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1.  FLASH proton density imaging for improved surface coil intensity correction in quantitative and semi-quantitative SSFP perfusion cardiovascular magnetic resonance 
A low excitation flip angle (α < 10°) steady-state free precession (SSFP) proton-density (PD) reference scan is often used to estimate the B1-field inhomogeneity for surface coil intensity correction (SCIC) of the saturation-recovery (SR) prepared high flip angle (α = 40-50°) SSFP myocardial perfusion images. The different SSFP off-resonance response for these two flip angles might lead to suboptimal SCIC when there is a spatial variation in the background B0-field. The low flip angle SSFP-PD frames are more prone to parallel imaging banding artifacts in the presence of off-resonance. The use of FLASH-PD frames would eliminate both the banding artifacts and the uneven frequency response in the presence of off-resonance in the surface coil inhomogeneity estimate and improve homogeneity of semi-quantitative and quantitative perfusion measurements.
B0-field maps, SSFP and FLASH-PD frames were acquired in 10 healthy volunteers to analyze the SSFP off-resonance response. Furthermore, perfusion scans preceded by both FLASH and SSFP-PD frames from 10 patients with no myocardial infarction were analyzed semi-quantitatively and quantitatively (rest n = 10 and stress n = 1). Intra-subject myocardial blood flow (MBF) coefficient of variation (CoV) over the whole left ventricle (LV), as well as intra-subject peak contrast (CE) and upslope (SLP) standard deviation (SD) over 6 LV sectors were investigated.
In the 6 out of 10 cases where artifacts were apparent in the LV ROI of the SSFP-PD images, all three variability metrics were statistically significantly lower when using the FLASH-PD frames as input for the SCIC (CoVMBF-FLASH = 0.3 ± 0.1, CoVMBF-SSFP = 0.4 ± 0.1, p = 0.03; SDCE-FLASH = 10 ± 2, SDCE-SSFP = 32 ± 7, p = 0.01; SDSLP-FLASH = 0.02 ± 0.01, SDSLP-SSFP = 0.06 ± 0.02, p = 0.03). Example rest and stress data sets from the patient pool demonstrate that the low flip angle SSFP protocol can exhibit severe ghosting artifacts originating from off-resonance banding artifacts at the edges of the field of view that parallel imaging is not able to unfold. These artifacts lead to errors in the quantitative perfusion maps and the semi-quantitative perfusion indexes, such as false positives. It is shown that this can be avoided by using FLASH-PD frames as input for the SCIC.
FLASH-PD images are recommended as input for SCIC of SSFP perfusion images instead of low flip angle SSFP-PD images.
PMCID: PMC4331176
Myocardial perfusion; Quantitative perfusion; Cardiovascular magnetic resonance; Surface coil correction; Proton density frames; Balanced Steady State Free Precession (SSFP); Fast Low Angle SHot (FLASH)
2.  Quantitative pixel-wise measurement of myocardial blood flow: The impact of surface coil-related field inhomogeneity and a comparison of methods for its correction 
Surface coil-related field inhomogeneity potentially confounds pixel-wise quantitative analysis of perfusion CMR images. This study assessed the effect of surface coil-related field inhomogeneity on the spatial variation of pixel-wise myocardial blood flow (MBF), and assessed its impact on the ability of MBF quantification to differentiate ischaemic from remote coronary territories. Two surface coil intensity correction (SCIC) techniques were evaluated: 1) a proton density-based technique (PD-SCIC) and; 2) a saturation recovery steady-state free precession-based technique (SSFP-SCIC).
26 subjects (18 with significant CAD and 8 healthy volunteers) underwent stress perfusion CMR using a motion-corrected, saturation recovery SSFP dual-sequence protocol. A proton density (PD)-weighted image was acquired at the beginning of the sequence. Surface coil-related field inhomogeneity was approximated using a third-order surface fit to the PD image or a pre-contrast saturation prepared SSFP image. The estimated intensity bias field was subsequently applied to the image series. Pixel-wise MBF was measured from mid-ventricular stress images using the two SCIC approaches and compared to measurements made without SCIC.
MBF heterogeneity in healthy volunteers was higher using SSFP-SCIC (24.8 ± 4.1%) compared to PD-SCIC (20.8 ± 3.0%; p = 0.009), however heterogeneity was significantly lower using either SCIC technique compared to analysis performed without SCIC (36.2 ± 6.3%). In CAD patients, the difference in MBF between remote and ischaemic territories was minimal when analysis was performed without SCIC (0.06 ± 0.91 mL/min/kg), and was substantially lower than with either PD-SCIC (0.50 ± 0.63 mL/min/kg; p = 0.013) or with SSFP-SCIC (0.63 ± 0.89 mL/min/kg; p = 0.005). In 6 patients, MBF quantified without SCIC was artifactually higher in the stenosed coronary territory compared to the remote territory. PD-SCIC and SSFP-SCIC had similar differences in MBF between remote and ischaemic territories (p = 0.145).
