375-383.
Background
We conducted a pilot phase II study to evaluate the efficacy and safety of S-1 as a first-line, S-1 plus cisplatin as a second-line, and weekly paclitaxel as a third-line therapy for advanced gastric cancer.
Patients and methods
Between 2002 and 2005, 19 patients were enrolled in this study. Chemotherapy consisted of either 60 mg/m2 of S-1 for 4 weeks at 6 weeks interval, a combination of 60 mg/m2 S-1 for 3 weeks and 60 mg/m2 cisplatin on day 8 at 5 weeks interval, or 60 mg/m2 paclitaxel at day 1, 8, 15, at 4 weeks interval. The regimen was repeated until the occurrence of unacceptable toxicities, disease progression, or patient refusal. The primary end point was the overall survival.
Results
The response rates were 33.3%, 12.5%, and 0% after the first, second, and third line chemotherapy, respectively. The mean overall survival time was 994 days. The median survival time could not be calculated because 12 out of 19 patients were still alive when the study was concluded. Regarding hematological toxicity, the major adverse effect was leukopenia, which reached grades 3–4 in all lines of chemotherapy investigated. In addition, regarding non-hematological toxicities, the major adverse effect was anorexia, which reached grade 3–4 in the second line chemotherapy, and no deaths were attributable to the adverse effects of the drugs.
Conclusion
This sequential therapy was an effective treatment for advanced gastric cancer with acceptable toxic side-effects. We considered this sequential therapy to be effective because of the smooth switch to the next regimen.
PMCID: PMC3161668  PMID: 21892302
advanced gastric cancer; recurrent gastric cancer; S-1; cisplatin; paclitaxel; phase II study; first-line chemotherapy; second-line chemotherapy; third-line chemotherapy
23.  Occurrence of the European Subgroup of Subtype I BK Polyomavirus in Japanese-Americans Suggests Transmission outside the Family▿  
Journal of Virology  2007;81(23):13254-13258.
To examine the mode of transmission of BK polyomavirus (BKV), urine samples were collected from Japanese-Americans in Los Angeles and from other southern Californians. Subtype I was the main subtype found in samples from both groups. The subtype I subgroup Ib-2, which is predominant in Europe, was the primary subgroup detected in second-generation Japanese-Americans and in southern Californians; however, the Ic subgroup prevalent in native Japanese was rare in these populations. Since the European subgroup (Ib-2) predominated in the studied geographic area, the findings demonstrate that transmission outside the family is common in the spread of BKV, unlike previous findings for JC polyomavirus.
doi:10.1128/JVI.01018-07
PMCID: PMC2169127  PMID: 17855530
24.  Genetic determinants of exceptional human longevity: insights from the Okinawa Centenarian Study 
Age  2006;28(4):313-332.
Centenarians represent a rare phenotype appearing in roughly 10–20 per 100,000 persons in most industrialized countries but as high as 40–50 per 100,000 persons in Okinawa, Japan. Siblings of centenarians in Okinawa have been found to have cumulative survival advantages such that female centenarian siblings have a 2.58-fold likelihood and male siblings a 5.43-fold likelihood (versus their birth cohorts) of reaching the age of 90 years. This is indicative of a strong familial component to longevity. Centenarians may live such extraordinarily long lives in large part due to genetic variations that either affect the rate of aging and/or have genes that result in decreased susceptibility to age-associated diseases. Some of the most promising candidate genes appear to be those involved in regulatory pathways such as insulin signaling, immunoinflammatory response, stress resistance or cardiovascular function. Although gene variants with large beneficial effects have been suggested to exist, only APOE, an important regulator of lipoproteins has been consistently associated with a longer human lifespan across numerous populations. As longevity is a very complex trait, several issues challenge our ability to identify its genetic influences, such as control for environmental confounders across time, the lack of precise phenotypes of aging and longevity, statistical power, study design and availability of appropriate study populations. Genetic studies on the Okinawan population suggest that Okinawans are a genetically distinct group that has several characteristics of a founder population, including less genetic diversity, and clustering of specific gene variants, some of which may be related to longevity. Further work on this population and other genetic isolates would be of significant interest to the genetics of human longevity.
doi:10.1007/s11357-006-9020-x
PMCID: PMC3259160  PMID: 22253498
longevity; genetics; centenarians; Okinawa; longevity genes
25.  Polymorphisms, Mutations, and Amplification of the EGFR Gene in Non-Small Cell Lung Cancers 
PLoS Medicine  2007;4(4):e125.
