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1.  Hepcidin regulation in prostate and its disruption in prostate cancer 
Cancer research  2015;75(11):2254-2263.
Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer, and is associated with accelerated disease progression in prostate cancer patients. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression.
PMCID: PMC4454355  PMID: 25858146
2.  Distinctive Patterns of Initially Presenting Metastases and Clinical Outcomes According to the Histological Subtypes in Stage IV Non-Small Cell Lung Cancer 
Medicine  2016;95(6):e2795.
This study was designed to compare the primary patterns of metastases and clinical outcomes between adenocarcinoma (Adenoca) and squamous cell carcinoma (SQ) in initially diagnosed stage IV non-small cell lung cancer (NSCLC).
Between June 2007 and June 2013, a total of 427 eligible patients were analyzed. These patients were histologically confirmed as Adenoca or SQ and underwent systemic imaging studies, including 18F-fluorodeoxyglucose positron emission tomography/computed tomography and brain imaging. Synchronous metastatic sites were categorized into 7 areas, and whole-body metastatic scores were calculated from 1 to 7 by summation of each involved region. We compared the patient, tumor, and metastatic characteristics according to the histological subtypes, and examined clinical outcomes.
The enrolled study cohort comprised 81% (n = 346) Adenoca patients and 19% (n = 81) SQ patients. The median age of the study population was 65 years (range, 30–94 years), and 263 (61.6%) patients were male. The most common metastatic sites were thoracic lymph nodes (LNs) (84.3%), followed by lung to lung/lymphangitic spread (59%) and bone (54.8%). The distribution of patient characteristics revealed that age ≥65 years (69.1% vs 50.6%; P = 0.003) and male sex (84% vs 56.4%; P < 0.001) were more frequently found in SQ patients. Regarding metastatic features, bone metastasis (60.4% vs 30.9%; P < 0.001), lung to lung/lymphangitic metastasis (63% vs 42%; P = 0.001), and brain metastasis (35% vs 16%; P = 0.001) were significantly and more frequently found in Adenoca patients. Patients with high metastatic scores (score 3–6) were more frequently found to have Adenoca (91.6% vs 73.4%; P < 0.001). In multivariate prognostic evaluation, sex (P = 0.001), age (P < 0.001), histology (P < 0.001), LN status (P = 0.032), pleural/pericardial metastasis (P = 0.003), abdomen/pelvis metastasis (P < 0.001), axilla/neck metastasis (P = 0.006), and treatment factors (P < 0.001) remained independent prognostic factors affecting overall survival.
We observed distinctive patterns of primary metastases and clinical outcomes according to the histological subtypes in stage IV NSCLC. Future studies need to disclose the underlying mechanism of these unique metastatic features and tumor biologies.
PMCID: PMC4753937  PMID: 26871841
3.  Dedicated Spine Trauma Clinical Quality Registries: A Systematic Review 
Global Spine Journal  2013;3(4):265-272.
Study Design Systematic review.
Objective We assessed the current state of spine registries by collecting spine trauma data and assessing their compliance to defined registry standards of being clinical quality. We ascertained if these registries collected spinal cord injury data alone or with spine column trauma data.
Methods A systematic review was performed using MEDLINE and Embase databases for articles describing dedicated spinal cord and spine column databases published between January 1990 and April 2011. Correspondence with these registries was performed via e-mail or post. When no correspondence was possible, the registries were analyzed with best information available.
Results Three hundred eight full-text articles were reviewed. Of 41 registries identified, 20 registries fulfilled the criteria of being clinical quality. The main reason for failure to attain clinical quality designation was due to the unavailability of patient outcomes. Eight registries collected both spine column and spinal cord injury data with 33 collecting only traumatic spinal cord injury data.
Conclusion There is currently a paucity of clinical quality spine trauma registries. Clinical quality registries are important tools for demonstrating trends and outcomes, monitoring care quality, and resolving controversies in the management of spine trauma. An international spine trauma data set (containing both spinal cord and spine column injury data) and standardized approach to recording and analysis are needed to allow international multicenter collaboration and benchmarking.
PMCID: PMC3854593  PMID: 24436881
clinical quality; registry; database; spine trauma; benchmarking
4.  Transcriptional repression of CDKN2D by PML/RARα contributes to the altered proliferation and differentiation block of acute promyelocytic leukemia cells 
Wang, Y | Jin, W | Jia, X | Luo, R | Tan, Y | Zhu, X | Yang, X | Wang, X | Wang, K
Cell Death & Disease  2014;5(10):e1431-.
