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Variables dropped at earlier stages were re-evaluated for inclusion in the final model. Normality of residuals and absence of heteroscedasticity were checked. All tests were two-sided. Statistical analysis was performed using the R package Version 2. 10 [42] and SAS 9. 2 software (SAS Institute, Cary, NC, USA).
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10.1186/s12874-016-0138-y
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Strengths and weaknesses of the study (internal validity) This study is the first to quantify the implementation duration of hospital-based pediatric clinical studies and to identify factors independently related to this duration. A limitation of the study is that the data were collected in 2009, which prevented us from describing more recent data. However, the issues highlighted by our study are not specific to this time period and are still relevant today. Although it is a retrospective study, there were no missing data and the sources of data were reliable. It would have been of interest to analyze the association between the risk level associated to the studies and their implementation duration; however, this data was not available. Nevertheless, the type of study closely reflects this information since the interventional pediatric studies are usually classified as high risk and the observational studies as low risk.
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10.1186/s12874-016-0138-y
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Acknowledgments We thank Céline Feger , Karine Landgren-Hugentobler , and Maya Dorsey for language editing, and the Clinical Trial Units , particularly Dr. Caroline Elie , for helping us to collect the data. This work was supported by a research grant from the Département de la Recherche Clinique et du Développement, Assistance Publique-Hôpitaux de Paris ( RSR08001 ). The funding agency was not involved in the study design, data analysis, or manuscript preparation.
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10.1186/s12874-016-0138-y
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78 1 [1;1] 1 Monocentric study 12 (17) 5 (15. 6) 14 (40) 0. 01 4 (80) 1 International study 1 (1) 0 (0) 4 (11) 0. 006 0 (0) 0 Rare disease 50 (69) 20 (63) 27 (77) 0. 43 4 (67) 0 Chronic disease 60 (83) 22 (69) 26 (74) 0. 22 4 (67) 0 Study population 0. 003 0 Pediatric 30 (42) 19 (59) 27 (77) 6 (100) Mixed (children and adults) 42 (58) 13 (41) 8 (23) 0 (0) Number of patients to include 150 [82;388] 128 [66;240] 60 [36;100] 0. 12 20 [20;25] 3 Length of participation, months 0. 2 [0. 03;12] 4 [0. 2;17] 8 [0. 5;24] 0. 61 0. 1 [0. 03;2] 2 Referenced in ClinicalTrials. gov 21 (29) 32 (100) 24 (69) <0. 001 1 (17) 0 Values shown are median[25 thpercentile; 75 th percentile] and number (percentage).
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Materials and Methods A retrospective study was carried out on data from a review of the records of 66 patients operated on by the same surgeon, from 2006 to 2014, at the Otorhinolaryngoiatric Unit of the "Centre Hospitalier des Escartons" of Briancon, France, with antrostomy for unilateral maxillary sinusitis and suspicion of a fungus ball. Inclusion criteria were patients who had been given a definitive anatomopathological and/or histological diagnosis of fungus ball of maxillary sinus, patients without conditions at diagnosis that could affect the development of sinonasal fungal disease (such as immunodeficiency, allergy, or previous endoscopic nasal surgery), and patients who had been operated on by the same chosen surgeon. The study group included 25 patients who met the inclusion criteria.
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10.1155/2016/4169523
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0%) for 2 days, 9 (36. 0%) for 3 days, 5 (20. 0%) for 4 days, and 4 (16. 0%) for 5 days. There was only one postsurgical complication (4. 0%), an epistaxis on the 5th postsurgical day, which was treated successfully by nasal packing for 48 hours. One other patient had a relapse at one year, treated with Caldwell-Luc approach with success. The follow-up ranged from 1 to 105 months, for a median of 46. 0 months. Positive follow-up outcome data were high. Indeed, the median SNOT-20 score was 3. 0, ranging from 0 to 35, where 0 is the absence of symptoms and 100 is the highest number of symptoms ever recorded. Table 1 reports the data from the clinical notes of the 25 patients. Table 2 reports the comparison between the gauze technique and the classical-technique in terms of age, surgery timing, hospitalization length, SNOT-20 results, timing of unpacking, and number of postoperatory complications.
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10.1155/2016/4169523
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Acknowledgments The authors thank Sara Tunesi for her statistical advices, Barbara Wade for her linguistic revision, and Davide Mariotto for his digital design support.
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10.1155/2016/4169523
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GFR measurement and estimation GFR was assessed through a continuous 51 Cr-EDTA (GE Healthcare, Little Chalfont, UK) infusion method. A priming dose of 0. 5 lCi/kg body weight of 51 Cr-EDTA was injected intravenously, followed by a constant 51 Cr-EDTA infusion. After allowing 1 h for equilibration of the tracer in the extracellular fluid, urine was collected and discarded. Average renal 51 Cr-EDTA clearance was assessed during six consecutive 30-min clearance periods. Blood was drawn at the midpoint of each clearance period with the last collection 300 min after injection of the priming dose. The radioactivity measurements in 1-mL plasma samples and in urine samples were carried out on a Packard Cobra 3-inch crystal c-ray well counter (PerkinElmer, Waltham, MA). Inulin clearance and iohexol clearance are described in Data S1.
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10.1111/ajt.13908
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1 , C and D). mRNA levels of AMPK ␣-, -, and ␥-subunits in liver and skeletal muscle were not different in AMPK␥2 NI and AMPK␥2 RG mice as compared with AMPK␥2 WT mice (data not shown). Taken together, these data demonstrate that the AMPK␥2 NI and AMPK␥2 RG knock-in mutations had no effect on expression of ␥2 subunit and other AMPK subunits. Two main isoforms of acetyl-CoA carboxylase (ACC), ACC1 and ACC2, are downstream targets of activated AMPK. Phosphorylation of ACC inhibits its enzymatic activity, and this effect is widely employed to reflect the activity of AMPK (22). We therefore used phosphorylated ACC (pACC) (both ACC1 and ACC2) to monitor AMPK activity. In skeletal muscle, pACC was significantly increased in AMPK␥2 NI and AMPK␥2 RG compared with AMPK␥2 WT mice (Fig. 1, E and F ).