Background
The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers.
Methods and Findings
We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP −216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%–54.7%) of mutant tumors compared with 25.9% (20.6%–31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%–88.4%) than in wild-type tumors (43.5%, 31.8%–55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles.
Conclusions
The three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs −216 and −191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.
Masaharu Nomura and colleagues examine the distribution ofEGFR polymorphisms in different populations and find differences that might explain different responses to tyrosine kinase inhibitors in lung cancer patients.
Editors' Summary
Background.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC), which has a very low cure rate. Recently, however, “targeted” therapies have brought new hope to patients with NSCLC. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genetic material. Chemotherapy drugs treat cancer by killing these rapidly dividing cells, but, because some normal tissues are sensitive to these agents, it is hard to kill the cancer completely without causing serious side effects. Targeted therapies specifically attack the changes in cancer cells that allow them to divide uncontrollably, so it might be possible to kill the cancer cells selectively without damaging normal tissues. Epidermal growth factor receptor (EGRF) was one of the first molecules for which a targeted therapy was developed. In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive the uncontrolled growth of some cancers, including NSCLC.
Why Was This Study Done?
Molecules that inhibit the tyrosine kinase activity of EGFR (for example, gefitinib) dramatically shrink some NSCLCs, particularly those in East Asian patients. Tumors shrunk by tyrosine kinase inhibitors (TKIs) often (but not always) have mutations in EGFR's tyrosine kinase. However, not all tumors with these mutations respond to TKIs, and other genetic changes—for example, amplification (multiple copies) of the EGFR gene—also affect tumor responses to TKIs. It would be useful to know which genetic changes predict these responses when planning treatments for NSCLC and to understand why the frequency of these changes varies between ethnic groups. In this study, the researchers have examined three polymorphisms—differences in DNA sequences that occur between individuals—in the EGFR gene in people with and without NSCLC. In addition, they have looked for associations between these polymorphisms, which are present in every cell of the body, and the EGFR gene mutations and allelic imbalances (genes occur in pairs but amplification or loss of one copy, or allele, often causes allelic imbalance in tumors) that occur in NSCLCs.
What Did the Researchers Do and Find?
The researchers measured how often three EGFR polymorphisms (the length of a repeat sequence called CA-SSR1, and two single nucleotide variations [SNPs])—all of which probably affect how much protein is made from the EGFR gene—occurred in normal tissue and NSCLC tissue from East Asians and individuals of European descent. They also looked for mutations in the EGFR tyrosine kinase and allelic imbalance in the tumors, and then determined which genetic variations and alterations tended to occur together in people with the same ethnicity. Among many associations, the researchers found that shorter alleles of CA-SSR1 and the minor forms of the two SNPs occurred less often in East Asians than in individuals of European descent. They also confirmed that EGFR kinase mutations were more common in NSCLCs in East Asians than in European-descent individuals. Furthermore, mutations occurred more often in tumors with allelic imbalance, and in tumors where there was allelic imbalance and an EGFR mutation, the mutant allele was amplified more often than the wild-type allele.
What Do These Findings Mean?
The researchers use these associations between gene variants and tumor-associated alterations to propose a model to explain the ethnic differences in mutational frequencies and responses to TKIs seen in NSCLC. They suggest that because of the polymorphisms in the EGFR gene commonly seen in East Asians, people from this ethnic group make less EGFR protein than people from other ethnic groups. This would explain why, if a threshold level of EGFR is needed to drive cells towards malignancy, East Asians have a high frequency of amplified EGFR tyrosine kinase mutations in their tumors—mutation followed by amplification would be needed to activate EGFR signaling. This model, though speculative, helps to explain some clinical findings, such as the frequency of EGFR mutations and of TKI sensitivity in NSCLCs in East Asians. Further studies of this type in different ethnic groups and in different tumors, as well as with other genes for which targeted therapies are available, should help oncologists provide personalized cancer therapies for their patients.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040125.
US National Cancer Institute information on lung cancer and on cancer treatment for patients and professionals
MedlinePlus encyclopedia entries on NSCLC
Cancer Research UK information for patients about all aspects of lung cancer, including treatment with TKIs
Wikipedia pages on lung cancer, EGFR, and gefitinib (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040125
PMCID: PMC1876407  PMID: 17455987

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