Cell proliferation and differentiation are highly coordinated processes. These two processes are disrupted during leukemogenesis, resulting in differentiation block and uncontrolled proliferation in leukemia. To understand the mechanisms disrupting the coordination between the two processes in acute promyelocytic leukemia (APL), we investigated the regulatory mechanism of the negative cell cycle regulator CDKN2D by the promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion protein and the role of CDKN2D in cell differentiation and proliferation. We found that CDKN2D expression in APL cells was significantly lower than that in normal promyelocytes. By chromatin immunoprecipitation and luciferase reporter assays, we showed that PML/RARα directly bound to and inhibited the transactivation of the CDKN2D promoter. Further evidence by the truncated and mutated CDKN2D promoters revealed that the everted repeat 8 (ER8) motif on the promoter was the binding site of PML/RARα. Forced expression of CDKN2D induced G0/G1 phase arrest and partial granulocytic differentiation in APL-derived NB4 cells, suggesting the function of CDKN2D in regulating both cell proliferation and granulocytic differentiation. Furthermore, all-trans retinoic acid (ATRA) significantly induced CDKN2D expression in APL cells and knockdown of CDKN2D expression during ATRA treatment partially blocked the ATRA-induced differentiation and cell cycle arrest. Collectively, our data indicate that CDKN2D repression by PML/RARα disrupts both cell proliferation and differentiation in the pathogenesis of APL, and induced expression of CDKN2D by ATRA alleviates the disruption of both processes to ensure treatment efficiency. This study provides a mechanism for coupling proliferation and differentiation in leukemic cells through the action of CDKN2D.
PMCID: PMC4649503  PMID: 25275592
5.  Finding the Missing Link in Lovastatin Biosynthesis: LovG is the Thioesterase Required for Dihydromonacolin L Release and Lovastatin Nonaketide Synthase Turnover 
Angewandte Chemie (International ed. in English)  2013;52(25):10.1002/anie.201302406.
The cryptic thioesterase LovG is found to be responsible for product release from the lovastatin nonaketide synthase (LNKS or LovB). The same enzyme also helps improving turnover of LovB through hydrolysis of incorrectly tailored intermediates.
PMCID: PMC3844545  PMID: 23653178
Lovastatin; Biosynthesis; Polyketide Synthase; Product Release; Thioesterase
6.  Amyloid-β1–42 oligomer accelerates senescence in adult hippocampal neural stem/progenitor cells via formylpeptide receptor 2 
Cell Death & Disease  2013;4(11):e924-.
The failure of adult hippocampal neurogenesis is increasingly considered as an important factor in the pathological correlates for memory decline in Alzheimer's disease (AD). Loss of adult-born neurons and abnormalities of neural stem/progenitor cells (NSPCs) within the dentate gyrus (DG) of adult hippocampus might contribute to this process. In this study, we showed that amyloid-β1–42 (Aβ42) oligomer triggers senescent phenotype of NSPCs in vitro. Oligomerized Aβ42 induced the production of senescence-associated biomarkers p16 and senescence-associated β-galactosidase (SA-β-gal) in adult mouse hippocampal NSPCs, as well as inhibited cells proliferation and differentiation. In the DG of amyloid precursor protein/presenilin1 (APP/PS1) transgenic mice, the number of senescent NSPCs was significantly increased and senescence-associated protein p16 was upregulated. Formylpeptide receptor 2 (FPR2), one of Aβ42 functional receptors, may be involved in NSPCs senescence. The FPR2 antagonist WRW4 significantly inhibited NSPCs senescence induced by Aβ42. In addition, the activation of p38 mitogen-activated protein kinase (MAPK) in response to the accumulation of reactive oxygen species (ROS) was involved in NSPCs senescence induced by Aβ42. WRW4 inhibited the accumulation of ROS and the activation of p38 MAPK in NSPCs. Our data suggest that Aβ42 accelerates NSPCs senescence via FPR2-dependent activation of its downstream ROS-p38 MAPK signaling, which limits the function of NSPCs and contributes to failure of neurogenesis. This is the first demonstration of NSPCs senescence response to Aβ42.
