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1.  Adjuvant Chemotherapy Correlates with Improved Survival after Radical Cystectomy in Patients with pT3b (Macroscopic Perivesical Tissue Invasion) Bladder Cancer 
Journal of Cancer  2015;6(8):750-758.
Object: To elucidate the impact of adjuvant chemotherapy (ACH) on survival in patients with urothelial carcinoma of the bladder (UCB) diagnosed with pT3 disease (perivesical tissue invasion).
Methods: We reviewed the clinical data of 424 patients who underwent radical cystectomy (RC) with bilateral pelvic lymphadenectomy for UCB in our institution between 1991 and 2012. None of the patients received neoadjuvant chemotherapy. Of all patients, 101 (23.8%) were diagnosed with pT3 disease: pT3a in 43 patients and pT3b in 58 patients. The Kaplan-Meier method with the log-rank test was used to estimate and compare overall survival (OS) and cancer-specific survival (CSS) between groups. Multivariate Cox proportional hazard models were used to identify predictors of OS and CSS.
Results: Five-year OS (48.5% vs. 45.6%) and CSS (56.1% vs. 56.2%) were similar in the pT3a and pT3b groups (p = 0.658 and 0.840, respectively). In all pT3 patients, ACH administration was an independent predictor for OS (p = 0.018), but not CSS (p = 0.623) on multivariate analyses. On multivariate analysis according to the pT3 sub-stage, ACH was significantly associated with improved OS (hazard ratio [HR] 0.35; 95% confidence interval [CI] 0.17-0.72, p = 0.004) and CSS (HR 0.33; 95% CI 0.10-0.85, p = 0.022) in only the pT3b group.
Conclusion: Our data suggest that in pT3b disease, characterized by macroscopic perivesical tissue invasion, patients may obtain an OS benefit from the administration of ACH. Thus, our findings provide evidence for establishing appropriate indications for ACH in muscle invasive UCB.
PMCID: PMC4504111  PMID: 26185537
urothelial carcinoma; radical cystectomy; perivesical tissue invasion; adjuvant chemotherapy; survival
2.  Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples 
PLoS ONE  2016;11(2):e0148047.
There is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology.
This paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9) processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY). Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies) and their sequencing data is subjected to a novel analytical pipeline.
Results are presented at family and genus level. The Kullback-Leibler divergence (DKL), a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst) average DKL while the V4 gave the lowest (best) average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria.
The results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at different time points throughout a clinical protocol.
PMCID: PMC4734828  PMID: 26829716
3.  Systemic Inflammatory Response Based on Neutrophil-to-Lymphocyte Ratio as a Prognostic Marker in Bladder Cancer 
Disease Markers  2016;2016:8345286.
A growing body of evidence suggests that systemic inflammatory response (SIR) in the tumor microenvironment is closely related to poor oncologic outcomes in cancer patients. Over the past decade, several SIR-related hematological factors have been extensively investigated in an effort to risk-stratify cancer patients to improve treatment selection and to predict posttreatment survival outcomes in various types of cancers. In particular, one readily available marker of SIR is neutrophil-to-lymphocyte ratio (NLR), which can easily be measured on the basis of absolute neutrophils and absolute lymphocytes in a differential white blood cell count performed in the clinical setting. Many investigators have vigorously assessed NLR as a potential prognostic biomarker predicting pathological and survival outcomes in patients with urothelial carcinoma (UC) of the bladder. In this paper, we aim to present the prognostic role of NLR in patients with UC of the bladder through a thorough review of the literature.
PMCID: PMC4736380  PMID: 26880857
4.  Knowledge and Practice Behaviors Regarding Urinary Incontinence Among Korean Healthcare Providers in Long-term Care Hospitals 
The purpose of this study was to assess the current management status of patients with urological issues and to examine the level of knowledge and practice behaviors regarding urinary incontinence (UI) among Korean healthcare providers in long-term care hospitals.
This study used a cross-sectional descriptive design with a written questionnaire to assess knowledge and practice behaviors of 756 healthcare providers in 11 long-term care hospitals in Korean metropolitan areas.
A total 42.6% of participants reported that more than 50% of patients had urologic issues, and that 68.1% of patients were regularly sent to urologists; no participants reported an on-site urologist in their facility. Participants identified collaboration with other hospitals and regular consultations by urologists as important factors in improving urologic care. Although the overall UI knowledge score was upper intermediate, a knowledge deficit was found for risk factors of UI. The knowledge level of physicians was significantly higher than that of other healthcare providers. Practice behaviors of nurses seemed to be better than those of other healthcare providers.
Systematic collaboration between healthcare providers and urologic specialists, enhancing staff competence, and patient-tailored intervention should be recommended to improve quality of care for patients with urologic issues in long-term care hospitals.
PMCID: PMC4703933  PMID: 26739180
Urinary Incontinence; Knowledge; Professional Practice; Health Personnel; Long-Term Care
5.  The clinical application of the sliding loop technique for renorrhaphy during robot-assisted laparoscopic partial nephrectomy: Surgical technique and outcomes 
Korean Journal of Urology  2015;56(11):762-768.