This study demonstrates that surface coil-related field inhomogeneity can confound pixel-wise MBF quantification. Whilst a PD-based SCIC led to a more homogenous correction than a saturation recovery SSFP-based technique, this did not result in an appreciable difference in the differentiation of ischaemic from remote coronary territories and thus either method could be applied.
PMCID: PMC4323126
Cardiovascular magnetic resonance; Perfusion; Myocardial blood flow; Quantification; Field inhomogeneity; Surface coil intensity correction
3.  Trans-ethnic genome-wide association study of colorectal cancer identifies a new susceptibility locus in VTI1A 
Nature communications  2014;5:4613.
The genetic basis of sporadic colorectal cancer (CRC) is not well explained by known risk polymorphisms. Here we perform a meta-analysis of two genome-wide association studies in 2,627 cases and 3,797 controls of Japanese ancestry and 1,894 cases and 4,703 controls of African ancestry, to identify genetic variants that contribute to CRC susceptibility. We replicate genome-wide statistically significant associations (P < 5×10−8) in 16,823 cases and 18,211 controls of European ancestry. This study reveals a new pan-ethnic CRC risk locus at 10q25 (rs12241008, intronic to VTI1A; P=1.4×10−9), providing additional insight into the etiology of CRC and highlighting the value of association mapping in diverse populations.
PMCID: PMC4180879  PMID: 25105248
6.  In Vivo Microscopy Reveals Extensive Embedding of Capillaries within the Sarcolemma of Skeletal Muscle Fibers 
To provide insight into mitochondrial function in vivo, we evaluated the 3D spatial relationship between capillaries, mitochondria, and muscle fibers in live mice.
3D volumes of in vivo murine Tibialis anterior muscles were imaged by multi-photon microscopy (MPM). Muscle fiber type, mitochondrial distribution, number of capillaries, and capillary-to-fiber contact were assessed. The role of myoglobin-facilitated diffusion was examined in myoglobin knockout mice. Distribution of GLUT4 was also evaluated in the context of the capillary and mitochondrial network.
MPM revealed that 43.6 ± 3.3% of oxidative fiber capillaries had ≥ 50% of their circumference embedded in a groove in the sarcolemma, in vivo. Embedded capillaries were tightly associated with dense mitochondrial populations lateral to capillary grooves and nearly absent below the groove. Mitochondrial distribution, number of embedded capillaries, and capillary-to-fiber contact were proportional to fiber oxidative capacity and unaffected by myoglobin knockout. GLUT4 did not preferentially localize to embedded capillaries.
Embedding capillaries in the sarcolemma may provide a regulatory mechanism to optimize delivery of oxygen to heterogeneous groups of muscle fibers. We hypothesize that mitochondria locate to paravascular regions due to myofibril voids created by embedded capillaries, not to enhance the delivery of oxygen to the mitochondria.
PMCID: PMC4185432  PMID: 25279425
Two-photon microscopy; 3D quantitative imaging; muscle mitochondria; myoglobin; vasculature; microcirculation; GLUT4
7.  Extensively drug-resistant Acinetobacter baumannii in a Thai hospital: a molecular epidemiologic analysis and identification of bactericidal Polymyxin B-based combinations 
Limited knowledge of the local molecular epidemiology and the paucity of new effective antibiotics has resulted in an immense challenge in the control and treatment of extensively drug-resistant (XDR) Acinetobacter baumannii infections in Thailand. Antimicrobial combination regimens may be the only feasible treatment option in such cases. We sought to characterize the local molecular epidemiology and assess the bactericidal activity of various antibiotics individually and in combination against XDR A. baumannii in a Thai hospital.
All XDR A. baumannii isolates from Thammasat University Hospital were collected between October 2010 and May 2011. Susceptibility testing was conducted according to reference broth dilution methods. Pulse-field gel electrophoresis was used to genotype the isolates. Carbapenemase genes were detected using polymerase chain reaction. In vitro testing of clinically-relevant concentrations of imipenem, meropenem, doripenem, rifampicin and tigecycline alone and in combination with polymyxin B was conducted using multiple combination bactericidal testing.
Forty-nine polymyxin B-susceptible XDR A. baumannii isolates were identified. blaOXA-23 and blaOXA-51 genes were detected in all isolates. Eight clonally related clusters were identified, resulting in the initiation of several infection control measures. Imipenem, meropenem, doripenem, rifampicin, and tigecycline in combination with PB respectively, exhibited bactericidal killing in 100%, 100%, 98.0%, 100% and 87.8% isolates respectively at 24 hours.