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10.1074/jbc.m116.738591
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Biochemistry, Genetics and Molecular Biology
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Slides were digitized using a Nanozoomer 2. 0 HT digital slide scanner (Hamamatsu). Statistical Analyses-All data are shown as the means Ϯ S. E. Statistical significance was calculated by one-way analysis of variance and multiple comparisons. Asterisks denote statistical significance of the AMPK␥2 NI and AMPK␥2 RG groups compared with the AMPK␥2 WT group: *, p Յ 0. 05; **, p Յ 0. 01; ***, p Յ 0. 001.
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10.1074/jbc.m116.738591
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Acknowledgments-We acknowledge Dr. Grahame Hardie ( Cell Signaling and Immunology, College of Life Sciences, University of Dundee ) for suggesting that we generate knock-in mice harboring AMPK mutations associated with WPW syndrome, which helped us to understand the causality of WPW syndrome and led to unexpected findings in the kidney. Dr. Lan Yi assisted with cryosection and oil red O staining of kidneys. Dr. Zhu Chen provided suggestions on manuscript revision.
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10.1074/jbc.m116.738591
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Data analyses The field data formed a spatial point pattern. We performed exploratory analysis using the Ripley's reduced second moment function K(r) (Ripley, 1981). A test based on 1000 simulations of complete spatial randomness (CSR) was carried out in order to discriminate between random, aggregated and regular point patterns. A map of HTOF density was derived from the kernel smoothed intensity function of the observed point pattern (Diggle, 2003) and expressed in tree per ha. All computations were carried out using the R language (R Core Team, 2014) and the R package spatstat (Baddeley and Turner, 2005).
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10.18182/tjf.28744
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Agricultural and Biological Sciences
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Acknowledgments We would like to acknowledge support for our work from the Région Centre (project ADRIEN ) and the INRA meta-program SMaCH -Sustainable Management of Crop Health (project SESAME ). We are grateful to A. Roques , C. Robinet , J. Garcia and F. Goussard ( INRA Orléans ) for stimulating discussions.
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10.18182/tjf.28744
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In this paper, we assessed precisely the length of incubation period of MERS-CoV infections using two different datasets from Saudi Arabia and from South Korea and showed that the incubation period of MERS-CoV infections appeared to vary depending on the location of the outbreak.
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10.1038/srep35839
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Sources of data. For the outbreak in South Korea, we retrieved publicly available data from multiple sources, including the Korea Center for Disease Control and Prevention, the Korean Ministry of Health and Welfare, the World Health Organization, and local Korean news reports to compile a line list of all confirmed cases that had been reported by 27 July 2015. We used the most updated information from official reports that have been published by the Center for Disease Control and Prevention and the Ministry of Health and Welfare on a daily basis during the outbreak. The official reports included a brief description of each of all confirmed cases, including demographic characteristics (e. g. , age and sex), dates of exposure, onset of symptoms and outcome. The information on exposure was mostly recorded as intervals of 2 to 15 days during which transmission was thought to have occurred rather than exact dates of presumed transmission.
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10.1038/srep35839
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Acknowledgements This research was supported by the Harvard Center for Communicable Disease Dynamics from the National Institute of General Medical Sciences (grant no. U54 GM088558 ), and a commissioned grant from the Health and Medical Research Fund, Food and Health Bureau, Government of the Hong Kong Special Administrative Region. The funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish.
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10.1038/srep35839
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Mycobacterium abscessus multiplies similarly inside wild-type and cystic fibrosis conductance transmembrane regulator defective Mf We next evaluated the intracellular growth of both variants in murine and human Mf by infecting cells at an MOI of 1 for 3 h (see Experimental procedures). That M. abscessus S and R survive in murine (figure 2a ) and human (figure 2b ) Mf is in line with previous reports [26, 28]. However, it was not possible to directly compare the data obtained for the S and R variants because we systematically observed important differences in the mycobacterial uptake (up to a one log 10 difference for the R variant). Determination of bacterial doubling time in Mf was 14. 3 + 0. 8 h for the R variant and 19. 4 + 0. 6 h for the S variant (figure 2a ,b) compared with 6. 0 + 0.
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10.1098/rsob.160185
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Subsequently, cells were fixed with 2% paraformaldehyde (Delta microscopy) for 10 min, permeabilized with 0. 1% Triton X100/PBS for 5 min and blocked with 4% BSA (Euromedex, France), 2% goat serum (Sigma, USA) in PBS for 1 h. Cells were incubated with rabbit anti-LC3 (MBL, France) overnight in blocking buffer, washed and then incubated for 3 h with secondary antibody coupled to Alexa-568. Observation was done using a Zeiss LSM 510 Inv confocal microscope and images were processed with IMAGEJ software. A total of 100 bacteria were counted per time point.
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10.1098/rsob.160185
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Cells were washed directly before performing fluorescence imaging. Measurement of the ratio of 450 nm (blue)/ 535 nm (green) fluorescence allowed determining whether the bacteria established a phagosome-cytosol communication. Reading was performed by a fluorescence microscope with AutoPlay (Nikon) for the automatic acquisition of at least 50 cells per well and image analyses were performed using a dedicated algorithm using METAMORPH software [47]. The experiment was repeated twice with similar results.
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10.1098/rsob.160185
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Statistical analyses Fisher's exact test and the Student's t-test were used for all comparisons (GraphPad PRISM version 6. 0d; GraphPad Software, Inc). A p-value less than 0. 05 was considered significant (n. s. ¼ non-significant, *p , 0. 05; **p , 0. 01; ***p , 0. 001; ****p , 0. 0001).