To report the initial clinical outcomes of the newly devised sliding loop technique (SLT) used for renorrhaphy in patients who underwent robot-assisted laparoscopic partial nephrectomy (RALPN) for small renal mass.
Materials and Methods
We reviewed the surgical videos and medical charts of 31 patients who had undergone RALPN with the SLT renorrhaphy performed by two surgeons (CWJ and CK) between January 2014 and October 2014. SLT renorrhaphy was performed after tumor excision and renal parenchymal defect repair. Assessed outcomes included renorrhaphy time (RT), warm ischemic time, perioperative complications, and perioperative renal function change. RT was defined as interval from the end of bed suture to the renal artery declamping.
In all patients, sliding loop renorrhaphy was successfully conducted without conversions to radical nephrectomy or open approaches. Mean renorrhaphy and warm ischemic time were 9.0 and 22.6 minutes, respectively. After completing renorrhaphy, there were no adverse events such as dehiscence of approximated renal parenchyma, renal parenchymal tearing, or significant bleeding. Furthermore, no postoperative complications or significant renal function decline were observed as of the last follow-up for all patients. The limitations of this study include the small volume case series, the retrospective nature of the study, and the heterogeneity of surgeons.
From our initial clinical experience, SLT may be an efficient and safe renorrhaphy method in real clinical practice. Further large scale, prospective, long-term follow-up, and direct comparative studies with other techniques are required to confirm the clinical applicability of SLT.
PMCID: PMC4643172  PMID: 26568794
Nephrectomy; Renorrhphy; Small renal mass; Technique
6.  Highly efficient organic light emitting diodes formed by solution processed red emitters with evaporated blue common layer structure 
Scientific Reports  2015;5:15903.
We prepared highly-efficient solution-processed red phosphorescent organic light emitting diodes (PHOLEDs) with a blue common layer structure that can reasonably confine the triplet excitons inside of the red emission layer (EML) with the assistance of a bipolar exciton blocking layer. The red PHOLEDs containing EML with a 7 : 3 ratio of 11-(4,6-diphenyl-[1,3,5]triazin-2-yl)-12-phenyl-11,12-dihydro-11,12-diaza-indeno[2,1-a]fluorene (n-type host, NH) : 4-(3-(triphenylen-2-yl)phenyl)dibenzo[b,d]thiophene (p-type host, PH) doped with 5% Iridium(III) bis(2-(3,5-dimethylphenyl)quinolinato-N,C2’)tetramethylheptadionate (Red Dopant, RD) produced the highest current and power efficiencies at 23.4 cd/A and 13.6 lm/W, with a 19% external quantum efficiency at 1000 cd/m2. To the best of our knowledge, such efficiency was the best among those that have been obtained from solution-processed small molecular red PHOLEDs. In addition, the host molecules utilized in this study have no flexible spacers, such as an alkyl chain, which normally deteriorate the stability of the device.
PMCID: PMC4626861  PMID: 26514274
7.  A Representational Similarity Analysis of the Dynamics of Object Processing Using Single-Trial EEG Classification 
PLoS ONE  2015;10(8):e0135697.
The recognition of object categories is effortlessly accomplished in everyday life, yet its neural underpinnings remain not fully understood. In this electroencephalography (EEG) study, we used single-trial classification to perform a Representational Similarity Analysis (RSA) of categorical representation of objects in human visual cortex. Brain responses were recorded while participants viewed a set of 72 photographs of objects with a planned category structure. The Representational Dissimilarity Matrix (RDM) used for RSA was derived from confusions of a linear classifier operating on single EEG trials. In contrast to past studies, which used pairwise correlation or classification to derive the RDM, we used confusion matrices from multi-class classifications, which provided novel self-similarity measures that were used to derive the overall size of the representational space. We additionally performed classifications on subsets of the brain response in order to identify spatial and temporal EEG components that best discriminated object categories and exemplars. Results from category-level classifications revealed that brain responses to images of human faces formed the most distinct category, while responses to images from the two inanimate categories formed a single category cluster. Exemplar-level classifications produced a broadly similar category structure, as well as sub-clusters corresponding to natural language categories. Spatiotemporal components of the brain response that differentiated exemplars within a category were found to differ from those implicated in differentiating between categories. Our results show that a classification approach can be successfully applied to single-trial scalp-recorded EEG to recover fine-grained object category structure, as well as to identify interpretable spatiotemporal components underlying object processing. Finally, object category can be decoded from purely temporal information recorded at single electrodes.
PMCID: PMC4546653  PMID: 26295970
8.  Trends in the Use of Chemotherapy before and after Radical Cystectomy in Patients with Muscle-invasive Bladder Cancer in Korea 
Journal of Korean Medical Science  2015;30(8):1150-1156.
