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1.  A variational Bayes discrete mixture test for rare variant association 
Genetic epidemiology  2014;38(1):21-30.
Recently, many statistical methods have been proposed to test for associations between rare genetic variants and complex traits. Most of these methods test for association by aggregating genetic variations within a predefined region, such as a gene. Although there is evidence that “aggregate” tests are more powerful than the single marker test, these tests generally ignore neutral variants and therefore are unable to identify specific variants driving the association with phenotype. We propose a novel aggregate rare-variant test that explicitly models a fraction of variants as neutral, tests associations at the gene-level, and infers the rare-variants driving the association. Simulations show that in the practical scenario where there are many variants within a given region of the genome with only a fraction causal our approach has greater power compared to other popular tests such as the Sequence Kernel Association Test (SKAT), the Weighted Sum Statistic (WSS), and the collapsing method of Morris and Zeggini (MZ). Our algorithm leverages a fast variational Bayes approximate inference methodology to scale to exome-wide analyses, a significant computational advantage over exact inference model selection methodologies. To demonstrate the efficacy of our methodology we test for associations between von Willebrand Factor (VWF) levels and VWF missense rare-variants imputed from the National Heart, Lung, and Blood Institute’s Exome Sequencing project into 2,487 African Americans within the VWF gene. Our method suggests that a relatively small fraction (~10%) of the imputed rare missense variants within VWF are strongly associated with lower VWF levels in African Americans.
PMCID: PMC4030763  PMID: 24482836
Exome sequencing study; approximate inference; von Willebrand Factor genetics
2.  Infant-Directed Visual Prosody: Mothers’ Head Movements and Speech Acoustics 
Interaction studies  2014;15(1):38-54.
Acoustical changes in the prosody of mothers’ speech to infants are distinct and near universal. However, less is known about the visible properties mothers’ infant-directed (ID) speech, and their relation to speech acoustics. Mothers’ head movements were tracked as they interacted with their infants using ID speech, and compared to movements accompanying their adult-directed (AD) speech. Movement measures along three dimensions of head translation, and three axes of head rotation were calculated. Overall, more head movement was found for ID than AD speech, suggesting that mothers exaggerate their visual prosody in a manner analogous to the acoustical exaggerations in their speech. Regression analyses examined the relation between changing head position and changing acoustical pitch (F0) over time. Head movements and voice pitch were more strongly related in ID speech than in AD speech. When these relations were examined across time windows of different durations, stronger relations were observed for shorter time windows (< 5 sec). However, the particular form of these more local relations did not extend or generalize to longer time windows. This suggests that the multimodal correspondences in speech prosody are variable in form, and occur within limited time spans.
PMCID: PMC4166504  PMID: 25242907
3.  No Evidence for Genome-Wide Interactions on Plasma Fibrinogen by Smoking, Alcohol Consumption and Body Mass Index: Results from Meta-Analyses of 80,607 Subjects 
Baumert, Jens | Huang, Jie | McKnight, Barbara | Sabater-Lleal, Maria | Steri, Maristella | Chu, Audrey Y. | Trompet, Stella | Lopez, Lorna M. | Fornage, Myriam | Teumer, Alexander | Tang, Weihong | Rudnicka, Alicja R. | Mälarstig, Anders | Hottenga, Jouke-Jan | Kavousi, Maryam | Lahti, Jari | Tanaka, Toshiko | Hayward, Caroline | Huffman, Jennifer E. | Morange, Pierre-Emmanuel | Rose, Lynda M. | Basu, Saonli | Rumley, Ann | Stott, David J. | Buckley, Brendan M. | de Craen, Anton J. M. | Sanna, Serena | Masala, Marco | Biffar, Reiner | Homuth, Georg | Silveira, Angela | Sennblad, Bengt | Goel, Anuj | Watkins, Hugh | Müller-Nurasyid, Martina | Rückerl, Regina | Taylor, Kent | Chen, Ming-Huei | de Geus, Eco J. C. | Hofman, Albert | Witteman, Jacqueline C. M. | de Maat, Moniek P. M. | Palotie, Aarno | Davies, Gail | Siscovick, David S. | Kolcic, Ivana | Wild, Sarah H. | Song, Jaejoon | McArdle, Wendy L. | Ford, Ian | Sattar, Naveed | Schlessinger, David | Grotevendt, Anne | Franzosi, Maria Grazia | Illig, Thomas | Waldenberger, Melanie | Lumley, Thomas | Tofler, Geoffrey H. | Willemsen, Gonneke | Uitterlinden, André G. | Rivadeneira, Fernando | Räikkönen, Katri | Chasman, Daniel I. | Folsom, Aaron R. | Lowe, Gordon D. | Westendorp, Rudi G. J. | Slagboom, P. Eline | Cucca, Francesco | Wallaschofski, Henri | Strawbridge, Rona J. | Seedorf, Udo | Koenig, Wolfgang | Bis, Joshua C. | Mukamal, Kenneth J. | van Dongen, Jenny | Widen, Elisabeth | Franco, Oscar H. | Starr, John M. | Liu, Kiang | Ferrucci, Luigi | Polasek, Ozren | Wilson, James F. | Oudot-Mellakh, Tiphaine | Campbell, Harry | Navarro, Pau | Bandinelli, Stefania | Eriksson, Johan | Boomsma, Dorret I. | Dehghan, Abbas | Clarke, Robert | Hamsten, Anders | Boerwinkle, Eric | Jukema, J. Wouter | Naitza, Silvia | Ridker, Paul M. | Völzke, Henry | Deary, Ian J. | Reiner, Alexander P. | Trégouët, David-Alexandre | O'Donnell, Christopher J. | Strachan, David P. | Peters, Annette | Smith, Nicholas L.