PMCID: PMC3847315  PMID: 24263098
Aβ42; adult hippocampal neural stem/progenitor cells; senescence; FPR2; ROS; p38 MAPK
8.  Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity 
Mi, Y-J | Hou, B | Liao, Q-M | Ma, Y | Luo, Q | Dai, Y-K | Ju, G | Jin, W-L
Cell Death and Differentiation  2012;19(7):1175-1186.
Nogo-A is originally identified as an inhibitor of axon regeneration from the CNS myelin. Nogo-A is mainly expressed by oligodendrocytes, and also by some neuronal subpopulations, particularly in the developing nervous system. Although extensive studies have uncovered regulatory roles of Nogo-A in neurite outgrowth inhibition, precursor migration, neuronal homeostasis, plasticity and neurodegeneration, its cell-autonomous functions in neurons are largely uncharacterized. Here, we show that HIV-1 trans-activating-mediated amino-Nogo-A protein transduction into cultured primary cortical neurons achieves an almost complete neuroprotection against oxidative stress induced by exogenous hydrogen peroxide (H2O2). Endogenously expressed neuronal Nogo-A is significantly downregulated upon H2O2 treatment. Furthermore, knockdown of Nogo-A results in more susceptibility to acute oxidative insults and markedly increases neuronal death. Interacting with peroxiredoxin 2 (Prdx2), amino-Nogo-A reduces reactive oxygen species (ROS) generation and extracellular signal-regulated kinase phosphorylation to exert neuroprotective effects. Structure–function mapping experiments reveal that, out of NiG-Δ20, a novel region comprising residues 290–562 of amino-Nogo-A is indispensable for preventing oxidative neuronal death. Moreover, mutagenesis analysis confirms that cysteine residues 424, 464 and 559 are involved in the inhibition of ROS generation and neuroprotective role of amino-Nogo-A. Our data suggest that neuronal Nogo-A might play a cell-autonomous role in improving neuronal survival against oxidative insult through interacting with Prdx2 and scavenging of ROS.
PMCID: PMC3374081  PMID: 22261619
Nogo-A; ROS; neuroprotection; Prdx2; cortical neurons
9.  Regulation of UDP-glucuronosyltransferase (UGT) 1A1 by progesterone and its impact on labetalol elimination 
We recently reported the increased oral clearance of labetalol in pregnant women. To elucidate the mechanism of the elevated oral clearance, we hypothesize that female hormones, at the high concentrations attainable during pregnancy, enhance hepatic metabolism of labetalol.Labetalol glucuronidation, the major elimination pathway of labetalol, was characterized by screening six recombinant human UGTs (UGT1A1, 1A4, 1A6, 1A9, 2B4, and 2B7) for their capacity to catalyze labetalol glucuronidation.The effect of female hormones (progesterone, estradiol, estriol, or estrone) on the promoter activities of relevant UDP glucuronosyltransferases (UGT) was investigated using a luciferase reporter assay in HepG2 cells. The involvement of estrogen receptor α (ERα) and pregnane X receptor (PXR) was examined by co-transfecting ERα- or PXR-constructs.UGT1A1 and UGT2B7 were identified as the major UGT enzymes producing labetalol glucuronides (trace amount of glucuronide conjugate was formed by UGT1A9). The activities of the UGT1A1 promoter containing PXR response elements were enhanced by progesterone but not by estrogens, indicating PXR-mediated induction of UGT1A1 promoter activity by progesterone. Results from semi-quantitative real-time PCR assays are consistent with the above findings. This effect of progesterone on UGT1A1 promoter activities was concentration-dependent.Promoter activities of UGT2B7 were not affected by either estrogens or progesterone.These results suggest a potential role for progesterone in regulating labetalol elimination by modulating expression of UGT1A1, leading to enhanced drug metabolism during pregnancy.
PMCID: PMC3691104  PMID: 18098064
Pregnancy; labetalol glucuronidation; UGT; metabolism; female hormones; induction
10.  Gamma band activity associated with BCI performance: simultaneous MEG/EEG study 
While brain computer interface (BCI) can be employed with patients and healthy subjects, there are problems that must be resolved before BCI can be useful to the public. In the most popular motor imagery (MI) BCI system, a significant number of target users (called “BCI-Illiterates”) cannot modulate their neuronal signals sufficiently to use the BCI system. This causes performance variability among subjects and even among sessions within a subject. The mechanism of such BCI-Illiteracy and possible solutions still remain to be determined. Gamma oscillation is known to be involved in various fundamental brain functions, and may play a role in MI. In this study, we investigated the association of gamma activity with MI performance among subjects. Ten simultaneous MEG/EEG experiments were conducted; MI performance for each was estimated by EEG data, and the gamma activity associated with BCI performance was investigated with MEG data. Our results showed that gamma activity had a high positive correlation with MI performance in the prefrontal area. This trend was also found across sessions within one subject. In conclusion, gamma rhythms generated in the prefrontal area appear to play a critical role in BCI performance.