We investigated trends in perioperative chemotherapy use, and determined factors associated with neoadjuvant chemotherapy (NAC) and adjuvant chemotherapy (AC) use in Korean patients with muscle-invasive bladder cancer (MIBC). We recruited 1,324 patients who had MIBC without nodal invasion or metastases and had undergone radical cystectomies (RC) between 2003 and 2013. The study's cut-off time for AC was three months after surgery, and the study's timespan was divided into three periods based on NAC use, namely, 2003-2005, 2006-2009, and 2010-2013. Complete remission was defined as histologically confirmed T0N0M0 after RC. NAC and AC were administered to 7.3% and 18.1% of the patients, respectively. The median time interval between completing NAC and undergoing RC was 32 days and the mean number of cycles was 3.2. The median time interval between RC and AC was 43 days and the mean number of cycles was 4.1. Gemcitabine and cisplatin were most frequently used in combination for NAC (49.0%) and AC (74.9%). NAC use increased significantly from 4.6% between 2003 and 2005 to 8.4% between 2010 and 2013 (P < 0.05), but AC use did not increase. Only 1.9% of patients received NAC and AC. Complete remission after NAC was achieved in 12 patients (12.5%). Multivariable modeling revealed that an advanced age, the earliest time period analyzed, and clinical tumor stage ≤ cT2 bladder cancer were negatively associated with NAC use (P < 0.05). While NAC use has slowly increased over time, it remains an underutilized therapeutic approach in Korean clinical practice.
PMCID: PMC4520947  PMID: 26240494
Urinary Bladder Neoplasms; Neoadjuvant Therapy; Adjuvant; Chemotherapy; Cystectomy
9.  Predictors of Neurocognitive Syndromes in Combat Veterans 
Cureus  null;7(7):e293.
Traumatic brain injury, depression and posttraumatic stress disorder (PTSD) are neurocognitive syndromes often associated with impairment of physical and mental health, as well as functional status. These syndromes are also frequent in military service members (SMs) after combat, although their presentation is often delayed until months after their return. The objective of this prospective cohort study was the identification of independent predictors of neurocognitive syndromes upon return from deployment could facilitate early intervention to prevent disability. We completed a comprehensive baseline assessment, followed by serial evaluations at three, six, and 12 months, to assess for new-onset PTSD, depression, or postconcussive syndrome (PCS) in order to identify baseline factors most strongly associated with subsequent neurocognitive syndromes. On serial follow-up, seven participants developed at least one neurocognitive syndrome: five with PTSD, one with depression and PTSD, and one with PCS. On univariate analysis, 60 items were associated with syndrome development at p < 0.15. Decision trees and ensemble tree multivariate models yielded four common independent predictors of PTSD: right superior longitudinal fasciculus tract volume on MRI; resting state connectivity between the right amygdala and left superior temporal gyrus (BA41/42) on functional MRI; and single nucleotide polymorphisms in the genes coding for myelin basic protein as well as brain-derived neurotrophic factor. Our findings require follow-up studies with greater sample size and suggest that neuroimaging and molecular biomarkers may help distinguish those at high risk for post-deployment neurocognitive syndromes.
PMCID: PMC4524772  PMID: 26251769
traumatic brain injury; depression; posttraumatic stress disorder; combat military veterans; neuroimaging; biomarkers
10.  Lymph node density as a prognostic variable in node-positive bladder cancer: a meta-analysis 
BMC Cancer  2015;15:447.
Although lymph node (LN) status and the LN burden determine the outcome of bladder cancer patients treated with cystectomy, compelling arguments have been made for the incorporation of LN density into the current staging system. Here, we investigate the relationship between LN density and clinical outcome in patients with LN-positive disease, following radical cystectomy for bladder cancer.
PubMed, SCOPUS, the Institute for Scientific Information Web of Science, and the Cochrane Library were searched to identify relevant published literature.
Fourteen studies were included in the meta-analysis, with a total number of 3311 patients. Of these 14 publications, 6 studies, (533 patients), 10 studies (2966 patients), and 5 studies (1108 patients) investigated the prognostic association of LN density with disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS), respectively. The pooled hazard ratio (HR) for DFS was 1.45 (95 % confidence interval [CI], 1.10–1.91) without heterogeneity (I2 = 0 %, p = 0.52). Higher LN density was significantly associated with poor DSS (pooled HR, 1.53; 95 % CI, 1.23–1.89). However, significant heterogeneity was found between studies (I2 = 66 %, p = 0.002). The pooled HR for OS was statistically significant (pooled HR, 1.45; 95 % CI, 1.11–1.90) without heterogeneity (I2 = 42 %, p = 0.14). The results of the Begg and Egger tests suggested that publication bias was not evident in this meta-analysis.
The data from this meta-analysis indicate that LN density is an independent predictor of clinical outcome in LN-positive patients. LN density may be useful in future staging systems, thus allowing better prognostic classification of LN-positive bladder cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1448-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4450458  PMID: 26027955
Bladder cancer; Meta-analysis; Lymph node density; Prognosis; Radical cystectomy
11.  Histological variant as the significant predictor of survival in patients with lymph node positive urothelial carcinoma of the bladder 
Scientific Reports  2015;5:9626.
The aim of this study was to evaluate the impact of histological variants of urothelial carcinoma (UC) on survival outcomes in patients with lymph node (LN) positive UC of the bladder. We reviewed and analyzed the clinical data from 424 patients who underwent radical cystectomy (RC) with pelvic lymph node dissection (PLND) for UC of the bladder and who did not receive neoadjuvant chemotherapy in our institution between 1991 and 2012. In total, 92 patients (21.7%) had histologically confirmed LN positive disease. In the LN negative group (332 patients), histological variants of UC were not a significant predictor in univariate analysis. However, in the LN positive group, histological variants of UC were a significant independent prognostic factor of overall survival (hazard ratio (HR) 3.54; 95% confidence interval (CI) 1.77–7.08, p < 0.001) and cancer specific survival (HR 3.66; 95% CI 1.69–7.90, p = 0.001) in both uni-variate and multivariate Cox regression analyses. The presence of histological variants of UC may indicate a worse prognosis in LN positive patients after RC with PLND for UC of the bladder and more aggressive adjuvant therapy may be required for the improvement of postoperative survival.