PLoS ONE  2014;9(12):e111156.
Plasma fibrinogen is an acute phase protein playing an important role in the blood coagulation cascade having strong associations with smoking, alcohol consumption and body mass index (BMI). Genome-wide association studies (GWAS) have identified a variety of gene regions associated with elevated plasma fibrinogen concentrations. However, little is yet known about how associations between environmental factors and fibrinogen might be modified by genetic variation. Therefore, we conducted large-scale meta-analyses of genome-wide interaction studies to identify possible interactions of genetic variants and smoking status, alcohol consumption or BMI on fibrinogen concentration. The present study included 80,607 subjects of European ancestry from 22 studies. Genome-wide interaction analyses were performed separately in each study for about 2.6 million single nucleotide polymorphisms (SNPs) across the 22 autosomal chromosomes. For each SNP and risk factor, we performed a linear regression under an additive genetic model including an interaction term between SNP and risk factor. Interaction estimates were meta-analysed using a fixed-effects model. No genome-wide significant interaction with smoking status, alcohol consumption or BMI was observed in the meta-analyses. The most suggestive interaction was found for smoking and rs10519203, located in the LOC123688 region on chromosome 15, with a p value of 6.2×10−8. This large genome-wide interaction study including 80,607 participants found no strong evidence of interaction between genetic variants and smoking status, alcohol consumption or BMI on fibrinogen concentrations. Further studies are needed to yield deeper insight in the interplay between environmental factors and gene variants on the regulation of fibrinogen concentrations.
PMCID: PMC4281156  PMID: 25551457
4.  Pharmacogenetic meta-analysis of genome-wide association studies of LDL cholesterol response to statins 
Postmus, Iris | Trompet, Stella | Deshmukh, Harshal A. | Barnes, Michael R. | Li, Xiaohui | Warren, Helen R. | Chasman, Daniel I. | Zhou, Kaixin | Arsenault, Benoit J. | Donnelly, Louise A. | Wiggins, Kerri L. | Avery, Christy L. | Griffin, Paula | Feng, QiPing | Taylor, Kent D. | Li, Guo | Evans, Daniel S. | Smith, Albert V. | de Keyser, Catherine E. | Johnson, Andrew D. | de Craen, Anton J. M. | Stott, David J. | Buckley, Brendan M. | Ford, Ian | Westendorp, Rudi G. J. | Eline Slagboom, P. | Sattar, Naveed | Munroe, Patricia B. | Sever, Peter | Poulter, Neil | Stanton, Alice | Shields, Denis C. | O’Brien, Eoin | Shaw-Hawkins, Sue | Ida Chen, Y.-D. | Nickerson, Deborah A. | Smith, Joshua D. | Pierre Dubé, Marie | Matthijs Boekholdt, S. | Kees Hovingh, G. | Kastelein, John J. P. | McKeigue, Paul M. | Betteridge, John | Neil, Andrew | Durrington, Paul N. | Doney, Alex | Carr, Fiona | Morris, Andrew | McCarthy, Mark I. | Groop, Leif | Ahlqvist, Emma | Bis, Joshua C. | Rice, Kenneth | Smith, Nicholas L. | Lumley, Thomas | Whitsel, Eric A. | Stürmer, Til | Boerwinkle, Eric | Ngwa, Julius S. | O’Donnell, Christopher J. | Vasan, Ramachandran S. | Wei, Wei-Qi | Wilke, Russell A. | Liu, Ching-Ti | Sun, Fangui | Guo, Xiuqing | Heckbert, Susan R | Post, Wendy | Sotoodehnia, Nona | Arnold, Alice M. | Stafford, Jeanette M. | Ding, Jingzhong | Herrington, David M. | Kritchevsky, Stephen B. | Eiriksdottir, Gudny | Launer, Leonore J. | Harris, Tamara B. | Chu, Audrey Y. | Giulianini, Franco | MacFadyen, Jean G. | Barratt, Bryan J. | Nyberg, Fredrik | Stricker, Bruno H. | Uitterlinden, André G. | Hofman, Albert | Rivadeneira, Fernando | Emilsson, Valur | Franco, Oscar H. | Ridker, Paul M. | Gudnason, Vilmundur | Liu, Yongmei | Denny, Joshua C. | Ballantyne, Christie M. | Rotter, Jerome I. | Adrienne Cupples, L. | Psaty, Bruce M. | Palmer, Colin N. A. | Tardif, Jean-Claude | Colhoun, Helen M. | Hitman, Graham | Krauss, Ronald M. | Wouter Jukema, J | Caulfield, Mark J.
Nature Communications  2014;5:5068.