Molecular epidemiologic analysis can aid the early detection of infection outbreak within the institution, resulting in the rapid containment of the outbreak. Imipenem/meropenem/rifampicin in combination with polymyxin B demonstrated consistent bactericidal effect against 49 blaOXA-23-harbouring XDR A. baumannii clinical isolates, suggesting a role of combination therapy in the treatment of these infections.
PMCID: PMC4314787  PMID: 25648393
Combination therapy; Carbapenem resistance; Acinetobacter baumannii
8.  Diagnostic Accuracy of Fully Quantitative, Semi-Quantitative, and Qualitative Assessment of Stress Perfusion CMR Compared to Quantitative Coronary Angiography 
JACC. Cardiovascular imaging  2014;7(1):14-22.
Our primary objective was to determine the sensitivity, specificity, and accuracy of fully quantitative stress perfusion CMR versus a reference standard of quantitative coronary angiography (QCA). We hypothesized that fully quantitative analysis of stress perfusion CMR would have high diagnostic accuracy for identifying significant coronary artery stenosis and exceed the accuracy of semi-quantitative measures of perfusion and qualitative interpretation.
Relatively few studies apply fully quantitative CMR perfusion measures to patients with coronary disease and comparisons to semi-quantitative and qualitative methods are limited.
Dual bolus dipyridamole stress perfusion CMR exams were performed in 67 patients with clinical indications for assessment of myocardial ischemia. Stress perfusion images alone were analyzed with a fully quantitative method (QP) and 3 semi-quantitative methods including contrast enhancement ratio (CER), upslope index (SLP), and upslope integral (INT). Comprehensive exams (cine imaging, stress/rest perfusion, late gadolinium enhancement) were analyzed qualitatively with two methods including the Duke Algorithm and standard clinical interpretation. A 70% or greater stenosis by QCA was considered abnormal.
The optimum diagnostic threshold for QP determined by receiver operating characteristic curve occurred when endocardial flow decreased to <50% of mean epicardial flow which yielded a sensitivity of 87% and specificity of 93%. The area under the curve (AUC) for QP was 0.92 which was superior to semi-quantitative methods: CER 0.78, SLP 0.82, and INT 0.75 (p=0.011, p=0.019, p=0.004 versus QP, respectively). AUC for QP was also superior to qualitative methods: Duke Algorithm 0.70 and clinical interpretation 0.78 (p<0.001 and p<0.001 versus QP, respectively).
Fully quantitative stress perfusion CMR has high diagnostic accuracy for detecting obstructive CAD. QP outperforms semi-quantitative measures of perfusion and qualitative methods that incorporate a combination of cine, perfusion, and late gadolinium enhancement imaging. These findings suggest a potential clinical role for quantitative stress perfusion CMR.
PMCID: PMC4186701  PMID: 24433707
Cardiac Magnetic Resonance; Myocardial Perfusion; Myocardial Ischemia; Quantitative Perfusion; Stress Testing
9.  Multidrug-Resistant Tuberculosis Outbreak in Gaming Centers, Singapore, 2012 
Emerging Infectious Diseases  2015;21(1):179-180.
PMCID: PMC4285258  PMID: 25531726
multidrug-resistant tuberculosis; MDR TB; outbreak; whole-genome sequencing; cyber café; contact tracing; bacteria; Singapore; gaming centers; tuberculosis and other mycobacteria
10.  COX-1 (PTGS1) and COX-2 (PTGS2) polymorphisms, NSAID interactions, and risk of colon and rectal cancer in two independent populations 
Cancer causes & control : CCC  2013;24(12):2059-2075.
Nonsteroidal anti-inflammatory drugs (NSAIDs) target the prostaglandin H synthase enzymes, cyclooxygenase (COX)-1 and -2, and reduce colorectal cancer risk. Genetic variation in the genes encoding these enzymes may be associated with changes in colon and rectal cancer risk and in NSAID efficacy.
We genotyped candidate polymorphisms and tagSNPs in PTGS1 (COX-1) and PTGS2 (COX-2) in a population-based case-control study (Diet, Activity and Lifestyle Study, DALS) of colon cancer (n=1470 cases/1837 controls) and rectal cancer (n=583/775), and independently among cases and controls from the Colon Cancer Family Registry (CCFR; colon n= 959/1535, rectal n= 505/839).