PMCID: PMC4427059  PMID: 25959144
12.  The prognostic impact of perioperative blood transfusion on survival in patients with bladder urothelial carcinoma treated with radical cystectomy 
Korean Journal of Urology  2015;56(4):295-304.
The aim of our study was to assess the influence of perioperative blood transfusion (PBT) on survival outcomes following radical cystectomy (RC) and pelvic lymph node dissection (PLND).
Materials and Methods
We reviewed and analyzed the clinical data of 432 patients who underwent RC for bladder cancer from 1991 to 2012. PBT was defined as the transfusion of allogeneic red blood cells during RC or postoperative hospitalization.
Of all patients, 315 patients (72.9%) received PBT. On multivariate logistic regression analysis, female gender (p=0.015), a lower preoperative hemoglobin level (p=0.003), estimated blood loss>800 mL (p<0.001), and performance of neoadjuvant chemotherapy (p<0.001) were independent risk factors related to requiring perioperative transfusions. The receipt of PBT was associated with increased overall mortality (hazard ratio, 1.91; 95% confidence interval, 1.25-2.94; p=0.003) on univariate analysis, but its association was not confirmed by multivariate analysis (p=0.058). In transfused patients, a transfusion of >4 packed red blood cell units was an independent predictor of overall survival (p=0.007), but not in cancer specific survival.
Our study was not conclusive to detect a clear association between PBT and survival after RC. However, the efforts should be made to continue limiting the overuse of transfusion especially in patients who are expected to have a high probability of PBT, such as females and those with a low preoperative hemoglobin level and history of neoadjuvant chemotherapy.
PMCID: PMC4392029  PMID: 25874043
Blood transfusion; Cystectomy; Survival; Urinary bladder neoplasms
13.  Disease-Free Survival at 2 and 3 Years is a Significant Early Surrogate Marker Predicting the 5-Year Overall Survival in Patients Treated with Radical Cystectomy for Urothelial Carcinoma of the Bladder: External Evaluation and Validation in a Cohort of Korean Patients 
Frontiers in Oncology  2015;5:246.
We aimed to externally validate the association of 2- and 3-year disease-free survival (DFS) with 5-year overall survival (OS) in patients treated with radical cystectomy (RC) for urothelial carcinoma (UC) of the bladder.
Materials and Methods
We reviewed the clinical data of 422 patients who underwent RC for UC of the bladder in our institution between 1991 and 2012. Survival curves were plotted with the Kaplan–Meier method. The Kappa statistic and Kendall tau-b test were used to assess the agreements between 2- and 3-year DFS and 5-year OS.
In the entire study population, 2- and 3-year DFS and 5-year OS rates were 76.4, 71.5, and 67.4%, respectively. All Kappa and Kendall’s tau-b test values for agreements between 2- and 3-year DFS and 5-year OS were more than 0.40, indicating moderate agreement for all patients and in each patient subgroup selected according to specific variables (all p-values <0.05). Kaplan–Meier analysis for DFS and Cox-proportional hazard models for landmark analysis at each time point indicated that most recurrences occurred within 3 years after surgery. The 5-year OS rates of patients who were recurrence-free at each time point gradually increased to more than 95% in an extended recurrence-free interval of 12–36 months.
Our external validation results support the existing finding that 2- and 3-year DFS can be a valid early surrogate end point to predict 5-year OS after RC in patients with UC of the bladder.
PMCID: PMC4625059  PMID: 26579498
urothelial carcinoma; radical cystectomy; surrogate; disease-specific survival; overall survival
14.  MicroRNA-22 and promoter motif polymorphisms at the Chga locus in genetic hypertension: functional and therapeutic implications for gene expression and the pathogenesis of hypertension 
Human Molecular Genetics  2013;22(18):3624-3640.
Hypertension is a common hereditary syndrome with unclear pathogenesis. Chromogranin A (Chga), which catalyzes formation and cargo storage of regulated secretory granules in neuroendocrine cells, contributes to blood pressure homeostasis centrally and peripherally. Elevated Chga occurs in spontaneously hypertensive rat (SHR) adrenal glands and plasma, but central expression is unexplored. In this report, we measured SHR and Wistar–Kyoto rat (control) Chga expression in central and peripheral nervous systems, and found Chga protein to be decreased in the SHR brainstem, yet increased in the adrenal and the plasma. By re-sequencing, we systematically identified five promoter, two coding and one 3′-untranslated region (3′-UTR) polymorphism at the SHR (versus WKY or BN) Chga locus. Using HXB/BXH recombinant inbred (RI) strain linkage and correlations, we demonstrated genetic determination of Chga expression in SHR, including a cis-quantitative trait loci (QTLs) (i.e. at the Chga locus), and such expression influenced biochemical determinants of blood pressure, including a cascade of catecholamine biosynthetic enzymes, catecholamines themselves and steroids. Luciferase reporter assays demonstrated that the 3′-UTR polymorphism (which disrupts a microRNA miR-22 motif) and promoter polymorphisms altered gene expression consistent with the decline in SHR central Chga expression. Coding region polymorphisms did not account for changes in Chga expression or function. Thus, we hypothesized that the 3′-UTR and promoter mutations lead to dysregulation (diminution) of Chga in brainstem cardiovascular control nuclei, ultimately contributing to the pathogenesis of hypertension in SHR. Accordingly, we demonstrated that in vivo administration of miR-22 antagomir to SHR causes substantial (∼18 mmHg) reductions in blood pressure, opening a novel therapeutic avenue for hypertension.