PMCID: PMC3853408  PMID: 24367322
BCI-illiteracy; motor imagery BCI; performance prediction; gamma activity; MEG; EEG
11.  Acute appendicitis: relationships between CT-determined severities and serum white blood cell counts and C-reactive protein levels 
The British Journal of Radiology  2011;84(1008):1115-1120.
The aim of this study was to evaluate the relationships between the severity of appendicitis as depicted on CT and blood inflammatory markers of serum white blood cell (WBC) count and C-reactive protein (CRP).
CT images in 128 patients (109 surgically proven and 19 with clinically excluded appendicitis) were retrospectively reviewed. Two radiologists by consensus evaluated and scored (using a 0, 1 or 2 point scale) severities based on CT-determined appendiceal diameters, appendiceal wall changes, caecal changes, periappendiceal inflammatory stranding and phlegmon or abscess formation. We investigated whether CT findings were significantly related to elevated WBC counts or CRP levels and performed the correlations of WBC counts and CRP levels with CT severity scores. Patients were also subjectively classified using four grades from normal (Grade I) to perforated appendicitis (Grade IV) on the basis of CT findings to evaluate differences in WBC counts and CRP levels between grades.
Only appendiceal wall changes and the phlegmon or abscess formation were related to elevated WBC counts and CRP levels, respectively (p<0.05). CT severity scores were found to be more strongly correlated with CRP levels (r = 0.669) than with WBC counts (r = 0.222). On the basis of CT grades, the WBC counts in Grade I were significantly lower than in other grades (p<0.001), whereas CRP levels in Grade IV were significantly higher than in other grades (p<0.001).
CRP levels were found to correlate with CT-determined acute appendicitis severity and could be a useful predictor for perforated appendicitis, whereas WBC counts might be useful to detect early acute appendicitis.
PMCID: PMC3473821  PMID: 21123307
12.  Spinous process fractures in osteoporotic thoracolumbar vertebral fractures 
The British Journal of Radiology  2011;84(1007):1046-1049.
To evaluate the incidence and pattern of spinous process fractures (SPFs) in patients with osteoporotic compression fractures (OCFs) of the thoracolumbar spine.
Spinal MRI or CT of 398 female patients (age range 50–89 years, mean age 70 years) who had OCFs in the thoracolumbar spine were retrospectively reviewed. The incidence, location and imaging results for the SPFs were evaluated.
Of the 398 patients who had thoracolumbar OCFs, 14 (3.5%) had SPF. In six patients with single compression fractures, the SPF occurred at the level just above the vertebral compression fracture. In six out of seven patients with multiple continuous compression fractures, the SPF occurred just one level above the uppermost level of the compression fracture. The remaining one patient who had thoracolumbar spinal fixation at T12–L2 with continuous compression fractures in T12–L5 had a SPF in L2. In one patient who had multiple compression fractures in discontinuous levels (fractures at T10 and L1, respectively), the SPF occurred at T12. The directions of the fractures were vertical or oblique vertical (perpendicular to the long axis of the spinous process) in all cases.
In the presence of an OCF in the thoracolumbar spine, a SPF was found in 3.5% of cases, and most of the fractures were located just one level above the compression fracture. Therefore, in patients who have OCF, the possibility of a SPF in the level just above the compression fracture should be considered.
PMCID: PMC3473709  PMID: 21343317
13.  Identification of New Differentially Methylated Genes That Have Potential Functional Consequences in Prostate Cancer 
PLoS ONE  2012;7(10):e48455.
Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.
PMCID: PMC3485209  PMID: 23119026
14.  Oxidative dna damage of lymphocytes in peripheral blood and ascites in cancer patients 
Current Oncology  2012;19(Suppl 2):eS10-eS14.