In PTGS2, a functional polymorphism (−765G>C; rs20417) was associated with a 2-fold increased rectal cancer risk (p=0.05) in the DALS study. This association replicated with a significant nearly 5-fold increased risk of rectal cancer in the CCFR study (ORCC vs GG=4.88; 95%CI=1.54–15.45; ORGC vs GG=1.36; 95%CI: 0.95–1.94). Genotype-NSAID interactions were observed in the DALS study for PTGS1 and rectal cancer risk, and for PTGS2 and colon cancer risk, but were no longer significant after correcting for multiple comparisons and did not replicate in the CCFR. No significant associations between PTGS1 polymorphisms and colon or rectal cancer risk were observed.
These findings suggest that polymorphisms in PTGS2 may be associated with rectal cancer risk and impact the protective effects of NSAIDs.
PMCID: PMC3913564  PMID: 24022467
colorectal cancer; PTGS; COX; genetic association; NSAID; aspirin; polymorphism
11.  Frailty Models for Familial Risk with Application to Breast Cancer 
In evaluating familial risk for disease we have two main statistical tasks: assessing the probability of carrying an inherited genetic mutation conferring higher risk; and predicting the absolute risk of developing diseases over time, for those individuals whose mutation status is known. Despite substantial progress, much remains unknown about the role of genetic and environmental risk factors, about the sources of variation in risk among families that carry high-risk mutations, and about the sources of familial aggregation beyond major Mendelian effects. These sources of heterogeneity contribute substantial variation in risk across families. In this paper we present simple and efficient methods for accounting for this variation in familial risk assessment. Our methods are based on frailty models. We implemented them in the context of generalizing Mendelian models of cancer risk, and compared our approaches to others that do not consider heterogeneity across families. Our extensive simulation study demonstrates that when predicting the risk of developing a disease over time conditional on carrier status, accounting for heterogeneity results in a substantial improvement in the area under the curve of the receiver operating characteristic. On the other hand, the improvement for carriership probability estimation is more limited. We illustrate the utility of the proposed approach through the analysis of BRCA1 and BRCA2 mutation carriers in the Washington Ashkenazi Kin-Cohort Study of Breast Cancer.
PMCID: PMC3963469  PMID: 24678132
familial risk prediction; frailty model; multivariate survival; ROC analysis; risk index; breast cancer
12.  Rare and low-frequency coding variants in CXCR2 and other genes are associated with hematological traits 
Nature genetics  2014;46(6):629-634.
Hematological traits are important clinical parameters. To test the role of rare and low-frequency coding variants on hematological traits, we analyzed hemoglobin, hematocrit, white blood cell (WBC) and platelet count in 31,340 individuals genotyped on an exome array. We identified several missense variants of CXCR2 associated with reduced WBC count (gene-based P=2.6×10−13). In a separate family-based re-sequencing study, we identified a novel loss-of-function CXCR2 frameshift mutation in a pedigree with congenital neutropenia that abolished ligand-induced CXCR2 signal transduction and chemotaxis. We also identified novel missense or splice site variants in key hematopoiesis regulators (EPO, TRF2, HBB, TUBB1, SH2B3) associated with blood cell traits. Finally, we were able to detect associations between the rare somatic JAK2 p.Val617Phe mutation and platelet count (P=3.9×10−22) as well as hemoglobin (P=0.002), hematocrit (P=9.5×10−7) and WBC (P=3.1×10−5). In conclusion, exome arrays complement GWAS in identifying new variants that contribute to complex human traits.
PMCID: PMC4050975  PMID: 24777453
13.  Imputation of coding variants in African Americans: better performance using data from the exome sequencing project 
Bioinformatics  2013;29(21):2744-2749.
Summary: Although the 1000 Genomes haplotypes are the most commonly used reference panel for imputation, medical sequencing projects are generating large alternate sets of sequenced samples. Imputation in African Americans using 3384 haplotypes from the Exome Sequencing Project, compared with 2184 haplotypes from 1000 Genomes Project, increased effective sample size by 8.3–11.4% for coding variants with minor allele frequency <1%. No loss of imputation quality was observed using a panel built from phenotypic extremes. We recommend using haplotypes from Exome Sequencing Project alone or concatenation of the two panels over quality score-based post-imputation selection or IMPUTE2’s two-panel combination.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3799474  PMID: 23956302
14.  Time series analysis of demographic and temporal trends of tuberculosis in Singapore 
BMC Public Health  2014;14(1):1121.
Singapore is an intermediate tuberculosis (TB) incidence country, with a recent rise in TB incidence from 2008, after a fall in incidence since 1998. This study identified population characteristics that were associated with the recent increase in TB cases, and built a predictive model of TB risk in Singapore.