PMCID: PMC3749858  PMID: 23674521
15.  Multifactorial, Site-Specific Recurrence Models after Radical Cystectomy for Urothelial Carcinoma: External Validation in a Cohort of Korean Patients 
PLoS ONE  2014;9(6):e100491.
The aim of this study was to evaluate the accuracy of site-specific recurrence models after radical cystectomy in the Korean population.
Materials and Methods
We conducted a review of an electronic medical record of 572 patients who underwent radical cystectomy for urothelial carcinoma of the bladder. Primary end point was the site-specific recurrence after radical cystectomy.
The median follow-up in the validation cohort was 42.3 months (interquartile range: 23.0–89.3 months). During the follow-up period, there were 165 patients (28.8%), 85 (14.9%), 31 (5.4%), and 78 (13.6%) who recurred in abdomen/pelvis, thoracic region, upper urinary tract, and bone, respectively. The c-indices of abdomen/pelvis, thoracic region, upper urinary tract, and bone models 3 years after radical cystectomy were 0.69 (95% confidence interval [CI], 0.65–0.73), 0.69 (95% CI, 0.64–0.75), 0.61 (95% CI, 0.52–0.69), and 0.65 (95% CI, 0.59–0.71), respectively. Kaplan-Meier curves demonstrated that models discriminated well and log-rank test were all highly significant (all p<0.001), except upper urinary tract model (p = 0.366). Decision curve analysis revealed that the use of prediction models for abdomen/pelvis, thoracic region, and bone recurrence was associated with net benefit gains relative to the treat-all strategy, but not the model for upper urinary tract recurrence.
Abdomen/pelvis, thoracic region, and bone models demonstrate moderate discrimination, adequate calibration, and meaningful net benefit gains, whereas upper urinary tract model does not seem applicable to patients from Asia because it has suboptimal accuracy.
PMCID: PMC4061079  PMID: 24937260
16.  Quantitative Trait Loci Affecting Atherosclerosis at the Aortic Root Identified in an Intercross between DBA2J and 129S6 Apolipoprotein E-Null Mice 
PLoS ONE  2014;9(2):e88274.
Apolipoprotein E-null mice on a DBA/2J genetic background (DBA-apoE) are highly susceptible to atherosclerosis in the aortic root area compared with those on a 129S6 background (129-apoE). To explore atherosclerosis-responsible genetic regions, we performed a quantitative trait locus (QTL) analysis using 172 male and 137 female F2 derived from an intercross between DBA-apoE and 129-apoE mice. A genome-wide scan identified two significant QTL for the size of lesions at the root: one is Ath44 on Chromosome (Chr) 1 at 158 Mb, and the other Ath45 on Chr 2 at 162 Mb. Ath44 co-localizes with but appears to be independent of a previously reported QTL, Ath1, while Ath45 is a novel QTL. DBA alleles of both Ath44 and Ath45 confer atherosclerosis-susceptibility. In addition, a QTL on Chr 14 at 73 Mb was found significant only in males, and 129 allele conferring susceptibility. Further analysis detected female-specific interactions between a second QTL on Chr 1 at 73 Mb and a QTL on Chr 3 at 21 Mb, and between Chr 7 at 84 Mb and Chr 12 at 77 Mb. These loci for the root atherosclerosis were independent of QTLs for plasma total cholesterol and QTLs for triglycerides, but a QTL for HDL (Chr 1 at 126 Mb) overlapped with the Ath44. Notably, haplotype analysis among 129S6, DBA/2J and C57BL/6 genomes and their gene expression data narrowed the candidate regions for Ath44 and Ath45 to less than 5 Mb intervals where multiple genome wide associations with cardiovascular phenotypes have also been reported in humans. SNPs in or near Fmo3, Sele and Selp for Ath44, and Lbp and Pkig for Ath45 were suggested for further investigation as potential candidates underlying the atherosclerosis susceptibility.
PMCID: PMC3930552  PMID: 24586312
17.  Heredity and cardiometabolic risk: naturally occurring polymorphisms in the human neuropeptide Y2 receptor promoter disrupt multiple transcriptional response motifs 
Journal of hypertension  2013;31(1):123-133.