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A. -L. R. , A. V. , A. B. , R. S. , A. B. , L. L. , T. D. , L. M. , M. R. and F. G. -M. carried out the microbiology, bacterial-cells interaction, confocal microscopy, electronic microscopy (A. V. with C. d. C. ). J. -L. H. , L. K. , C. d. C. , I. V. , R. B. , L. M. and J. -L. G. participated in the design of the study and drafted the manuscript; A. -L. R. , A. B. , R. S. , L. M. , I. V. , R. B. and J. -L. H. carried out the statistical analyses; J. -L. H. , L. K. , R. B. , C. d. C. and I. V. conceived the study, designed the study, coordinated the study and drafted the manuscript. All authors gave final approval for publication. European Community FP7 Marie Curie Career Integration Grant Europe (autophagtuberculosis 293416), University of Toulouse and Vaincre la Mucoviscidose. A. V. would like to thank Infectiopole Sud for financial support.
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10.1098/rsob.160185
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Acknowledgements. We greatly acknowledge Dr S. Canaan for critical reading of the manuscript and help in formatting the TIFF files of EM figures and V. Dubois for formatting the figures. We wish to acknowledge TRI-Genotoul Imaging facility (Toulouse, France ). The authors wish to thank Jean Paul Chauvin , head of this EM facility, for expert technical assistance with the electron microscopes and the digital camera.
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10.1098/rsob.160185
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Functional maps of BOLD signal were computed using Statistical Parametric Mapping (SPM8, Wellcome Trust Centre for Neuroimaging, University College London, UK) run in MATLAB R2012a (MathWorks, Natick, MA). For first-level single-subject analysis, functional images were preprocessed for slice timing and motion corrections, and smoothed with a 6×6×6 3D Gaussian kernel [25]. For second-level group analysis, anatomical and functional volumes were co-registered, spatially normalized into the Montreal Neurological Institute (MNI) template, and the preprocessed functional images were smoothed with an 8×8×8 3D Gaussian kernel. Voxel-wise analysis of BOLD signal consisted of modeling the activation and baseline conditions with a boxcar function convolved with the canonical hemodynamic response function using a general linear model [26] with motion parameters as covariates and applying a 128 Hz high-pass filter [27].
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10.1371/journal.pone.0152614
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Cortical Terminations of WM Fascicles Image datasets were co-registered (mutual information algorithm) using iPlan Stereotaxy 3. 0 software. The FA, color-coded direction, and BOLD-cluster maps were co-registered with T1-weighted MR images (anatomical reference). Accuracy of registration was carefully reviewed (visual analysis of merged images and test-retests) using the following landmarks: putamen, pallidum, corpus callosum (whole body and major and minor forceps), anterior and posterior limbs of the internal capsule, cerebellar contour, tentorium of the posterior fossa, sylvian region, upper brainstem contour, ventricular system (frontal horns and trigone), interhemispheric fissure and main cerebral gyrations. For each individual, we generated 3D surface renderings of the brain from T1-weighted MR images used to determine the cortical terminations of WM fascicles within the frontal, parietal, temporal and occipital lobes of the right and left hemispheres.
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10.1371/journal.pone.0152614
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Connections between WM Fascicles and BOLD Clusters Structure-function relationships were investigated by analyzing WM fascicle-BOLD cluster connections at individual level (each subject being its own anatomical reference) within cortical territories known as essential language areas in order to figure out which WM fascicles are related to language processing. A connection was determined when fibers went through a cluster, regardless of number of fibers involved (Fig 3 ). Since fMRI clusters were located within the gray matter and fascicles terminated in the subcortical WM, each cluster was enlarged by 4 mm in the depth direction using a "scaling object" tool (iPlan Stereotaxy 3. 0) to encompass the gray-white matter interface.
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10.1371/journal.pone.0152614
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Acknowledgments We thank Bruno Pereira for his help with statistical analysis and our talented MRI technologists for their engaged involvement in data acquisition.
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10.1371/journal.pone.0152614
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TRIPOD (Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis) methodology was used for the development, the Quality Assurance and the description of the experimental procedures. Conclusion: In an internationally approved quality assurance framework, ML seems promising in predicting the outcome of patients that would benefit or not of the PII. Once confirmed in larger and/or multi-centric databases, ML could support the physician in tailoring the treatment and in deciding if deliver or not the PII. www. impactjournals.
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10.18632/oncotarget.10749
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Research Paper of the PII, but all the patients receiving it are exposed to the potential risk of its acute and late toxicity. Moreover, a study by Crowley et al. supports the use, for selected cases of ACC, of smaller than standard radiation fields, avoiding PII, in order to reduce acute and late toxicity [6] , an attitude of particular interest in patients presenting an intrinsic higher risk of toxicity (e. g. HIV+ patients, elderly patients…) [7, 8]. Unfortunately, none of the available classical statistical techniques or predictive models allow the identification of patients presenting a higher risk of inguinal microscopic invasion (for example, higher than 5%). Predictive models based on the Machine Learning (ML) and Artificial Intelligence (AI) techniques are being more and more adopted in the medical and bio-molecular field, as these methods have many attractive theoretic properties, specifically, the ability of analysing very large datasets and to detect non predefined relations such as nonlinear effects and/or interactions [9, 10].
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10.18632/oncotarget.10749
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Importance of the considered features By applying the Information gain technique (see Methods section) to the considered dataset of patients, we found that 8 of the considered features carried a significant amount of information for the correct classification of the patients' outcome: PS, T and N stage, uTNM, Stage of the tumor, cTNM stage, tumor site, no symptoms or pain or tenesmus at diagnosis, histology and method used for the histologic definition, the presence of positive inguinal nodes, the administration of neoadjuvant CT, the treatment of an anal canal cancer relapsing after an initial surgery. In order to validate such results, we generate new predictive models using the same ML techniques, based only on the features selected by the Information gain technique. The mean accuracy was not worsened (data not shown).