Retrospective time series analysis was used to study TB notification data collected from 1995 to 2011 from the Singapore Tuberculosis Elimination Program (STEP) registry. A predictive model was developed based on the data collected from 1995 to 2010 and validated using the data collected in 2011.
There was a significant difference in demographic characteristics between resident and non-resident TB cases. TB risk was higher in non-residents than in residents throughout the period. We found no significant association between demographic and macro-economic factors and annual incidence of TB with or without adjusting for the population-at-risk. Despite growing non-resident population, there was a significant decrease in the non-resident TB risk (p < 0.0001). However, there was no evidence of trend in the resident TB risk over this time period, though differences between different demographic groups were apparent with ethnic minorities experiencing higher incidence rates.
The study found that despite an increasing size of non-resident population, TB risk among non-residents was decreasing at a rate of about 3% per year. There was an apparent seasonality in the TB reporting.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2458-14-1121) contains supplementary material, which is available to authorized users.
PMCID: PMC4230736  PMID: 25359711
Time-series analysis; Tuberculosis
15.  Autophagy Facilitates Antibody-Enhanced Dengue Virus Infection in Human Pre-Basophil/Mast Cells 
PLoS ONE  2014;9(10):e110655.
Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.
Methodology/Principal Findings
We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4BC74A mutant.
Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.
PMCID: PMC4199741  PMID: 25329914
16.  Development of CpG-Oligodeoxynucleotides for Effective Activation of Rabbit TLR9 Mediated Immune Responses 
PLoS ONE  2014;9(9):e108808.
CpG-oligodeoxynucleotides (CpG-ODN) are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9) is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18–24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund’s adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody response. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.
PMCID: PMC4182578  PMID: 25269083
17.  Costs associated with febrile neutropenia in solid tumor and lymphoma patients – an observational study in Singapore 
The primary objective was to describe the total direct inpatient costs among solid tumor and lymphoma patients with chemotherapy-induced febrile neutropenia (FN) and the factors that were associated with higher direct cost. The secondary objective was to describe the out-of-pocket patient payments and the factors that were associated with higher out-of-pocket patient payments.
This was a single-center observational study conducted at the largest cancer center in Singapore. All of the adult cancer patients hospitalized due to FN from 2009 to 2012 were studied. The primary outcomes were the total hospital cost and the out-of-pocket patient payments (adjusted by government subsidy) per FN episode. Univariate analysis and multiple linear regression were conducted to identify the factors associated with higher FN costs.
Three hundred and sixty seven adult cancer patients were documented with FN-related hospitalizations. The mean total hospital cost was US$4,193 (95% CI: US$3,779-4,607) and the mean out-of-pocket patient payment was US$2,230 (95% CI: US$1,976-2,484), per FN episode. The factors associated with a higher total hospital cost were longer length of stay, severe sepsis, and lymphoma as underlying cancer. The out-of-pocket patient payment was positively associated with longer length of stay, severe sepsis, lymphoma diagnosed as underlying cancer, the therapeutic use of granulocyte colony-stimulating factor (GCSF), the private ward class, and younger patients.
The total hospital cost and out-of-pocket patient payments of FN management in lymphoma cases were substantial compared with other solid tumors. Factors associated with a higher FN management cost may be useful for developing appropriate strategies to reduce the cost of FN for cancer patients.
PMCID: PMC4263105  PMID: 25252614
Febrile neutropenia; Neutropenia; Fever; Cost analysis; Factors
18.  Bright Blood T2-Weighted MRI Has High Diagnostic Accuracy for Myocardial Hemorrhage in Myocardial Infarction: A Preclinical Validation Study in Swine 
Myocardial hemorrhage after myocardial infarction (MI) usually goes undetected. We investigated the diagnostic accuracy of bright blood T2-weighted cardiac magnetic resonance imaging (MRI) for myocardial hemorrhage in experimental MI.
Methods and Results
MI was created in swine by occluding the left anterior descending (n=10) or circumflex (n=5) coronary arteries for 90 minutes followed by reperfusion for ≤3 days (n=2), 10 days (n=7) or 60 days (n=6). MRI was performed at 1.5 Tesla using bright blood T2-prepared steady-state free-precession, T2* and early (1 min) and late (10 – 15 min) gadolinium enhancement (EGE, LGE, respectively) MRI. Left ventricular sections and histology were assessed for hemorrhage by an experienced cardiac pathologist blinded to the MRI data. Hypointense regions on T2-weighted and contrast-enhanced MRI were independently determined by 3 cardiologists experienced in MRI who were also blinded to the pathology results. Eighty ventricular pathologic sections were matched with MRI (n=68 for EGE MRI). All sections with evidence of MI (n=63 (79%)) also exhibited hyperintense zones consistent with edema on T2-weighted MRI and infarct on LGE MRI. Myocardial hemorrhage occurred in 49 left ventricular sections (61%) and corresponded with signal voids on 48 T2-weighted (98%) and 26 LGE-MRI (53%). Alternatively, signal voids occurred in the absence of hemorrhage in 3 T2-weighted (90% specificity) and 5 LGE MRI (84% specificity). On EGE MRI, 27/43 cases of early MVO corresponded with hemorrhage (63% sensitivity) while 5/25 defects occurred in the absence of hemorrhage (80% specificity). The positive and negative predictive values for pathological evidence of hemorrhage were 94% & 96% for T2-weighted, 84% & 55% for LGE MRI and 85% & 56% for EGE MRI.