Statins effectively lower LDL cholesterol levels in large studies and the observed interindividual response variability may be partially explained by genetic variation. Here we perform a pharmacogenetic meta-analysis of genome-wide association studies (GWAS) in studies addressing the LDL cholesterol response to statins, including up to 18,596 statin-treated subjects. We validate the most promising signals in a further 22,318 statin recipients and identify two loci, SORT1/CELSR2/PSRC1 and SLCO1B1, not previously identified in GWAS. Moreover, we confirm the previously described associations with APOE and LPA. Our findings advance the understanding of the pharmacogenetic architecture of statin response.
Statins are effectively used to prevent and manage cardiovascular disease, but patient response to these drugs is highly variable. Here, the authors identify two new genes associated with the response of LDL cholesterol to statins and advance our understanding of the genetic basis of drug response.
PMCID: PMC4220464  PMID: 25350695
5.  Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions 
Interface Focus  2013;3(5):20130007.
We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for subdiffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear deconvolution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy.
PMCID: PMC3915824  PMID: 24511385
high resolution; live-cell imaging; fluorescence microscopy; saturated excitation; confocal microscopy
6.  A Multi-Ethnic Meta-Analysis of Genome-Wide Association Studies in Over 100,000 Subjects Identifies 23 Fibrinogen-Associated Loci but no Strong Evidence of a Causal Association between Circulating Fibrinogen and Cardiovascular Disease 
Sabater-Lleal, Maria | Huang, Jie | Chasman, Daniel | Naitza, Silvia | Dehghan, Abbas | Johnson, Andrew D | Teumer, Alexander | Reiner, Alex P | Folkersen, Lasse | Basu, Saonli | Rudnicka, Alicja R | Trompet, Stella | Mälarstig, Anders | Baumert, Jens | Bis, Joshua C. | Guo, Xiuqing | Hottenga, Jouke J | Shin, So-Youn | Lopez, Lorna M | Lahti, Jari | Tanaka, Toshiko | Yanek, Lisa R | Oudot-Mellakh, Tiphaine | Wilson, James F | Navarro, Pau | Huffman, Jennifer E | Zemunik, Tatijana | Redline, Susan | Mehra, Reena | Pulanic, Drazen | Rudan, Igor | Wright, Alan F | Kolcic, Ivana | Polasek, Ozren | Wild, Sarah H | Campbell, Harry | Curb, J David | Wallace, Robert | Liu, Simin | Eaton, Charles B. | Becker, Diane M. | Becker, Lewis C. | Bandinelli, Stefania | Räikkönen, Katri | Widen, Elisabeth | Palotie, Aarno | Fornage, Myriam | Green, David | Gross, Myron | Davies, Gail | Harris, Sarah E | Liewald, David C | Starr, John M | Williams, Frances M.K. | Grant, P.J. | Spector, Timothy D. | Strawbridge, Rona J | Silveira, Angela | Sennblad, Bengt | Rivadeneira, Fernando | Uitterlinden, Andre G | Franco, Oscar H | Hofman, Albert | van Dongen, Jenny | Willemsen, G | Boomsma, Dorret I | Yao, Jie | Jenny, Nancy Swords | Haritunians, Talin | McKnight, Barbara | Lumley, Thomas | Taylor, Kent D | Rotter, Jerome I | Psaty, Bruce M | Peters, Annette | Gieger, Christian | Illig, Thomas | Grotevendt, Anne | Homuth, Georg | Völzke, Henry | Kocher, Thomas | Goel, Anuj | Franzosi, Maria Grazia | Seedorf, Udo | Clarke, Robert | Steri, Maristella | Tarasov, Kirill V | Sanna, Serena | Schlessinger, David | Stott, David J | Sattar, Naveed | Buckley, Brendan M | Rumley, Ann | Lowe, Gordon D | McArdle, Wendy L | Chen, Ming-Huei | Tofler, Geoffrey H | Song, Jaejoon | Boerwinkle, Eric | Folsom, Aaron R. | Rose, Lynda M. | Franco-Cereceda, Anders | Teichert, Martina | Ikram, M Arfan | Mosley, Thomas H | Bevan, Steve | Dichgans, Martin | Rothwell, Peter M. | Sudlow, Cathie L M | Hopewell, Jemma C. | Chambers, John C. | Saleheen, Danish | Kooner, Jaspal S. | Danesh, John | Nelson, Christopher P | Erdmann, Jeanette | Reilly, Muredach P. | Kathiresan, Sekar | Schunkert, Heribert | Morange, Pierre-Emmanuel | Ferrucci, Luigi | Eriksson, Johan G | Jacobs, David | Deary, Ian J | Soranzo, Nicole | Witteman, Jacqueline CM | de Geus, Eco JC | Tracy, Russell P. | Hayward, Caroline | Koenig, Wolfgang | Cucca, Francesco | Jukema, J Wouter | Eriksson, Per | Seshadri, Sudha | Markus, Hugh S. | Watkins, Hugh | Samani, Nilesh J | Wallaschofski, Henri | Smith, Nicholas L. | Tregouet, David | Ridker, Paul M. | Tang, Weihong | Strachan, David P. | Hamsten, Anders | O’Donnell, Christopher J.
Circulation  2013;128(12):10.1161/CIRCULATIONAHA.113.002251.