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10.18632/oncotarget.10749
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Literature sources and search In August 2015 we searched MEDLINE, PEDro, OSTMED. DR, and the Cochrane Library databases and as well as Google Scholar, the Journal of American Osteopathy Association (JAOA) and the International Journal of Osteopathic Medicine (IJOM) websites. The search strategy was as follows: • for reliability studies, we started with the combination of keywords ["reliability" OR "agreement" OR "reproducibility"] AND ["cranial" OR "craniosacral" OR "cranium" OR "primary respiratory mechanism"]. When the number of references exceeded 100 hits with the above equation, we added ["osteopathy" OR "osteopathic"]. • for efficacy studies, we used the combination of keywords ["cranial manipulation" OR "osteopathy in the cranial field" OR "cranial osteopathy" OR "craniosacral technique"] AND ["medicine" OR "treatment" OR "therapy" OR "technique" OR "manipulation" OR "osteopathy" OR "osteopathic"].
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10.1371/journal.pone.0167823
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Biochemistry, Genetics and Molecular Biology
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Study selection For inclusion in our review, studies had to meet the aforementioned eligibility criteria. For study selection, we considered all techniques claimed by the authors to belong to the field of cranial osteopathy or mentioned in the classical osteopathic literature. If in doubt, we considered the technique to be inside the field. Studies that described the use of techniques or diagnostic/therapeutic strategies from cranial osteopathy together with other diagnostic/ therapeutic modes but without performing subgroup analysis were excluded. The systematic selection process was composed of 3 steps. Firstly we made a selection by title. Duplications due to overlap in the coverage of the databases and off-topic studies were excluded. Secondly, the abstracts of each study were analyzed.
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Data extraction The data extracted included: study design (including randomization and blinding procedures), sample size and characteristics (such as age and/or disease or inclusion criteria), main outcomes and results obtained. For reliability studies we added information regarding examiners (e. g. , number, qualification, expertise) as well as the statistical methods used. For efficacy studies, we added the primary outcome to be evaluated and a precise description of the treatments applied.
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10.1371/journal.pone.0167823
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Biochemistry, Genetics and Molecular Biology
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The second study rated as having low risk of bias, by Haller et al. [44] aimed at investigating craniosacral therapy (CST) compared to sham treatment in patients with chronic non-specific neck pain. The primary outcome was pain intensity assessed with visual analog scale and 16 secondary outcomes were investigated. Data (between CST and sham groups) were compared immediately and three months after the intervention. The results showed statistical and clinically relevant differences in favor of CST for the primary outcome and seven of the secondary outcomes immediately after treatment. At three months the results remained statistically and clinically relevant for the primary outcome and statistical differences still existed for five of the secondary outcomes. While this study is methodologically relatively strong, it nevertheless has some limitations.
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Acknowledgments We thank Dr. Alison Foote from the "Publication in English" service of Grenoble-Alpes University Hospital for critically editing the manuscript.
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10.1371/journal.pone.0167823
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| DISCUSSION This study addresses the prevalence of self-reported TBI in a population of female prisoners in a French prison. Secondary aims included studying covariables known to be associated with TBI. The main findings were that the rate of self-reported history of TBI was high, with an overall prevalence of 21%, and violence was the most frequent causes of TBI among women. The majority of reported TBIs were moderate or severe (57%). Furthermore, the rate of epilepsy was high (6%). Among women who reported a TBI, perceived health was worse e 30% of missing data for juvenile females and >23% for adult females. and alcohol and psychotropic drugs use was more frequent than among those who did not report a TBI. This study is the first to report on the prevalence of TBI among females prisoners in France.
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10.1002/brb3.535
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| Limitations Our study is cross-sectional and as data were collected by means of an interview (self-report), the results should be interpreted with caution. As, however, the reliability of prisoners' responses is frequently challenged, it is interesting that a recent study involving 200 Australian prisoners highlighted the fact that inmates' responses to questionnaires corresponded closely to the reality (Schofield et al. , 2011). These findings run counter to the conventional wisdom that responses by the criminal population are dishonest and therefore unusable. One could also argue a recall bias, which is an error caused by differences in the accuracy or completeness of the recollections retrieved by participants regarding past events or experiences. Even if cases and controls were not of the same mean age (35 for cases, 29 among controls), findings about differences can be considered sound enough to be discussed because it is difficult to believe that anyone would not remember having sustained a TBI.
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10.1002/brb3.535
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ACKNOWLEDGMENTS The authors wish to express their gratitude to Prof. H Williams for his contribution to the creation of the questionnaire. We would like to thank Dr R. Katz and Mr. James Moore for having scrutinized the manuscript and Mme G. Bard for collecting the data, collating references, and formatting the text. We would also like to thank the physicians and nurses from the Fleury-Mérogis medical team who participated in the administration of the questionnaire. We also thank the anonymous reviewers for their thoughtful comments on the first version of the manuscript, which enabled us to improve it. The results of this work have already been presented at the SOFMER conference (Montpellier 2015).
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10.1002/brb3.535
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FUNDING INFORMATION This study was funded by the "Fondation des Gueules Cassées " and has been supported by the "Centre Ressources du Traumatisme Crânien Ile de France " and Sorbonne Univ Paris 06 , UMR 7371 , UMR S 1146 , Laboratoire d'Imagerie Biomédicale, F-75005, Paris, France.
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10.1002/brb3.535
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Image acquisition was performed under the Zeiss ZEN 2012 software and image processing was carried under the open-source FIJI and ICY software.