Patients with malignant ascites (ma) usually experience poor quality of life, and treatment of this symptom remains a challenge. Oxidative stress, which can cause oxidative damage to dna, plays a pivotal role in carcinogenesis; however, the relationship between oxidative stress and dna damage to tumour-associated lymphocytes (tals) in ma is unclear.
We measured the total antioxidant capacity (tac) of plasma and ma supernatant in 31 cancer patients with ma, and we used a comet assay to assess dna damage to both peripheral blood mononuclear cells (pbmcs) and tals. Measurements in age- and sex-matched healthy volunteers were used as controls.
The tac of plasma was remarkably lower in cancer patients (9.73 ± 1.96 U/mL) than in healthy control subjects (11.31 ± 1.50 U/mL, p < 0.001). The tac of ma supernatant (6.34 ± 1.57 U/mL) was significantly lower than that of plasma in cancer patients (7.42 ± 1.36 U/mL, p < 0.001). The comet percentage of pbmcs was higher in cancer patients (17.26% ± 6.04%) than in healthy control subjects (9.44% ± 4.47%, p < 0.01). In cancer patients, the comet percentage of tals (36.14% ± 17.85%) was significantly higher than that of pbmcs (17.26% ± 6.04%, p < 0.001). In cancer patients with ma, negative correlations were observed between plasma tac and dna damage to pbmcs (r = −0.505, p = 0.004) and between the tac of ma supernatant and the comet percentage of tals (r = −0.588, p = 0.001).
Results indicate the presence of significant oxidative damage to the dna of lymphocytes in peripheral blood and ascites from patients with ma, being especially higher in the cells from ascites. The lower tac of ma supernatant may be related to a higher degree of dna damage to tals. The present study suggests that an oxidant–antioxidant imbalance may be one of the mechanisms leading to the dna damage detected in peripheral blood and local tals in patients with ma, which may provide a novel approach to the treatment of ma.
PMCID: PMC3413254  PMID: 22876163
Malignant ascites; tumour-associated lymphocytes; total antioxidant capacity; dna damage
15.  The detection and discrimination of malignant and benign focal hepatic lesions: T2 weighted vs diffusion-weighted MRI 
The British Journal of Radiology  2011;84(1000):319-326.
The purpose of this study was to evaluate the use of diffusion-weighted imaging (DWI) for the detection and characterisation of focal hepatic lesions compared with the use of T2 weighted imaging.
45 patients with 97 hepatic lesions (51 malignant lesions and 46 benign lesions) were included in this retrospective study. Malignant hepatic lesions included 12 hepatocellular carcinomas, 26 metastases and 13 intrahepatic cholangiocarcinomas. Benign hepatic lesions included 19 haemangiomas and 27 cysts. The MRI protocol for the upper abdomen included T2 weighted images, in- and opposed-phase T1 weighted images and dynamic T1 weighted images. Breath-hold fat-suppressed single-shot echo planar DWI was performed with the following parameters: 1338/66; b factors, 0, 50 and 800 s mm–2. Two independent observers reviewed the T2 weighted images and the DWI to detect and to characterise the hepatic lesions.
For detection of malignant hepatic lesions, the use of DWI showed a significantly higher detection rate than the use of T2 weighted images (p<0.05). However, there was no significant difference between the use of DWI and T2 weighted images for benign hepatic lesions. For the differentiation between malignant and benign hepatic lesions, there was no significant difference in sensitivity, specificity and accuracy between the use of T2 weighted images and the use of DWI.
The use of DWI was better for the detection of malignant hepatic lesions than the use of T2 weighted images. However, for detection of benign hepatic lesions and characterisation of hepatic lesions, the use of DWI was equivalent to the use of T2 weighted images.
PMCID: PMC3473479  PMID: 20959371
16.  Combined Analysis of AFP and HCCR-1 as an Useful Serological Marker for Small Hepatocellular Carcinoma: A Prospective Cohort Study 
Disease markers  2012;32(4):265-271.
Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors in the world. The only serological marker widely used for the diagnosis of HCC is alpha-fetoprotein (AFP). Despite that AFP is widely used for the diagnosis of HCC, it has a limit as a serological marker due to its low sensitivity and specificity. The human cervical cancer proto-oncogene 1 (HCCR-1) was previously reported as a new biomarker for HCC. To further evaluate the HCCR-1 as a biomarker for HCC, we conducted the prospective cohort study. We evaluated the significance of simultaneous measurement of 2 tumor markers in the diagnosis of HCC in China, Japan and Korea. Two markers for HCC, AFP and HCCR-1, were measured in the sera obtained from 1,338 patients at the time of initial diagnosis of HCC. Of the 1338 HCC patients, 616 (46%) and 686 (51.3%) were sero-positive for AFP and HCCR-1, respectively. The positive rate for HCC was increased up to 74.1% in combined use of AFP and HCCR-1. Many cases (54%) for AFP-negative HCC were positive for HCCR-1 and vice versa. More importantly, the diagnostic rate for small HCC (< 2 cm) was significantly improved in the combined analysis of AFP and HCCR-1 to 56.9% although it was only 40.1% and 23.4% in the single analysis of HCCR-1 and AFP, respectively. Our result suggests that the HCCR-1 could be an useful biomarker for HCC while the diagnostic rate could be significantly improved in the combined use of HCCR-1 and AFP.
PMCID: PMC3826799  PMID: 22430193
Hepatocellular carcinoma; biomarker; AFP; HCCR-1
17.  Olfactory copy number association with age at onset of Alzheimer disease 
Neurology  2011;76(15):1302-1309.
Copy number variants (CNVs) have been recognized as a source of genetic variation that contributes to disease phenotypes. Alzheimer disease (AD) has high heritability for occurrence and age at onset (AAO). We performed a cases-only genome-wide CNV association study for age at onset of AD.
The discovery case series (n = 40 subjects with AD) was evaluated using array comparative genome hybridization (aCGH). A replication case series (n = 507 subjects with AD) was evaluated using Affymetrix array (n = 243) and multiplex ligation-dependent probe amplification (n = 264). Hazard models related onset age to CNV.
The discovery sample identified a chromosomal segment on 14q11.2 (19.3–19.5 Mb, NCBI build 36, UCSC hg18 March 2006) as a region of interest (genome-wide adjusted p = 0.032) for association with AAO of AD. This region encompasses a cluster of olfactory receptors. The replication sample confirmed the association (p = 0.035). The association was found for each APOE4 gene dosage (0, 1, and 2).
High copy number in the olfactory receptor region on 14q11.2 is associated with younger age at onset of AD.
PMCID: PMC3090061  PMID: 21482944
18.  Complete Reconstitution of a Highly-Reducing Iterative Polyketide Synthase 
Science (New York, N.Y.)  2009;326(5952):589-592.
Highly-reducing iterative polyketide synthases are large multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of LovB (the Lovastatin Nonaketide Synthase) from an engineered strain of Saccharomyces cerevisiae, and complete reconstitution of its catalytic function in the presence and absence of cofactors (NADPH, SAM) and its partner enzyme, the enoyl reductase LovC. The results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding the functions and structures of this family of enzymes.
PMCID: PMC2875069  PMID: 19900898
19.  Evaluation of combinatorial cis-regulatory elements for stable gene expression in chicken cells 
BMC Biotechnology  2010;10:69.
Recent successes in biotechnological application of birds are based on their unique physiological traits such as unlimited manipulability onto developing embryos and simple protein constituents of the eggs. However it is not likely that target protein is produced as kinetically expected because various factors affect target gene expression. Although there have been various attempts to minimize the silencing of transgenes, a generalized study that uses multiple cis-acting elements in chicken has not been made. The aim of the present study was to analyze whether various cis-acting elements can help to sustain transgene expression in chicken fibroblasts.
We investigated the optimal transcriptional regulatory elements for enhancing stable transgene expression in chicken cells. We generated eight constructs that encode enhanced green fluorescent protein (eGFP) driven by either CMV or CAG promoters (including the control), containing three types of key regulatory elements: a chicken lysozyme matrix attachment region (cMAR), 5'-DNase I-hypersensitive sites 4 (cHS4), and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Then we transformed immortalized chicken embryonic fibroblasts with these constructs by electroporation, and after cells were expanded under G418 selection, analyzed mRNA levels and mean fluorescence intensity (MFI) by quantitative real-time PCR and flow cytometry, respectively. We found that the copy number of each construct significantly decreased as the size of the construct increased (R2 = 0.701). A significant model effect was found in the expression level among various constructs in both mRNA and protein (P < 0.0001). Transcription with the CAG promoter was 1.6-fold higher than the CMV promoter (P = 0.027) and the level of eGFP expression activity in cMAR- or cHS4-flanked constructs increased by two- to three-fold compared to the control CMV or CAG promoter constructs. In addition, flow cytometry analysis showed that constructs having cis-acting elements decreased the level of gene silencing as well as the coefficient of variance of eGFP-expressing cells (P < 0.0001).