The neuropeptide Y2 G-protein-coupled receptor (NPY2R) relays signals from PYY or neuropeptide Y toward satiety and control of body mass. Targeted ablation of the NPY2R locus in mice yields obesity, and studies of NPY2R promoter genetic variation in more than 10 000 human participants indicate its involvement in control of obesity and BMI. Here we searched for genetic variation across the human NPY2R locus and probed its functional effects, especially in the proximal promoter.
Methods and results
Twin pair studies indicated substantial heritability for multiple cardiometabolic traits, including BMI, SBP, DBP, and PYY, an endogenous agonist at NPY2R. Systematic polymorphism discovery by resequencing across NPY2R uncovered 21 genetic variants, 10 of which were common [minor allele frequency (MAF) >5%], creating one to two linkage disequilibrium blocks in multiple biogeographic ancestries. In vivo, NPY2R haplotypes were associated with both BMI (P =3.75E–04) and PYY (P =4.01E–06). Computational approaches revealed that proximal promoter variants G-1606A, C-599T, and A-224G disrupt predicted IRF1 (A>G), FOXI1 (T>C), and SNAI1 (A>G) response elements. In neuroendocrine cells transfected with NPY2R promoter/luciferase reporter plasmids, all three variants and their resulting haplotypes influenced transcription (G-1606A, P <2.97E–06; C-599T, P <1.17E–06; A-224G, P <2.04E–06), and transcription was differentially augmented or impaired by coexpression of either the cognate full-length transcription factors or their specific siRNAs at each site. Endogenous expression of transcripts for NPY2R, IRF1, and SNAI1 was documented in neuroendocrine cells, and the NPY2R mRNA was differentially expressed in two neuroendocrine tissues (adrenal gland, brainstem) of a rodent model of hypertension and the metabolic syndrome, the spontaneously hypertensive rat.
We conclude that common genetic variation in the proximal NPY2R promoter influences transcription factor binding so as to alter gene expression in neuroendocrine cells, and consequently cardiometabolic traits in humans. These results unveil a novel control point, whereby cis-acting genetic variation contributes to control of complex cardiometabolic traits, and point to new transcriptional strategies for intervention into neuropeptide actions and their cardiometabolic consequences.
PMCID: PMC3615981  PMID: 23149563
autonomic; genetics; hypertension; nervous system; obesity
18.  Genes and environment: novel, functional polymorphism in the human cathepsin L (CTSL1) promoter disrupts a xenobiotic response element (XRE) to alter transcription and blood pressure 
Journal of hypertension  2012;30(10):1961-1969.
Cathepsin L (CTSL1) catalyzes the formation of peptides that influence blood pressure (BP). Naturally occurring genetic variation or targeted ablation of the Ctsl1 locus in mice yield cardiovascular pathology. Here, we searched for genetic variation across the human CTSL1 locus and probed its functional effects, especially in the proximal promoter.
Methods and results
Systematic polymorphism discovery by re-sequencing across CTSL1 in 81 patients uncovered 38 genetic variants, five of which were relatively common (MAF >5%), creating a single linkage disequilibrium block in multiple biogeographic ancestries. One of these five common variants lay in a functional domain of the gene: promoter C-171A (rs3118869), which disrupts a predicted xenobiotic response element (XRE; match C>A). In transfected CTSL1 promoter/luciferase reporter plasmids, C-171A allele influenced transcription (C>A, P = 3.36E-6), and transcription was also augmented by co-exposure to the aryl hydrocarbon receptor (AHR) complex (AHR:ARNT) in the presence of their ligand dioxin (P = 6.81E-8); allele (C vs. A) and AHR:ARNT/dioxin stimulus interacted to control gene expression (interaction P = 0.033). Endogenous Ctsl1, Ahr, and Arnt transcripts were present in chromaffin cells. Promoter functional C-171A genotype also predicted hypertension (P = 1.0E–3), SBP (P = 4.0E–4), and DBP (P = 3.0E–3), in an additive pattern for diploid genotypes (A/A > C/A > C/C) in 868 patients, and the results were extended by validation analysis into an independent population sample of 986 patients.
We conclude that common genetic variation in the proximal CTSL1 promoter, especially at position C-171A, is functional in cells, and alters transcription so as to explain the association of CTSL1 with BP in vivo. At the XRE, endogenous genetic variation plus exogenous aryl hydrocarbon stimulation interact to control CTSL1 gene expression. These results unveil a novel control point whereby heredity and environment can intersect to control a complex trait, and point to new transcriptional strategies for intervention into transmitter biosynthesis and its cardiovascular consequences.
PMCID: PMC3478326  PMID: 22871890
autonomic; genetics; hypertension; nervous system
19.  Connective Tissue Growth Factor (CTGF) Expression Modulates Response to High Glucose 
PLoS ONE  2013;8(8):e70441.