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10.1364/boe.7.002362
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Biochemistry, Genetics and Molecular Biology
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Spectral imaging GL261 murine glioma cells expressing DsRed2 (plasmid: pDsRed2-N1, Clontech,Mountain View, USA) were cultured as previously described [11, 16] on glass-bottom Petri dishes. Five microliters of a QD655 solution (QTracker 655, Molecular Probes; 8µl in 50 μl of PBS qsp) were added and images were acquired successively under an 800nm NLO excitation and a 1050nm OPO excitation with the same laser power. Fluorescence signals were collected from 411 to 696nm on a descanned 32-channel spectral detector with a λ-space of 8. 9nm. Spectral deconvolution was performed under the Zeiss Zen 2012 software.
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10.1364/boe.7.002362
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Importantly, our results on the excitation of QD655 and EYFP outline that the 2P excitation spectrum cannot be simply predicted from the monophoton excitation spectrum of the fluorophores. Several similar examples for QD, proteins and organic dyes can be found in online spectra database (http://www. spectra. arizona. edu/). Finally, because commercial solutions already offer the possibility to deliver the fundamental pump wavelength simultaneously with the tuned OPO wavelength, at powers compatible with in depth imaging, we show that 5) multicolor imaging of fluorescent transgenic mice enter a new era. This should greatly facilitate the investigation of many questions related to the pathophysiology of various diseases and to assess the effects of pharmacological treatments directly in the living animal.
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10.1364/boe.7.002362
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Acknowledgments The authors would like to acknowledge Celine Caravagna for contributing to surgical procedures and Gwenaelle Tardif for genotyping and cell culture. This work was supported by grants from Fondation de la Recherche Médicale ( ING20140129149 ) and from Agence Nationale de la Recherche ( ANR15-CE16-0009-01 ) to FD, from Site de Recherche Intégré en Cancérologie ( INCA-DGOS-INSERM 6038 ) and ARSEP Fondation to GR as well as a CIFRE PhD fellowship by Carl Zeiss France to AJ.
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10.1364/boe.7.002362
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Immunohistochemistry For Six1 immunostaining, SOL and gastrocnemius plantaris (GP) muscles were embedded in cryomatrix and quickly frozen in isopentane cooled with liquid nitrogen. Cryostat sections (10 μm) were fixed in 4 % PFA and washed in 1× PBS. The sections were treated with Antigen Unmasking Solution (H-3300, Vector Laboratories) at 95 °C for 10 min and washed in 1× PBS for three times. Sections were treated with 1 % H 2 O 2 solution for 20 min. After three washes in 1× PBS, they were permeabilized with 0. 1 % Triton X-100 for 20 min and left for 1 h in blocking solution (1× PBS, 1. 5 % goat serum, 0. 1 % Triton X-100). Rabbit polyclonal antibodies directed against Six1 (HPA001893, Sigma) (1/100 dilution), and dystrophin (NCL-DYS2, Leica Biosystems) (1/50 dilution) were applied overnight at 4 °C to the treated sections.
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10.1186/s13395-016-0102-x
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SDH/GPDH staining Fresh-frozen sections were incubated in 0. 2 M phosphate buffer (pH 7. 6) containing sodium succinate and nitroblue tetrazolium, NBT (N6876, Sigma Aldrich) for 30 min at 37 °C. Sections were then washed with water and mounted in glycerine gelatin medium. GPDH staining was performed by incubation of unfrozen muscle sections with α-glycerol phosphate as described [37]. For quantification of SDH and GPDH staining, the color images were converted to thresholded images at hue (121-208) and brightness (0-140) by a threshold tool of ImageJ software. The area of thresholded images was measured by ImageJ and normalized by the whole soleus muscle area.
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10.1186/s13395-016-0102-x
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ChIP experiments GP and tibialis anterior (TA) muscles of 2 months old female mice were minced with scissors immediately after harvesting and fixed in 1 % formaldehyde for 10 min. Formaldehyde was quenched by addition of 0. 125 M glycine, and muscles were washed twice in PBS. Muscles were then incubated on ice in lysis buffer (10 mM Tris-HCl pH 7. 9, 85 mM KCl, 0. 5 % NP40, protease inhibitors (Complete, Roche)) for 10 min and homogenized with a mortar and subsequently with a dounce homogenizer. Nuclei were obtained by centrifugation, incubated in SDS lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1 % SDS, protease inhibitors) for 10 min, and sonicated in a bioruptor apparatus (Diagenode). The debris was removed by centrifugation. Sonicated DNA was incubated with 1 μg of Six1 antibodies (HPA001893, Sigma) under rotation at 4 °C overnight.
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10.1186/s13395-016-0102-x
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The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for quality controls. The analysis of some of these metrics and the study of the distribution of raw data show no outlier experiment. RMA normalization was performed using R and normalized data was subjected to statistical tests.
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Western blot Cell or tissue lysates of GP and SOL from cSix1KO and control mice (20-40 μg) were denatured in Laemmli buffer, separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Membranes were blocked in 50 mM Tris-HCl pH 7. 6, 137 mM NaCl and 0. 1 % (v/v) Tween-20 containing 10 % (w/v) skimmed milk or 5 % (w/v) BSA for 1 h at room temperature and incubated overnight at 4 °C with the indicated primary antibodies (Complex I, NADH dehydrogenase, ab14713, Abcam (Cambridge, UK); Complex II, succinate dehydrogenase, ab109865, Abcam (Cambridge, UK); cytochrome bc1 complex, ab110252, Abcam (Cambridge, UK); Complex IV, cytochrome C oxidase, ab14744, Abcam (Cambridge, UK); Complex V, ATP synthase, ab14748, Abcam (Cambridge, UK); hexokinase II, Sc-6521 (Santa Cruz Biotechnology); glycogen synthase 1, CST #3893, Cell Signaling Technology; AS160, 07-741, Millipore; GLUT4, kind donation from Geoffrey Holman, University of Bath); lamin B, sc-6216 (Santa Cruz Biotechnology); β-tubulin, 05-661 (Millipore); Six1, HPA001893 (Sigma).