Bright blood T2-weighted MRI has high diagnostic accuracy for myocardial hemorrhage.
PMCID: PMC4158314  PMID: 21930836
myocardial infarction; magnetic resonance imaging; hemorrhage
19.  Germline Missense Variants in the BTNL2 Gene Are Associated with Prostate Cancer Susceptibility 
Rare, inherited mutations account for 5%–10% of all prostate cancer (PCa) cases. However, to date, few causative mutations have been identified.
To identify rare mutations for PCa, we performed whole-exome sequencing (WES) in multiple kindreds (n = 91) from 19 hereditary prostate cancer (HPC) families characterized by aggressive or early onset phenotypes. Candidate variants (n = 130) identified through family- and bioinformatics-based filtering of WES data were then genotyped in an independent set of 270 HPC families (n = 819 PCa cases; n = 496 unaffected relatives) for replication. Two variants with supportive evidence were subsequently genotyped in a population-based case-control study (n = 1,155 incident PCa cases; n = 1,060 age-matched controls) for further confirmation. All participants were men of European ancestry.
The strongest evidence was for two germline missense variants in the butyrophilin-like 2 (BTNL2) gene (rs41441651, p.Asp336Asn and rs28362675, p.Gly454Cys) that segregated with affection status in two of the WES families. In the independent set of 270 HPC families, 1.5% (rs41441651; P = 0.0032) and 1.2% (rs28362675; P = 0.0070) of affected men, but no unaffected men, carried a variant. Both variants were associated with elevated PCa risk in the population-based study (rs41441651: OR = 2.7; 95% CI, 1.27–5.87; P = 0.010; rs28362675: OR = 2.5; 95% CI, 1.16–5.46; P = 0.019).
Results indicate that rare BTNL2 variants play a role in susceptibility to both familial and sporadic prostate cancer.
Results implicate BTNL2 as a novel PCa susceptibility gene.
PMCID: PMC3769499  PMID: 23833122
20.  Effectiveness of seasonal influenza vaccinations against laboratory-confirmed influenza-associated infections among Singapore military personnel in 2010–2013 
Limited information is available about seasonal influenza vaccine effectiveness (VE) in tropical communities.
Virus subtype-specific VE was determined for all military service personnel in the recruit camp and three other non-recruit camp in Singapore's Armed Forces from 1 June 2009 to 30 June 2012.
Consenting servicemen underwent nasal washes, which were tested with RT-PCR and subtyped. The test positive case and test negative control design was used to estimate the VE. To estimate the overall effect of the programme on new recruits, we used an ecological time series approach.
A total of 7016 consultations were collected. The crude estimates for the VE of the triavalent vaccine against both influenza A(H1N1)pdm09 and influenza B were 84% (95% CI 78–88%, 79–86%, respectively). Vaccine efficacy against influenza A(H3N2) was markedly lower (VE 33%, 95% CI −4% to 57%). An estimated 70% (RR = 0·30; 95% CI 0·11–0·84), 39% (RR = 0·61;0·25–1·43) and 75% (RR = 0·25; 95% CI 0·11–0·50) reduction in the risk of influenza A(H1N1)pdm09, influenza A(H3N2) and influenza B infections, respectively, in the recruit camp during the post-vaccination period compared with during the pre-vaccination period was observed.
Overall, the blanket influenza vaccine programme in Singapore's Armed Forces has had a moderate to high degree of protection against influenza A(H1N1)pdm09 and influenza B, but not against influenza A(H3N2). Blanket influenza vaccination is recommended for all military personnel.
PMCID: PMC4181820  PMID: 24828687
influenza A(H1N1)pdm09; influenza A(H3N2); influenza B; influenza vaccination; vaccine effectiveness
21.  Coronary microvascular ischemia in hypertrophic cardiomyopathy - a pixel-wise quantitative cardiovascular magnetic resonance perfusion study 
Microvascular dysfunction in HCM has been associated with adverse clinical outcomes. Advances in quantitative cardiovascular magnetic resonance (CMR) perfusion imaging now allow myocardial blood flow to be quantified at the pixel level. We applied these techniques to investigate the spectrum of microvascular dysfunction in hypertrophic cardiomyopathy (HCM) and to explore its relationship with fibrosis and wall thickness.