Our current data show that an optimal combination of cis-acting elements and promoters/enhancers for sustaining gene expression in chicken cells is suggested. These results provide important information for avian transgenesis and gene function studies in poultry.
PMCID: PMC2949789  PMID: 20849657
20.  Transdifferentiation-inducing HCCR-1 oncogene 
BMC Cell Biology  2010;11:49.
Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules.
We report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell.
Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.
PMCID: PMC2909153  PMID: 20591135
21.  Effects of Caffeine on Exercise Performance in Sedentary Females 
The purpose of the study was to examine the effect of caffeine ingestion on total work, average power, oxygen consumption (VO2), respiratory exchange ratio (RER), ratings of perceived exertion (RPE), heart rate (HR) and energy expenditure (kJ) during stationary cycling at a standardised power output, as well as during a set time period where participants were required to cycle as fast as they could. Ten healthy, sedentary, female, non- regular caffeine users completed 15 min of stationary cycling at a standardised power output equating to 65% HRmax (Phase A), followed by 10 min of stationary cycling where they were required to cycled as fast as they could (Phase B) after ingesting 6.0 mg·kg-1 of caffeine or placebo 60 min prior to exercise. VO2 and energy expenditure were significantly higher at the end of Phase A (p = 0.008 and p = 0.011, respectively). All other variables examined in Phase A were similar between trials. In Phase B, there were no significant differences found for any variable assessed. While caffeine ingestion resulted in significant increases in VO2 and energy expenditure during steady-state exercise, it did not improve cycling performance during a 10 min trial where participants were required to cycle as fast as they could.
Key pointsA 6.0 mg·kg-1 dose of caffeine did not improve work done (J·kg-1) or mean power (W) during 10 min of self-paced stationery cycling in sedentary female participants.A 6.0 mg·kg-1 dose of caffeine significantly increased VO2 and energy expenditure (kJ) during 15 min of steady-state stationery cycling in sedentary female participants.A 6.0 mg·kg-1 dose of caffeine did not significantly affect RPE, RER or HR during 15 min of steady-state cycling or 10 min of cycling performed as fast as the participant could achieve, when compared to placebo, in sedentary female participants.
PMCID: PMC3761740  PMID: 24149684
Sub-maximal exercise; rating of perceived exertion; energy expenditure; weight maintenance
22.  Epidermal growth factor induces HCCR expression via PI3K/Akt/mTOR signaling in PANC-1 pancreatic cancer cells 
BMC Cancer  2010;10:161.
Human cervical cancer oncoprotein 1 (HCCR-1), reported as a negative regulator of p53, is over-expressed in a variety of human cancers. However, it is yet unknown whether HCCR-1 plays any role in pancreatic cancer development. The aim of this study was to investigate the effect of epidermal growth factor on the expression of HCCR in pancreatic cancer cells, and to explore if PI3K/Akt/mTOR signaling pathway mediated this expression.
A polyclonal antibody against HCCR protein was raised by immunizing Balb/c mice with the purified recombinant protein pMBPc-HCCR. Tissue samples were constructed on a tissue chip, and the expression of HCCR was investigated by immunohistochemistry assay and Western blotting. Pancreatic cell line, PANC-1 cells were stably transfected with plasmids containing sense-HCCR-1 fragment and HCCR siRNA fragment. MTT and transwell assay were used to investigate the proliferation and invasion of stable tansfectants. The specific inhibitor of PI3K and mTOR was used to see if PI3K/mTOR signal transduction was involved in the induction of HCCR gene expression. A Luciferase assay was used to see if Akt can enhance the HCCR promoter activity.
HCCR was up-regulated in pancreatic tumor tissues (mean Allred score 4.51 ± 1.549 vs. 2.87 ± 2.193, P < 0.01), especially with high expression in poorly differentiated pancreatic cancer. The growth of cells decreased in HCCR-1 siRNA transfected cells compared with vector transfectants. The number of invasion cells was significantly lower in HCCR-1 siRNA transfected cells (24.4 ± 9.9) than that in vector transfectants (49.1 ± 15.4). Treatment of PANC-1 cells with epidermal growth factor increased HCCR protein level in a dose- and time-dependent manner. However, application of LY294002 and rapamycin caused a dramatic reduction of epidermal growth factor-induced HCCR expression. Over-expression of exogenous constitutively active Akt increased the HCCR promoter activity; in contrast, dominant negative Akt decreased the promoter activity.