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10.1186/s13395-016-0102-x
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Microarray data accession number Microarray data have been deposited in the Gene Expression Omnibus as accession no. GSE50023.
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Acknowledgements We thank V. Moncollin at ENS Lyon for help with the adult muscle ChIP experiments, the imaging facility at Institute Cochin for technical assistance, the sequencing and genomic platform at Institute Cochin for microarray experiments and F. Dumont and S. Jacques for advice. We Thank Dr. Daniel Metzger for the gift of the HSA-CRE ert2 mouse line. We thank Dr. Sophie Gautron and Dr. Stefano Schiaffino for critical reading of the manuscript and Dr Pascale Bossard for helpful discussions. I. S. is supported by ANR , The Uehara Memorial Foundation and JSPS Postdoctoral Fellowships for Research Abroad. Financial support was provided by the Institut National de la Santé et la Recherche Médicale (INSERM) , the "Association Française contre les Myopathies" (AFM) , the Centre National de la Recherche Scientifique (CNRS) , and the Agence Nationale pour la Recherche ( ANR RPV09108KKA ).
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Lymph node metastases Negative EUS-FNA of regional lymph nodes and negative staging laparotomy/ hand-assisted-laparoscopy with biopsy of regional lymph nodes Abbreviations: CA 19-9; carbohydrate antigen 19-9, EUS-FNA; Endoscopic ultrasonography-fine needle aspiration. doi:10. 1371/journal. pone. 0156127. t001 was extracted from the ELTR database containing all patients that were transplanted between 1990 and 2010 for hCCA. There were 249 patients from 57 European centers. Twenty-seven centers transplanted 2 patients. The list provided only basic variables, insufficient for indepth analyses. Therefore, all centers were contacted with a request to participate in the study. Centers were preferably addressed in their own language (English, Italian, French, Swedish, Dutch). Each center was asked to upload additional information regarding patient and tumor.
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10.1371/journal.pone.0156127
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Statistics Statistical analyses were carried out using IBM SPSS Statistics, (IBM, Armonk, New York, USA). The results are expressed as the means ±SD. Comparison of means was performed with the Student t-test for independent samples. Comparison of categorical variables was performed with the Chi-Square test and Fisher's exact probability test. Five-year survival rates were calculated using the Kaplan-Meier method and the differences between groups were calculated using the log rank test. Univariate analyses were conducted for patient survival by Kaplan-Meier estimates of survival probabilities and the log-rank test for comparisons. A Cox proportional hazard regression model was used to analyze associations with patient survival in multivariable analysis. P values were two-sided and values of less than 0.
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10.1371/journal.pone.0156127
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Results and Discussion Twenty-one centers uploaded data of 173 patients in the electronic database, resulting in a response rate of 69%. All patients were transplanted between 1990 and 2010. Twenty-six patients were excluded from the database, 12 because they were erroneously coded in the ELTR (the indication for transplantation was not hilar cholangiocarcinoma) and 14 because hCCA was incidentally found after liver transplantation. A study group of 147 patients remained. Eighty-two patients were transplanted in the first decade between 1990 and 2000 and 65 patients were transplanted between 2000 and 2010. The status of the distal bile duct margin was established in 137 patients and was tumor free (R0 resection) in 125 patients (91. 2%). Mean follow-up was 4. 1 years (± 5. 0).
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10.1371/journal.pone.0156127
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We determined the use of mTOR inhibitors in postoperative immunosuppressive regimens because of their potential anticancer effect. mTOR inhibitors were used in 11% of cases in group A versus 13% in group B (P = 0. 77). Data on the presence of PSC was available for 25 patients in group A: six patients (24%) had underlying PSC.
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10.1371/journal.pone.0156127
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Acknowledgments Professor René Adam is custodian of the European Liver Transplant Registry (ELTR). Dr. William F. Bennet is an ELTR Board member. We are much obliged to the following collaborators of this study: • Dr. Frank Lehner and Professor Jurgen Klempnauer from the department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany. • Professor Bo-Göran Ericzon from the department of Transplantation Surgery, Center for Surgical Sciences, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden. • Professor Roland Croner from the department of Surgery, University Hospital Erlangen, Erlangen, Germany. • Professor Johann Pratschke and Dr. Robert Sucher from the Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria • Dr.
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10.1371/journal.pone.0156127
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Acknowledgments Professor René Adam is custodian of the European Liver Transplant Registry (ELTR). Dr. William F. Bennet is an ELTR Board member. We are much obliged to the following collaborators of this study: • Dr. Frank Lehner and Professor Jurgen Klempnauer from the department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany. • Professor Bo-Göran Ericzon from the department of Transplantation Surgery, Center for Surgical Sciences, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden. • Professor Roland Croner from the department of Surgery, University Hospital Erlangen, Erlangen, Germany. • Professor Johann Pratschke and Dr. Robert Sucher from the Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria • Dr.
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10.1371/journal.pone.0156127
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Then, libraries were constructed and captured by using SureSelect Human All Exon v5 kit (Agilent), following the manufacturer's protocol. Paired-end (2 × 151 bases) sequencing was performed on a NextSeq500 device (Illumina). Obtained sequences were aligned and annotated with the human Hg19 genome based on the SureSelect Human All Exon v5 manifest by using Burrows-Wheeler Aligne (BWA) and Genome Analysis toolkit (GATK) algorithms. Only sequences with a read depth of 10 × and a mutation allele frequency superior to 5% were conserved for the analysis. The analysis is fostered on 137 clinically relevant genes related to biological pathways linked to cancer predisposition or available targeted therapies (Supplementary Table 1 ). Copy number variations were studied by using Control-FREEC software as described [5, 6].