Estimates of the heritability of plasma fibrinogen concentration, an established predictor of cardiovascular disease (CVD), range from 34 to 50%. Genetic variants so far identified by genome-wide association (GWA) studies only explain a small proportion (< 2%) of its variation.
Methods and Results
We conducted a meta-analysis of 28 GWA studies, including more than 90,000 subjects of European ancestry, the first GWA meta-analysis of fibrinogen levels in 7 African Americans studies totaling 8,289 samples, and a GWA study in Hispanic-Americans totaling 1,366 samples. Evaluation for association of SNPs with clinical outcomes included a total of 40,695 cases and 85,582 controls for coronary artery disease (CAD), 4,752 cases and 24,030 controls for stroke, and 3,208 cases and 46,167 controls for venous thromboembolism (VTE). Overall, we identified 24 genome-wide significant (P<5×10−8) independent signals in 23 loci, including 15 novel associations, together accounting for 3.7% of plasma fibrinogen variation. Gene-set enrichment analysis highlighted key roles in fibrinogen regulation for the three structural fibrinogen genes and pathways related to inflammation, adipocytokines and thyrotrophin-releasing hormone signaling. Whereas lead SNPs in a few loci were significantly associated with CAD, the combined effect of all 24 fibrinogen-associated lead SNPs was not significant for CAD, stroke or VTE.
We identify 23 robustly associated fibrinogen loci, 15 of which are new. Clinical outcome analysis of these loci does not support a causal relationship between circulating levels of fibrinogen and CAD, stroke or VTE.
PMCID: PMC3842025  PMID: 23969696
Fibrinogen; cardiovascular disease; genome-wide association study
7.  Ceruloplasmin and Heart Failure in the Atherosclerosis Risk in Communities (ARIC) Study 
Circulation. Heart failure  2013;6(5):936-943.
Ceruloplasmin (Cp) decreases nitric oxide bioavailability in blood and has been associated with cardiovascular disease (CVD) in clinical studies. We assessed the association between Cp and incident heart failure (HF), death and CVD in the Atherosclerosis Risk in Communities (ARIC) Study.
Methods and Results
Cp was measured at ARIC visit 4 (1996–1998). We studied 9,240 individuals without HF or CVD at ARIC visit 4, and followed them for a mean of 10.5 years. Genome-wide association study was performed to identify genetic determinants of Cp levels and evaluate their association with incident HF. Cp levels (mean±standard deviation) were higher in women vs men (335±79 vs 258±44 mg/L, p<0.0001), women on vs not on hormone-replacement therapy (398±89 vs 291±60 mg/L, p<0.0001) and African Americans vs Caucasians (299±63 vs 293±74 mg/L, p=0.0005). After adjusting for traditional risk factors, high-sensitivity C-reactive protein, N-terminal pro–B-type natriuretic peptide, and high-sensitivity cardiac troponin T, higher levels of Cp were associated with HF (hazard ratio [HR] 1.44, 95% confidence interval [CI] 1.13–1.83) and mortality (HR 1.38, 95% CI 1.11–1.63). A locus on the ceruloplasmin gene on chromosome 3 was significantly associated with Cp levels (normal 295.56±77.60mg/L, heterozygote 316.72±88.02mg/L; homozygote 331.04±85.40mg/L, p=8.3×10−) but not with incident HF. After adjustment for traditional risk factors Cp levels were also weekly associated with CVD.
Cp was associated with incident, HF mortality and CVD in the ARIC population. A single locus on chromosome 3 was associated with Cp levels but not with HF.
PMCID: PMC3908901  PMID: 23861484
ceruloplasmin; heart failure; cardiovascular disease; single nucleotide polymorphism
8.  Transforming Growth Factor Beta-1 and Incidence of Heart Failure in Older Adults: The Cardiovascular Health Study 
Cytokine  2012;60(2):341-345.
Transforming growth factor-beta1 (TGF-B1) is a highly pleiotropic cytokine whose functions include a central role in the induction of fibrosis.
To investigate the hypothesis that elevated plasma levels of TGF-B1 are positively associated with incident heart failure (HF).
Participants and Methods
The hypotheses were tested using a two-phase case-control study design, ancillary to the Cardiovascular Health Study – a longitudinal, population-based cohort study. Cases were defined as having an incident HF event after their 1992-93 exam and controls were free of HF at follow-up. TGF-B1 was measured using plasma collected in 1992-93 and data from 89 cases and 128 controls were used for analysis. The association between TGF-B1 and risk of HF was evaluated using the weighted likelihood method, and odds ratios (OR) for risk of HF were calculated for TGF-B1 as a continuous linear variable and across quartiles of TGF-B1.
The OR for HF was 1.88 (95% confidence intervals [CI] 1.26 to 2.81) for each nanogram increase in TGF-B1, and the OR for the highest quartile (compared to the lowest) of TGF-B1 was 5.79 (95% CI 1.65 – 20.34), after adjustment for age, sex, C-reactive protein, platelet count and digoxin use. Further adjustment with other covariates did not change the results.
Higher levels of plasma TGF-B1 were associated with an increased risk of incident heart failure among older adults. However, further study is needed in larger samples to confirm these findings.