CMR perfusion imaging was undertaken during adenosine-induced hyperemia and again at rest in 35 patients together with late gadolinium enhancement (LGE) imaging. Myocardial blood flow (MBF) was quantified on a pixel-by-pixel basis from CMR perfusion images using a Fermi-constrained deconvolution algorithm. Regions-of-interest (ROI) in hypoperfused and hyperemic myocardium were identified from the MBF pixel maps. The myocardium was also divided into 16 AHA segments.
Resting MBF was significantly higher in the endocardium than in the epicardium (mean ± SD: 1.25 ± 0.35 ml/g/min versus 1.20 ± 0.35 ml/g/min, P < 0.001), a pattern that reversed with stress (2.00 ± 0.76 ml/g/min versus 2.36 ± 0.83 ml/g/min, P < 0.001). ROI analysis revealed 11 (31%) patients with stress MBF lower than resting values (1.05 ± 0.39 ml/g/min versus 1.22 ± 0.36 ml/g/min, P = 0.021). There was a significant negative association between hyperemic MBF and wall thickness (β = −0.047 ml/g/min per mm, 95% CI: −0.057 to −0.038, P < 0.001) and a significantly lower probability of fibrosis in a segment with increasing hyperemic MBF (odds ratio per ml/g/min: 0.086, 95% CI: 0.078 to 0.095, P = 0.003).
Pixel-wise quantitative CMR perfusion imaging identifies a subgroup of patients with HCM that have localised severe microvascular dysfunction which may give rise to myocardial ischemia.
PMCID: PMC4145339  PMID: 25160568
Hypertrophic cardiomyopathy; Perfusion; Cardiovascular magnetic resonance; Microvascular dysfunction; Sudden cardiac death
22.  The Incidence of Japanese Encephalitis in Taiwan—A Population-Based Study 
A mass Japanese encephalitis (JE) vaccination program targeting children was launched in Taiwan in 1968, and the number of pediatric JE cases substantially decreased thereafter. The aim of this study was to elucidate the long-term trend of JE incidence, and to investigate the age-specific seroprevalence of JE-neutralizing antibodies.
Methodology/Principal Findings
A total of 2,948 laboratory-confirmed JE cases that occurred between 1966 and 2012 were analyzed using a mandatory notification system managed by the Centers for Disease Control, Taiwan. A total of 6,594 randomly-sampled serum specimens obtained in a nationwide population-based survey in 2002 were analyzed to estimate the seroprevalence of JE-neutralizing antibodies in the general population. The average annual JE incidence rate of the group aged 30 years and older was 0.167 cases per 100,000 people between 2001 and 2012, which was higher than the 0.052 cases per 100,000 people among those aged under 30 years. These seroepidemiological findings indicate that the cohort born between 1963 and 1975, who generally received two or three doses of the vaccine and were administered the last booster dose more than 20 years ago, exhibited the lowest positive rate of JE-neutralizing antibodies (54%). The highest and second highest antibody rates were observed, respectively, in the oldest unvaccinated cohort (86%) and in the youngest cohort born between 1981 and 1986, who received four doses 10–15 years ago (74%).
Over the past decade, the main age group of the confirmed JE cases in Taiwan shifted from young children to adults over 30 years of age. People who were born between 1963 and 1975 exhibited the lowest seroprevalence of JE-neutralizing antibodies. Thus, the key issue for JE control in Taiwan is to reduce adult JE cases through a cost-effective analysis of various immunization strategies.
Author Summary
JE is one of the major public health problems in Asian and the Western Pacific regions, and most cases occur in children under the age of 14 years. A JE virus infection can cause severe sequelae such as an impairment of language ability, cognitive ability, or movement. Because humans are a dead-end host of the JE virus, the disease cannot be transmitted among people. Vaccination is currently the most effective method for preventing JE, and children in most endemic areas are vaccinated. After decades of mass vaccination, the number of confirmed JE cases has considerably declined in Taiwan, Japan, and South Korea. Most JE cases have occurred in adults rather than children in these countries, thus, the disease must be controlled by reducing the number of adult JE cases. Therefore, a prevention policy for the adult and elderly population should be implemented in the near future.
PMCID: PMC4109885  PMID: 25058573
23.  SBERIA: Set Based gene EnviRonment InterAction test for rare and common variants in complex diseases 
Genetic epidemiology  2013;37(5):452-464.