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10.3748/wjg.v22.i48.10680
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It was interesting to see that among the 8 altered genes (Table 1 ), we observed a SMAD4 stop mutation, which is frequently found in CRC. An activating mutation of AKT1 was observed (Q79K). It could be targetable by protein kinase B (AKT)/mTor inhibitors. Surprisingly, we observed a constitutive Chek2 mutation (R117G), a gene involved in the homologous repair process. This mutation is cited in the public database for conferring a predisposition to cancer. Moreover, the analysis of copy number variation showed a loss of heterozygosity in chromosome 22 (from 29091114 to 29130709) (Figure 1A ). This analysis suggested a complete deletion of Check2 function in tumor cells. After discussion of the case at the molecular tumor board, the patient was proposed to receive off-label PARP inhibitor olaparib which previously showed efficiency in patients with Chek2 mutation in metastatic prostate cancer [7].
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10.3748/wjg.v22.i48.10680
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METHODS Electronic databases were systematically searched for original articles referring to the use of systemic mTOR inhibitors in vascular anomalies. PRISMA guidelines were followed for the systematic review.
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10.2340/00015555-2300
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Search strategy The search was performed by one author (MN) with the assistance of an information specialist (OL). Electronic databases MEDLINE via PubMed, CENTRAL, LILACS and EMBASE were searched on 12 November 2014, with no limitations on dates or language. To search for studies of mTOR inhibitors, the following keywords were used: "mTOR inhibitor", "sirolimus", "everolimus", "temsirolimus" and "deforolimus". To search for all vascular anomalies, the following keywords were used: "angioma", hemangioma", "Kasabach-Merritt", "hemangioendothelioma", "glomangioma", "vascular", "venous", "capillary", "lymphatic", "lymphedema", "lymphangioma" and "arteriovenous".
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Data extraction For each selected report, 2 authors (MN, AM) independently extracted information on the first author, publication year, journal, country/site, study design, characteristics of patients, type of vascular anomaly, number of lesions, associated complications, type of mTOR inhibitor, efficacy and side-effects, cointerventions and follow-up. Any disagreements were resolved by discussion. A data table was established for each patient. The extraction table was developed by 3 authors who are dermatologists familiar with vascular anomalies (MN, GL, AM). For missing data, the first author was contacted when possible.
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10.2340/00015555-2300
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Main results The aim of this systematic review was to assess the efficacy and safety of mTOR inhibitors for vascular anomalies in children and adults. Data were assessed for 84 patients; all children < 18 years. Sirolimus was the most frequent mTOR inhibitor used and was rapidly efficient in all cases, at a median of 2 weeks 95% CI (1-10 weeks). Sirolimus was well tolerated, the main side-effect being mouth sores, which led to treatment withdrawal in one case.
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10.2340/00015555-2300
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ACKNOWLEDGEMENTS The authors would like to thank Patricia Casanova and Olivier Lauze for their technical assistance. The authors declare no conflicts of interest.
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Tissue preparation for organ bath studies Bronchial rings were suspended horizontally on tissue hooks in an organ bath containing 5 ml of PSS (gassed with 95% O 2 , 5% CO 2 ) and maintained at 37 °C (pH 7. 40). Each preparation was connected to a force displacement transducer (it1, Emka Technologies, Paris, France). Isometric tension was measured and processed with a computerized system running IOX software (v2. 4. 2) and analyzed with Datanalyst (v2. 1. 0) software (EMKA technologies, Paris, France). Bronchi were suspended with an initial tension of 1. 5 g [15] and equilibrated for 60 min. During the equilibration period, the bath's PPS was changed after 10, 20 and 30 min. At the end of this period, bronchi were contracted with acetylcholine (3. 10 -3 M) to determine the maximal tone (Emax) developed during contraction in the absence of any treatment or pretreatment.
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10.1186/s12931-016-0464-y
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Drugs ACh, indomethacin, L-NAME, tetrodotoxin, Gd 3+ , nicardipine, capsazepine, and calphostin C were purchased from Sigma-Aldrich (St. Louis, MO, USA), tertiapin was bought from Bachem (Voisin le Bretonneux, France), 4 ), bronchial tone at rest immediately after the end of cyclic stretching; (5), post-stretching bronchial tone within 10 min following the cyclic stretching period; (6) maximal bronchial contraction in response to 3 mM ACh after cyclic stretching. In the control group, bronchial tone did not change significantly between points 4 and 6 (data not shown) (20) and SB-203580 was supplied by Calbiochem (San Diego, CA, USA). MK476 came from Merck Sharp & Chibret (Paris, France). Y27632 was purchased from Alexis Biochemicals (San Diego, CA, USA). All drugs (other than indomethacin, nicardipine and SB-203580) were dissolved in distilled water.
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The results were analyzed using Student's t test for paired and normally distributed data or using a Wilcoxon matched-pairs signed rank test if the data were not normally distributed. The standardized effect size |d| for the difference between the means was calculated in order to determine whether the observed effect of each pretreatment was small (|d| ≥ 0. 20), medium (|d| ≥ 0. 50) or large (|d| ≥ 0. 80), according to Cohen's conventions [20]. The 95% confidence interval (CI) for d was calculated as a measure of the uncertainty with regard to the true effect of each pretreatment. A P value < 0. 05 was considered to be statistically significant. Data analysis and statistical tests were performed using Statistica'99 software, version 5. 5, StatSoft, Tulsa, OK, USA).
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Availability of data and material The datasets supporting the conclusions of this article are available to the corresponding author.
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10.1186/s12931-016-0464-y
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Three-dimensional spheroid cell culture MiaPaCa-2 cells (5×10 3 /well) were seeded in 96-well plates containing Matrigel (Corning). The culture medium (containing appropriate concentration of Met or Mito-Met 10 ) was replaced every two days. At days 3, 7, and 14 the images were acquired using a Nikon Eclipse Ti inverted microscope (Nikon Inc. , NY). Spheroid-forming cells were counted using the Nikon NIS Elements imaging software.