Connective tissue growth factor (CTGF) is an important mediator of fibrosis; emerging evidence link changes in plasma and urinary CTGF levels to diabetic kidney disease. To further ascertain the role of CTGF in responses to high glucose, we assessed the consequence of 4 months of streptozotocin-induced diabetes in wild type (+/+) and CTGF heterozygous (+/−) mice. Subsequently, we studied the influence of glucose on gene expression and protein in mice embryonic fibroblasts (MEF) cells derived from wildtype and heterozygous mice. At study initiation, plasma glucose, creatinine, triglyceride and cholesterol levels were similar between non-diabetic CTGF+/+ and CTGF+/− mice. In the diabetic state, plasma glucose levels were increased in CTGF+/+ and CTGF+/− mice (28.2 3.3 mmol/L vs 27.0 3.1 mmol/L), plasma triglyceride levels were lower in CTGF+/− mice than in CTGF+/+ (0.7 0.2 mmol/L vs 0.5 0.1 mmol/L, p<0.05), but cholesterol was essentially unchanged in both groups. Plasma creatinine was higher in diabetic CTGF+/+ group (11.7±1.2 vs 7.9±0.6 µmol/L p<0.01), while urinary albumin excretion and mesangial expansion were reduced in diabetic CTGF+/− animals. Cortices from diabetic mice (both CTGF +/+ and CTGF +/−) manifested higher expression of CTGF and thrombospondin 1 (TSP1). Expression of nephrin was reduced in CTGF +/+ animals; this reduction was attenuated in CTGF+/− group. In cultured MEF from CTGF+/+ mice, glucose (25 mM) increased expression of pro-collagens 1, IV and XVIII as well as fibronectin and thrombospondin 1 (TSP1). In contrast, activation of these genes by high glucose was attenuated in CTGF+/− MEF. We conclude that induction of Ctgf mediates expression of extracellular matrix proteins in diabetic kidney. Thus, genetic variability in CTGF expression directly modulates the severity of diabetic nephropathy.
PMCID: PMC3741286  PMID: 23950936
20.  Mefloquine Exposure Induces Cell Cycle Delay and Reveals Stage-Specific Expression of the pfmdr1 Gene 
Drug-resistant Plasmodium falciparum malaria is a major public health problem. An elevated pfmdr1 gene copy number (CN) is known to decrease parasite sensitivity to the commonly used antimalarial mefloquine (MFQ). To understand the relationship between pfmdr1 CN and mefloquine resistance, we evaluated pfmdr1 transcript levels in three P. falciparum strains with different CNs in the presence and absence of MFQ. Parasite strains with multiple pfmdr1 gene copies exhibited higher relative transcript levels than single-copy parasites, and MFQ induced pfmdr1 expression above the levels without treatment in all three strains evaluated. Concomitant morphology analyses of the sampled cultures revealed that MFQ treatment of synchronized ring-stage parasites induced a delay in parasite maturation through the intraerythrocytic cycle. pfmdr1 expression peaks in the ring stage, and MFQ could be causing increased transcription by delaying parasite maturation. However, pretreatment with mefloquine did not affect the artemisinin in vitro half-maximal inhibitory concentration (IC50). These results suggest that MFQ-induced increases in pfmdr1 expression are the direct result of the maturation delay at the ring stage but that this change in expression does not affect the antimalarial activity of artemisinin.
PMCID: PMC3553685  PMID: 23208721
21.  Ketamine Does Not Produce Relief of Neuropathic Pain in Mice Lacking the β-Common Receptor (CD131) 
PLoS ONE  2013;8(8):e71326.
Neuropathic pain (NP) is a debilitating condition associated with traumatic, metabolic, autoimmune and neurological etiologies. Although the triggers for NP are diverse, there are common underlying pathways, including activation of immune cells in the spinal cord and up-regulation of the N-methyl-D-aspartate receptor (NMDAR). Ketamine, a well-known NDMAR antagonist, reduces neuropathic pain in a sustained manner. Recent study has shown that the novel 11-amino acid peptide erythropoietin derivative ARA290 produces a similar, long-lasting relief of NP. Here, we show that both drugs also have similar effects on the expression of mRNA of the NMDAR, as well as that of microglia, astrocytes and chemokine (C-C motif) ligand 2, all-important contributors to the development of NP. Although the effects of ketamine and ARA 290 on NP and its molecular mediators suggest a common mechanism of action, ARA 290 has no affinity for the NMDAR and acts specifically via the innate repair receptor (IRR) involved in tissue protection. We speculated therefore, that the IRR might be critically involved in the action of ketamine on neuropathic pain. To evaluate this, we studied the effects of ketamine and ARA 290 on acute pain, side effects, and allodynia following a spared nerve injury model in mice lacking the β-common receptor (βcR), a structural component of the IRR. Ketamine (50 mg/kg) and ARA 290 (30 µg/kg) produced divergent effects on acute pain: ketamine produced profound antinociception accompanied with psychomotor side effects, but ARA290 did not, in both normal and knock out mice. In contrast, while both drugs were antiallodynic in WT mice, they had no effect on NP in mice lacking the βcR. Together, these results show that an intact IRR is required for the effective treatment of NP with either ketamine or ARA 290, but is not involved in ketamine’s analgesic and side effects.
PMCID: PMC3731332  PMID: 23936499
22.  Reduced Alpha-Lipoic Acid Synthase Gene Expression Exacerbates Atherosclerosis in Diabetic Apolipoprotein E–Deficient Mice 
Atherosclerosis  2012;223(1):137-143.
To study the effects of reduced lipoic acid gene expression on diabetic atherosclerosis in apolipoprotein E null mice (Apoe−/−).