PMCID: PMC4143419  PMID: 22878343
transforming growth factor-beta; heart failure; fibrosis; growth factors; cardiac remodeling
9.  Race, Gender, and Mortality in Adults ≥65 Years of Age With Incident Heart Failure (from the Cardiovascular Health Study) 
The American journal of cardiology  2009;103(8):1120-1127.
In patients with heart failure (HF), mortality is lower in women versus men. However, it is unknown whether the survival advantage in women compared with men is present in both whites and African Americans with HF. The inception cohort consisted of adults ≥65 years with incident HF after enrollment in the CHS, a prospective population-based study of cardiovascular disease. Of 5,888 CHS subjects, 1,264 developed new HF and were followed up for 3 years. Subjects were categorized into 4 race-gender groups, and Cox proportional hazard regression models were used to examine whether 3-year total and cardiovascular mortality differed among the 4 groups after adjusting for sociodemographic factors, co-morbidities, and treatment. A gender-race interaction was also tested for each outcome. In subjects with incident HF, African Americans had more hypertension and diabetes than whites, and white men had more coronary heart disease than other gender-race groups. Receipt of cardiovascular treatments among the 4 groups was similar. Mortality rates after HF were lower in women compared with men (for white women, African-American women, African-American men, and white men, total mortality was 35.5, 33.6, 44.4, and 40.5/100 person-years, and cardiovascular mortality was 18.4, 19.5, 20.2, and 22.7/100 person-years, respectively). After adjusting for covariates, women had a 15% to 20% lower risk of total and cardiovascular mortality compared with men, but there was no significant difference in outcome by race. The gender-race interaction for either outcome was not significant. In conclusion, in older adults with HF, women had significantly better survival than men irrespective of race, suggesting that gender-based survival differences may be more important than race-based differences.
PMCID: PMC4122325  PMID: 19361600
10.  Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay 
Gastroenterology  2013;145(2):383-95.e1-21.
Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues.
We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs.
CD44+CD24loCD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78lo/− putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs.
We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+CD24loCD166+, GRP78lo/−, and c-Kit− facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+CD24−/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.
PMCID: PMC3781924  PMID: 23644405
Stemness; Differentiation; Single-Cell Sorting; Flow Cytometry Analysis
11.  ProBLM Web Server: Protein and Membrane Placement and Orientation Package 
The 3D structures of membrane proteins are typically determined without the presence of a lipid bilayer. For the purpose of studying the role of membranes on the wild type characteristics of the corresponding protein, determining the position and orientation of transmembrane proteins within a membrane environment is highly desirable. Here we report a geometry-based approach to automatically insert a membrane protein with a known 3D structure into pregenerated lipid bilayer membranes with various dimensions and lipid compositions or into a pseudomembrane. The pseudomembrane is built using the Protein Nano-Object Integrator which generates a parallelepiped of user-specified dimensions made up of pseudoatoms. The pseudomembrane allows for modeling the desolvation effects while avoiding plausible errors associated with wrongly assigned protein-lipid contacts. The method is implemented into a web server, the ProBLM server, which is freely available to the biophysical community. The web server allows the user to upload a protein coordinate file and any missing residues or heavy atoms are regenerated. ProBLM then creates a combined protein-membrane complex from the given membrane protein and bilayer lipid membrane or pseudomembrane. The user is given an option to manually refine the model by manipulating the position and orientation of the protein with respect to the membrane.
PMCID: PMC4122144  PMID: 25126110
12.  A genome-wide association study for venous thromboembolism: the extended Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium 
Genetic epidemiology  2013;37(5):512-521.
Venous thromboembolism (VTE) is a common, heritable disease resulting in high rates of hospitalization and mortality. Yet few associations between VTE and genetic variants, all in the coagulation pathway, have been established. To identify additional genetic determinants of VTE, we conducted a 2-stage genome-wide association study (GWAS) among individuals of European ancestry in the extended CHARGE VTE consortium. The discovery GWAS comprised 1,618 incident VTE cases out of 44,499 participants from six community-based studies. Genotypes for genome-wide single-nucleotide polymorphisms (SNPs) were imputed to ~2.5 million SNPs in HapMap and association with VTE assessed using study-design appropriate regression methods. Meta-analysis of these results identified two known loci, in F5 and ABO. Top 1,047 tag SNPs (p≤0.0016) from the discovery GWAS were tested for association in an additional 3,231 cases and 3,536 controls from three case-control studies. In the combined data from these two stages, additional genome-wide significant associations were observed on 4q35 at F11 (top SNP rs4253399, intronic to F11) and on 4q28 at FGG (rs6536024, 9.7 kb from FGG) (p<5.0×10−13 for both). The associations at the FGG locus were not completely explained by previously reported variants. Loci at or near SUSD1 and OTUD7A showed borderline yet novel associations (p<5.0×10-6) and constitute new candidate genes. In conclusion, this large GWAS replicated key genetic associations in F5 and ABO, and confirmed the importance of F11 and FGG loci for VTE. Future studies are warranted to better characterize the associations with F11 and FGG and to replicate the new candidate associations.
PMCID: PMC3990406  PMID: 23650146
venous thrombosis; genetics; genome-wide association; genetic epidemiology
13.  Sensitivities, Specificities, and Predictive Values of Microbiological Culture Techniques for the Diagnosis of Prosthetic Joint Infection 
BioMed Research International  2014;2014:180416.