EGF-induced HCCR-1 over-expression is mediated by PI3K/AKT/mTOR signaling which plays a pivotal role in pancreatic tumor progression, suggesting that HCCR-1 could be a potential target for cancer therapeutics.
PMCID: PMC2880295  PMID: 20423485
23.  TCF/β-catenin plays an important role in HCCR-1 oncogene expression 
BMC Molecular Biology  2009;10:42.
Oncogene HCCR-1 functions as a negative regulator of the p53 and contributes to tumorigenesis of various human tissues. However, it is unknown how HCCR-1 contributes to the cellular and biochemical mechanisms of human tumorigenesis.
In this study, we showed how the expression of HCCR-1 is modulated. The luciferase activity assay indicated that the HCCR-1 5'-flanking region at positions -166 to +30 plays an important role in HCCR-1 promoter activity. Computational analysis of this region identified two consensus sequences for the T-cell factor (TCF) located at -26 to -4 (Tcf1) and -136 to -114 (Tcf2). Mutation at the Tcf1 site led to a dramatic decrease in promoter activity. Mobility shift assays (EMSA) revealed that nuclear proteins bind to the Tcf1 site, but not to the Tcf2 site. LiCl, Wnt signal activator by GSK-3β inhibition, significantly increased reporter activities in wild-type Tcf1-containing constructs, but were without effect in mutant Tcf1-containing constructs in HEK/293 cells. In addition, endogenous HCCR-1 expression was also increased by treatment with GSK-3β inhibitor, LiCl or AR-A014418 in HEK/293 and K562 cells. Finally, we also observed that the transcription factor, TCF, and its cofactor, β-catenin, bound to the Tcf1 site.
These findings suggest that the Tcf1 site on the HCCR-1 promoter is a major element regulating HCCR-1 expression and abnormal stimulation of this site may induce various human cancers.
PMCID: PMC2693525  PMID: 19435525
24.  Quantification of glyceryl trinitrate effect through analysis of the synthesised ascending aortic pressure waveform 
Heart  2002;88(2):143-148.
Objective: To establish through analysis of the radial pressure pulse waveform the dose dependent effects of glyceryl trinitrate (GTN) on properties of different blood vessels.
Design: Radial pulse waveform was measured in randomised order before, during a five hour application of a GTN patch delivering 0.104–0.625 mg/h, and for two hours after patch removal. The radial pressure waveform (Millar applanation tonometer) was convolved into an ascending aortic wave using a generalised transfer function (SphygmoCor process) enabling measurement of aortic systolic, diastolic, pulse, mean, and augmented pressure and left ventricular ejection duration in addition to standard brachial cuff pressures.
Setting: Fu Wai and Ren Ming hospitals in Beijing, China.
Patients: 46 recumbent hospitalised patients aged 56 (9) years, awaiting electrophysiological or other diagnostic studies, fasting, and with other treatments suspended.
Major outcome measures: Conventional brachial pressure measures and data from the synthesised aortic pulse.
Results: There was no consistent change in heart rate or brachial pressures except for a decrease in systolic and pulse pressures (p < 0.01) at dose > 0.416 mg/h. In contrast, there were substantial and significant (p < 0.0001) decreases in aortic systolic, pulse, and augmented pressures at all doses, mean pressure (p < 0.001) at doses > 0.416 mg/h, and ejection duration (p < 0.001) at doses > 0.208 mg/h.
Conclusions: Pulse waveform analysis exposes dose dependent effects of GTN on the aortic waveform, suggesting muscular conduit arterial dilatation with reduced wave reflection at the lowest dose, arteriolar dilatation and decreased peripheral resistance at the highest dose, and venous dilatation at the intermediate dose.
PMCID: PMC1767201  PMID: 12117838
glyercyl trinitrate; pulsed waveform analysis; aortic pressure waveform
25.  Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells 
Jin, W | Wu, L | Liang, K | Liu, B | Lu, Y | Fan, Z
British Journal of Cancer  2003;89(1):185-191.
PMCID: PMC2394213  PMID: 12838322
breast cancer; drug resistance; Ras; Akt; MAPK

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