Methods and Results
Heterozygous lipoic acid synthase gene knockout mice (Lias+/−) crossed with Apoe−/− mice were used to evaluate the diabetic effect induced by streptozotocin on atherosclerosis in the aortic sinus of the heart. While diabetes markedly increased atherosclerotic plaque size in Apoe−/− mice, a small but significant effect of reduced expression of lipoic acid gene was observed in diabetic Lias+/−Apoe−/− mice. In the aortic lesion area, the Lias+/−Apoe−/− mice exhibited significantly increased macrophage accumulation and cellular apoptosis than diabetic Lias+/+Apoe−/− littermates. Plasma glucose, cholesterol, and interleukin-6 were also higher. These abnormalities were accompanied with increased oxidative stress including a decreased ratio of reduced glutathione/oxidized glutathione in erythrocytes, increased systemic lipid peroxidation, and increased Gpx1 and MCP1 gene expression in the aorta.
Decreased endogenous lipoic acid gene expression plays a role in development of diabetic atherosclerosis. These findings extend our understanding of the role of antioxidant in diabetic atherosclerosis.
PMCID: PMC3389304  PMID: 22658261
lipoic acid; Lias mouse model; atherosclerosis; diabetes; and apolipoprotein E null mice
23.  Pro- and anti-atherogenic effects of a dominant negative P465L mutation of PPARγ in apolipoprotein E-null mice 
The dominant-negative mutation, P467L, in Peroxisome Proliferator Activated Receptor gamma (PPARγ) affects adipose tissue distribution, insulin sensitivity and blood pressure in heterozygous humans. We hypothesized that the equivalent mutation, PPARγ-P465L, in mice will worsen atherosclerosis.
Methods and Results
ApolipoproteinE-null mice with and without PPARγ-P465L mutation were bred in 129S6 inbred genetic background. Mild hypertension and lipodystrophy of PPARγ-P465L persisted in the apoE-null background. Glucose homeostasis was normal, but plasma adiponectin was significantly lower and resistin was higher in PPARγ-P465L mice. Plasma cholesterol and lipoprotein distribution were not different, but plasma triglycerides tended to be reduced. Surprisingly, there were no overall changes in the atherosclerotic plaque size or composition. PPARγ-P465L macrophages had a small decrease in CD-36 mRNA and a small yet significant reduction in VLDL uptake in culture. In unloaded apoE-null macrophages with PPARγ-P465L, cholesterol uptake was reduced while apoAI-mediated efflux was increased. However, when cells were cholesterol loaded in presence of acetylated LDL, no genotype difference in uptake or efflux was apparent. A reduction of VCAM1 expression in aorta suggests a relatively anti-atherogenic vascular environment in mice with PPARγ-P465L.
Small, competing pro- and anti-atherogenic effects of PPARγ-P465L mutation result in unchanged plaque development in apoE-deficient mice.
PMCID: PMC3389794  PMID: 22539598
mouse model; macrophage; bone marrow transfer; PPAR gamma; atherosclerosis
24.  Aldosterone deficiency and mineralocorticoid receptor antagonism prevent angiotensin II-induced cardiac, renal, and vascular injury 
Kidney international  2012;82(6):643-651.
Angiotensin II causes cardiovascular injury in part by aldosterone-induced mineralocorticoid receptor activation, and it can also activate the mineralocorticoid receptor in the absence of aldosterone in vitro. Here we tested whether endogenous aldosterone contributes to angiotensin II/salt-induced cardiac, vascular, and renal injury by the mineralocorticoid receptor. Aldosterone synthase knockout mice and wild type littermates were treated with angiotensin II or vehicle plus the mineralocorticoid receptor antagonist spironolactone or regular diet while drinking 0.9- saline. Angiotensin II/salt caused hypertension in both the knockout and wild type mice; an effect significantly blunted in the knockout mice. Either genetic aldosterone deficiency or mineralocorticoid receptor antagonism reduced cardiac hypertrophy, aortic remodeling, and albuminuria, as well as cardiac, aortic, and renal plasminogen activator inhibitor-1 mRNA expression during angiotensin II treatment. Mineralocorticoid receptor antagonism reduced angiotensin II/salt-induced glomerular hypertrophy, but aldosterone deficiency did not. Combined mineralocorticoid receptor antagonism and aldosterone deficiency reduced blood urea nitrogen and restored nephrin immunoreactivity. Angiotensin II/salt also promoted glomerular injury through the mineralocorticoid receptor in the absence of aldosterone. Thus, mineralocorticoid antagonism may have protective effects in the kidney beyond aldosterone synthase inhibition.
PMCID: PMC3434275  PMID: 22622494
25.  Human Thrombomodulin Knockin Mice Reveal Differential Effects of Human Thrombomodulin on Thrombosis and Atherosclerosis 
We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo.
Methods and Results
Knockin mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knockin mice exhibited decreased protein C activation in the aorta (P < 0.01) and lung (P < 0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knockin mice compared with wild-type mice (P < 0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knockin mice (12 ± 3 minutes) than wild-type mice (31 ± 6 minutes; P < 0.05). No differences in serum cytokine levels were detected between knockin and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knockin mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis.
Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This “humanized” animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.
PMCID: PMC3202707  PMID: 21885846
thrombomodulin; protein C; inflammation; atherosclerosis; thrombosis

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