Background. Identifying the microorganism in a prosthetic joint infection is the key to appropriately targeting antimicrobial treatment. Despite the availability of various techniques, no single test is considered the definitive gold standard. Aim. Our aim was to determine the sensitivity, specificity, and positive/negative predictive values for a variety of culture techniques. Methods. We performed a retrospective case series of 219 patients undergoing revision surgery of their hip or knee replacement between May 2004 and February 2013. The patients were classified as either infected or noninfected according to criteria set out by the Musculoskeletal Infection Society. The number and type of samples taken intraoperatively varied between cases but included tissue samples and fluid sent in either blood culture vials or sterile containers. Results. The highest sensitivity was found with blood culture vials (0.85) compared to fluid in sterile containers (0.26) and tissues samples (0.32). Blood culture vials also had a better specificity and positive and negative predictive values profile. Conclusion. We conclude that, of the techniques studied, fluid in blood culture vials had the best profile for the correct identification of microorganisms and advocate its use.
PMCID: PMC4055545  PMID: 24963476
14.  Using Eye Tracking to Assess Reading Performance in Patients with Glaucoma: A Within-Person Study 
Journal of Ophthalmology  2014;2014:120528.
Reading is often cited as a demanding task for patients with glaucomatous visual field (VF) loss, yet reading speed varies widely between patients and does not appear to be predicted by standard visual function measures. This within-person study aimed to investigate reading duration and eye movements when reading short passages of text in a patient's worse eye (most VF damage) when compared to their better eye (least VF damage). Reading duration and saccade rate were significantly different on average in the worse eye when compared to the better eye (P < 0.001) in 14 patients with glaucoma that had median (interquartile range) between-eye difference in mean deviation (MD; a standard clinical measure for VF loss) of 9.8 (8.3 to 14.8) dB; differences were not related to the size of the difference in MD between eyes. Patients with a more pronounced effect of longer reading duration on their worse eye made a larger proportion of “regressions” (backward saccades) and “unknown” EMs (not adhering to expected reading patterns) when reading with the worse eye when compared to the better eye. A between-eye study in patients with asymmetric disease, coupled with eye tracking, provides a useful experimental design for exploring reading performance in glaucoma.
PMCID: PMC4026991  PMID: 24883203
15.  Gene expression in thiazide diuretic or statin users in relation to incident type 2 diabetes 
Thiazide diuretics and statins are used to improve cardiovascular outcomes, but may also cause type 2 diabetes (T2DM), although mechanisms are unknown. Gene expression studies may facilitate understanding of these associations. Participants from ongoing population-based studies were sampled for these longitudinal studies of peripheral blood microarray gene expression, and followed to incident diabetes. All sampled subjects were statin or thiazide users. Those who developed diabetes during follow-up comprised cases (44 thiazide users; 19 statin users), and were matched to drug-using controls who did not develop diabetes on several factors. Supervised normalization, surrogate variable analyses removed technical bias and confounding. Differentially-expressed genes were those with a false discovery rate Q-value<0.05. Among thiazide users, diabetes cases had significantly different expression of CCL14 (down-regulated 6%, Q-value=0.0257), compared with controls. Among statin users, diabetes cases had marginal but insignificantly different expression of ZNF532 (up-regulated 15%, Q-value=0.0584), CXORF21 (up-regulated 11%, Q-value=0.0584), and ZNHIT3 (up-regulated 19%, Q-value=0.0959), compared with controls. These genes comprise potential targets for future expression or mechanistic research on medication-related diabetes development.
PMCID: PMC3939004  PMID: 24596594
Type 2 diabetes; statins; thiazide diuretics; whole blood; gene expression; microarray; supervised normalization; surrogate variable analysis; chemokine ligand 14; zinc finger proteins
16.  Genome-Wide Association Study of Cardiac Structure and Systolic Function in African Americans: The Candidate Gene Association Resource (CARe) Study 
Using data from four community-based cohorts of African Americans (AA), we tested the association between genome-wide markers (SNPs) and cardiac phenotypes in the Candidate-gene Association REsource (CARe) study.
Methods and Results
Among 6,765 AA, we related age, sex, height and weight-adjusted residuals for nine cardiac phenotypes (assessed by echocardiogram or MRI) to 2.5 million SNPs genotyped using Genome-Wide Affymetrix Human SNP Array 6.0 (Affy6.0) and the remainder imputed. Within cohort genome-wide association analysis was conducted followed by meta-analysis across cohorts using inverse variance weights (genome-wide significance threshold=4.0 ×10−07). Supplementary pathway analysis was performed. We attempted replication in 3 smaller cohorts of African ancestry and tested look-ups in one consortium of European ancestry (EchoGEN). Across the 9 phenotypes, variants in 4 genetic loci reached genome-wide significance: rs4552931 in UBE2V2 (p=1.43 × 10−07) for left ventricular mass (LVM); rs7213314 in WIPI1 (p=1.68 × 10−07) for LV internal diastolic diameter (LVIDD); rs1571099 in PPAPDC1A (p= 2.57 × 10−08) for interventricular septal wall thickness (IVST); and rs9530176 in KLF5 (p=4.02 × 10−07) for ejection fraction (EF). Associated variants were enriched in three signaling pathways involved in cardiac remodeling. None of the 4 loci replicated in cohorts of African ancestry were confirmed in look-ups in EchoGEN.