Identification of gene-environment interaction (GxE) is important in understanding the etiology of complex diseases. However, partially due to the lack of power, there have been very few replicated GxE findings compared to the success in marginal association studies. The existing GxE testing methods mainly focus on improving the power for individual markers. In this paper, we took a different strategy and proposed a Set Based gene EnviRonment InterAction test (SBERIA), which can improve the power by reducing the multiple testing burdens and aggregating signals within a set. The major challenge of the signal aggregation within a set is how to tell signals from noise and how to determine the direction of the signals. SBERIA takes advantage of the established correlation screening for GxE to guide the aggregation of genotypes within a marker set. The correlation screening has been shown to be an efficient way of selecting potential GxE candidate SNPs in case-control studies for complex diseases. Importantly, the correlation screening in case-control combined samples is independent of the interaction test. With this desirable feature, SBERIA maintains the correct type I error level and can be easily implemented in a regular logistic regression setting. We showed that SBERIA had higher power than benchmark methods in various simulation scenarios, both for common and rare variants. We also applied SBERIA to real GWAS data of 10,729 colorectal cancer cases and 13,328 controls and found evidence of interaction between the set of known colorectal cancer susceptibility loci and smoking.
PMCID: PMC3713231  PMID: 23720162
gene-environment interaction; set based; correlation screening; GWAS; rare variants
24.  Developing Barbed Microtip-Based Electrode Arrays for Biopotential Measurement 
Sensors (Basel, Switzerland)  2014;14(7):12370-12386.
This study involved fabricating barbed microtip-based electrode arrays by using silicon wet etching. KOH anisotropic wet etching was employed to form a standard pyramidal microtip array and HF/HNO3 isotropic etching was used to fabricate barbs on these microtips. To improve the electrical conductance between the tip array on the front side of the wafer and the electrical contact on the back side, a through-silicon via was created during the wet etching process. The experimental results show that the forces required to detach the barbed microtip arrays from human skin, a polydimethylsiloxane (PDMS) polymer, and a polyvinylchloride (PVC) film were larger compared with those required to detach microtip arrays that lacked barbs. The impedances of the skin-electrode interface were measured and the performance levels of the proposed dry electrode were characterized. Electrode prototypes that employed the proposed tip arrays were implemented. Electroencephalogram (EEG) and electrocardiography (ECG) recordings using these electrode prototypes were also demonstrated.
PMCID: PMC4168478  PMID: 25014098
barbed microtips; contact impedance; detaching force; dry electrode; electrocardiography (ECG); electroencephalogram (EEG); silicon wet etching
25.  High Cytoplasmic Expression of Krüppel-like Factor 4 Is an Independent Prognostic Factor of Better Survival in Hepatocellular Carcinoma 
Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality in the world. Hepatocarcinogenesis is complex, with an extraordinary molecular heterogeneity. Krüppel-like factor 4 (KLF4) plays an important role in cell proliferation and differentiation, and it can function as a tumor suppressor or an oncoprotein, depending on tissue type. The role of KLF4 in HCC remains controversial. The aim of this study was to explore the clinical significance of KLF4 expression in HCC. The study included 205 patients with surgical resection. We performed immunostaining for KLF4 and Ki-67 to investigate the correlations of the clinicopathological parameters of HCC and to examine the proliferative index. KLF4 staining was observed in the cytoplasm of non-tumorous hepatocytes and tumor cells. We subdivided the immunohistological staining results for KLF4 into low expression (Staining 0 and 1+) and high expression (Staining 2+ and 3+) subgroups. The expression of KLF4 was significantly correlated with tumor differentiation (p = 0.001). The Ki-67 proliferative index was significantly lower in well-differentiated HCCs (0.781% ± 1.02% vs. 2.16% ± 3.14%, p = 0.012), but not significantly different between low-KLF4 expression and high-KLF4 expression (1.87% ± 2.93% vs. 2.51% ± 3.28%, p = 0.32). Kaplan–Meier analysis showed that a high expression of KLF4 was significantly correlated with a longer disease-specific survival (p = 0.019). Univariate and multivariate analyses showed that high KLF4 expression was an independent predictor of a better disease-specific survival (p = 0.017; hazard ratio = 0.398; 95% confidence interval: 0.19–0.85). High cytoplasmic expression of KLF4 was associated with better disease-specific survival and was an independently favorable prognostic factor in hepatocellular carcinoma. These promising results suggest that KLF4 may play an anti-oncogenic role in hepatocarcinogenesis.
PMCID: PMC4100128  PMID: 24897024
KLF4; Ki-67; hepatocellular carcinoma; survival; tissue microarray; immunohistochemical study

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