In the largest GWAS of cardiac structure and function to date in AA, we identified 4 genetic loci related to LVM, IVST, LVIDD and EF that reached genome-wide significance. Replication results suggest that these loci may represent unique to individuals of African ancestry. Additional large-scale studies are warranted for these complex phenotypes.
PMCID: PMC3591479  PMID: 23275298
echocardiography; ethnic; genome-wide association studies; Left atrium genetics; left ventricular mass genetics
17.  Association of Body Mass Index, Diabetes, Hypertension, and Blood Pressure Levels with Risk of Permanent Atrial Fibrillation 
After an initial episode of atrial fibrillation (AF), AF may recur and become permanent. AF progression is associated with higher morbidity and mortality. Understanding the risk factors for permanent AF could help identify people who would benefit most from interventions.
To determine whether body mass index (BMI), diabetes, hypertension, and blood pressure levels are associated with permanent AF among people whose initial AF episode terminated.
Population-based inception cohort study.
Enrollees in Group Health, an integrated health care system, aged 30–84 with newly diagnosed AF in 2001–2004, whose initial AF terminated within 6 months and who had at least 6 months of subsequent follow-up (N = 1,385).
Clinical characteristics were determined from medical records. Permanent AF was determined from medical records and ECG and administrative databases. Permanent AF was defined as AF present on two separate occasions 6–36 months apart, without any documented sinus rhythm between the two occasions. Cox proportional hazards models were used to estimate adjusted hazard ratios (HRs).
Five-year cumulative incidence of permanent AF was 24 %. Compared with normal BMI (18.5–24.9 kg/m2), BMI levels of 25.0–29.9 (overweight), 30.0–34.9 (obese 1), 35.0–39.9 (obese 2), and ≥ 40.0 kg/m2 (obese 3) were associated with HRs of permanent AF of 1.26 (95 % CI: 0.92, 1.72); 1.35 (0.96, 1.91); 1.50 (0.97, 2.33); and 1.79 (1.13, 2.84), adjusted for age, sex, diabetes, hypertension, blood pressure, coronary heart disease, valvular heart disease, heart failure, and prior stroke. Diabetes, hypertension, and blood pressure were not associated with permanent AF.
For people whose initial AF episode terminates, benefits of having lower BMI may include a lower risk of permanent AF. Risk of permanent AF was similar for people with and without diabetes or hypertension and across blood pressure levels.
Electronic supplementary material
The online version of this article (doi:10.1007/s11606-012-2220-4) contains supplementary material, which is available to authorized users.
PMCID: PMC3614136  PMID: 22972153
cohort study; anthropometry; electrocardiogram; atrial fibrillation
19.  What's on TV? Detecting age-related neurodegenerative eye disease using eye movement scanpaths 
Purpose: We test the hypothesis that age-related neurodegenerative eye disease can be detected by examining patterns of eye movement recorded whilst a person naturally watches a movie.
Methods: Thirty-two elderly people with healthy vision (median age: 70, interquartile range [IQR] 64–75 years) and 44 patients with a clinical diagnosis of glaucoma (median age: 69, IQR 63–77 years) had standard vision examinations including automated perimetry. Disease severity was measured using a standard clinical measure (visual field mean deviation; MD). All study participants viewed three unmodified TV and film clips on a computer set up incorporating the Eyelink 1000 eyetracker (SR Research, Ontario, Canada). Eye movement scanpaths were plotted using novel methods that first filtered the data and then generated saccade density maps. Maps were then subjected to a feature extraction analysis using kernel principal component analysis (KPCA). Features from the KPCA were then classified using a standard machine based classifier trained and tested by a 10-fold cross validation which was repeated 100 times to estimate the confidence interval (CI) of classification sensitivity and specificity.
Results: Patients had a range of disease severity from early to advanced (median [IQR] right eye and left eye MD was −7 [−13 to −5] dB and −9 [−15 to −4] dB, respectively). Average sensitivity for correctly identifying a glaucoma patient at a fixed specificity of 90% was 79% (95% CI: 58–86%). The area under the Receiver Operating Characteristic curve was 0.84 (95% CI: 0.82–0.87).
Conclusions: Huge data from scanpaths of eye movements recorded whilst people freely watch TV type films can be processed into maps that contain a signature of vision loss. In this proof of principle study we have demonstrated that a group of patients with age-related neurodegenerative eye disease can be reasonably well separated from a group of healthy peers by considering these eye movement signatures alone.
PMCID: PMC4228197  PMID: 25429267
eye movements; scanpaths; glaucoma; perimetry; eye tracking; KPCA; perception; diagnosis procedures
20.  Dye-Binding Assays for Evaluation of the Effects of Small Molecule Inhibitors on Amyloid (Aβ) Self-Assembly 
ACS Chemical Neuroscience  2012;3(11):807-819.
Dye-binding assays, such as those utilizing Congo red and thioflavin T, are among the most widely used tools to probe the aggregation of amyloidogenic biomolecules and for the evaluation of small molecule inhibitors of amyloid aggregation and fibrillization. A number of recent reports have indicated that these dye-binding assays could be prone to false positive effects when assessing inhibitors’ potential toward Aβ peptides, species involved in Alzheimer’s disease. Specifically, this review focuses on the application of thioflavin T for determining the efficiency of small molecule inhibitors of Aβ aggregation and addresses potential reasons that might be associated with the false positive effects in an effort to increase reliability of dye-binding assays.
PMCID: PMC3503347  PMID: 23173064
Alzheimer’s disease; amyloid peptide; small molecule inhibitor; dye-binding assay; thioflavin T; fluorescence
21.  Asymmetry in infants' selective attention to facial features during visual processing of infant-directed speech 
Two experiments used eye tracking to examine how infant and adult observers distribute their eye gaze on videos of a mother producing infant- and adult-directed speech. Both groups showed greater attention to the eyes than to the nose and mouth, as well as an asymmetrical focus on the talker's right eye for infant-directed speech stimuli. Observers continued to look more at the talker's apparent right eye when the video stimuli were mirror flipped, suggesting that the asymmetry reflects a perceptual processing bias rather than a stimulus artifact, which may be related to cerebral lateralization of emotion processing.
PMCID: PMC3769626  PMID: 24062705
infant-directed speech; eye-tracking; face perception; emotion; lateralization; language; speech perception
22.  A systematic review of the evidence for single stage and two stage revision of infected knee replacement 
Periprosthetic infection about the knee is a devastating complication that may affect between 1% and 5% of knee replacement. With over 79 000 knee replacements being implanted each year in the UK, periprosthetic infection (PJI) is set to become an important burden of disease and cost to the healthcare economy. One of the important controversies in treatment of PJI is whether a single stage revision operation is superior to a two-stage procedure. This study sought to systematically evaluate the published evidence to determine which technique had lowest reinfection rates.
A systematic review of the literature was undertaken using the MEDLINE and EMBASE databases with the aim to identify existing studies that present the outcomes of each surgical technique. Reinfection rate was the primary outcome measure. Studies of specific subsets of patients such as resistant organisms were excluded.
63 studies were identified that met the inclusion criteria. The majority of which (58) were reports of two-stage revision. Reinfection rated varied between 0% and 41% in two-stage studies, and 0% and 11% in single stage studies. No clinical trials were identified and the majority of studies were observational studies.
Evidence for both one-stage and two-stage revision is largely of low quality. The evidence basis for two-stage revision is significantly larger, and further work into direct comparison between the two techniques should be undertaken as a priority.
PMCID: PMC3734185  PMID: 23895421
Infection; Knee replacement; One stage; Two-stage; Arthroplasty; Revision
23.  DelPhi web server v2: incorporating atomic-style geometrical figures into the computational protocol 
Bioinformatics  2012;28(12):1655-1657.
Summary: A new edition of the DelPhi web server, DelPhi web server v2, is released to include atomic presentation of geometrical figures. These geometrical objects can be used to model nano-size objects together with real biological macromolecules. The position and size of the object can be manipulated by the user in real time until desired results are achieved. The server fixes structural defects, adds hydrogen atoms and calculates electrostatic energies and the corresponding electrostatic potential and ionic distributions.
Availability and implementation: The web server follows a client–server architecture built on PHP and HTML and utilizes DelPhi software. The computation is carried out on supercomputer cluster and results are given back to the user via http protocol, including the ability to visualize the structure and corresponding electrostatic potential via Jmol implementation. The DelPhi web server is available from
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3371833  PMID: 22531215
24.  Genetic Loci for Retinal Arteriolar Microcirculation 
PLoS ONE  2013;8(6):e65804.
Narrow arterioles in the retina have been shown to predict hypertension as well as other vascular diseases, likely through an increase in the peripheral resistance of the microcirculatory flow. In this study, we performed a genome-wide association study in 18,722 unrelated individuals of European ancestry from the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium and the Blue Mountain Eye Study, to identify genetic determinants associated with variations in retinal arteriolar caliber. Retinal vascular calibers were measured on digitized retinal photographs using a standardized protocol. One variant (rs2194025 on chromosome 5q14 near the myocyte enhancer factor 2C MEF2C gene) was associated with retinal arteriolar caliber in the meta-analysis of the discovery cohorts at genome-wide significance of P-value <5×10−8. This variant was replicated in an additional 3,939 individuals of European ancestry from the Australian Twins Study and Multi-Ethnic Study of Atherosclerosis (rs2194025, P-value = 2.11×10−12 in combined meta-analysis of discovery and replication cohorts). In independent studies of modest sample sizes, no significant association was found between this variant and clinical outcomes including coronary artery disease, stroke, myocardial infarction or hypertension. In conclusion, we found one novel loci which underlie genetic variation in microvasculature which may be relevant to vascular disease. The relevance of these findings to clinical outcomes remains to be determined.
PMCID: PMC3680438  PMID: 23776548
25.  Genetic variation in F3 (tissue factor) and the risk of incident venous thrombosis: meta-analysis of 8 studies 
PMCID: PMC3397243  PMID: 22340074
Venous thrombosis; tissue factor; F3; D-dimer; epidemiology; meta-analysis

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