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Biochemistry, Genetics and Molecular Biology
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Cytotoxicity assay To determine the cytotoxicity of Mito-Met analogs and other TPP + -conjugated compounds, we used the Sytox Green-based assay as described previously (16). MiaPaCa-2 cells were treated for 24 h, and dead cells were monitored in real time in the presence of 200 nM Sytox Green (Invitrogen) under an atmosphere of 5% CO 2 :95% air at 37°C. Data are represented as a percentage of dead cells after normalization to total cell number (measured with Sytox Green after a 3 h treatment with 0. 065% Triton X-100) for each group.
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Cell cycle analysis PANC1 cells (5×10 5 ) were seeded into 35 mm dishes and immediately treated with 10 mM Met or 10 μM Mito-Met 10 in complete media. At 48 h post-treatment, cells were fixed with 70% ethanol, permeabilized with 0. 25% Triton X-100 in PBS, and stained using a propidium iodide/RNase solution (20 μg/ml and 10 μg/ml, respectively). Data were collected on the BD-LSR II flow cytometer (BD Sciences) and analyzed using Flow-Jo (Flow-Jo LLC, Ashland, OR).
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10.1158/0008-5472.can-15-2534
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Over time, PDAC tumor bearing mice treated with Mito-Met 10 had lower tumor burden when assessed at early, middle, and later time points as visualized by bioluminescent imaging, with smaller tumors at the completion of the study (Fig. 6D ). Serum from these animals was collected, and hepatic and kidney toxicity tested using standard AST, ALT, AP, and BUN assays, respectively. As predicted from cell culture data (Fig. 3 ), neither Met nor Mito-Met 10 elicited toxicity in vivo (Supplemental Table 1 ). Following administration of Mito-Met 10 for two weeks in FC-1242luc orthotopic mice, we detected an increased accumulation of this compound in liver, kidney, spleen, and tumor tissues (Suppl. Fig. 11 ). Collectively, results from the in vivo experiments indicate a potent antitumor activity of Mito-Met 10 , with negligible off-target toxicity.
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Acknowledgments Financial support: This work was supported by grants from the NIH National Cancer Institute [ U01 CA178960 (to M. B. Dwinell and B. Kalyanaraman ) and R01 CA152810 (to B. Kalyanaraman)], the Medical College of Wisconsin Cancer Center (to M. B. Dwinell , B. Kalyanaraman and B. Johnson ) and Aix-Marseille Université CNRS (to M. Hardy and O. Ouari ).
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10.1158/0008-5472.can-15-2534
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Biochemistry, Genetics and Molecular Biology
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Infarct size measurement In regional IR experiments, at the end of reperfusion, the coronary artery was reoccluded. Evans blue dye (2%) was then injected through the cannulated aorta to delineate the area at risk (AR), which remained unstained by the Evans blue solution. The heart was removed and the left ventricle (LV) excised and frozen at -80°C for 15 min, and cut from apex to base into 2 mm-thick transverse slices (6 or 7 per heart). To delineate the IS, the slices were incubated at 37°C with buffered 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution for 10 min, and were then fixed in a buffered 10% formalin solution for 24 h before being photographed. AR and IS were quantified by a blinded observer using a computerized planimetric technique (ImageJ software, NIH, Bethesda, Maryland, USA).
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10.1371/journal.pone.0162632
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After washing, bound antibody was visualized by use of horseradish peroxidase-conjugated goat anti-rabbit or anti-sheep antibody (1:5000, Cell Signalling, Saint Quentin, Yvelines, France) for 1 hour at room temperature. Loading and quality transfer were evaluated by tubulin staining (1:1000, Santa cruz, La Jolla, CA, USA). Enhanced chemiluminescence was performed with the ECL Western blot detection kit (Amersham, GE Healthcare Europe GmbH, saclay, France) according to the manufacturer's instructions, and blots were exposed to BioRad Camera. Density of protein bands was computerized (ImageJ software, NIH, Bethesda, Maryland, USA). Data are normalized to tubulin (Sigma-Aldrich, Saint Quentin Fallavier, France) and expressed relative to sham group values.
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Biochemistry, Genetics and Molecular Biology
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Acknowledgments We wish to thank Dr C. Arnaud ( Laboratoire HP2, INSERM, Grenoble, France ), Pr C. Batandier ( Laboratoire de Bioénergetique Fondamentale Appliquée, Grenoble, France ) and Pr R. Clapier-Ventura ( Laboratoire de Signalisation et Physiopathologie Cardiovasculaire, UMR-S 1180, Paris, France ) for technical assistance and help and Dr Z. Benlasfar ( Institut Pasteur de Tunis, Tunisia ) for providing the viper venom. We are grateful to Pr H. Louzir , head of Pasteur Institute of Tunis , for support.
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10.1371/journal.pone.0162632
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The first objective was to track the labeled cells. To this end, a cryogenic probe (Cryoprobe TM , Bruker) was used for high resolution. The second objective was to evaluate the fistula healing by measuring the fistula orifice with the software, Paravision 5. 1, JIVE tool (Bruker), in the three orthogonal planes (Figure S1 ). This enabled the delimitation of the fistula orifice surface as a region of interest (ROI), for which the size was calculated (cm 2 ).
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10.7150/thno.14064
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The density of the microvascular network at the transplantation site and its periphery was also investigated by pCLE after the intravenous administration of FITC-dextran 70 kDa. Mosaicing TM software (Mauna Kea Technologies, France) was employed to enable a real-time display of the draft mosaic gathering the acquired and previous images. A dedicated module, Vessel Detection TM (Mauna Kea Technologies, France), was used to compute the functional capillary density (FCD) and diameter distribution. A diameter of interest (DOI) of 4 µm was chosen to avoid underestimation of small vessels that are the most important in tissue regeneration. The pCLE analysis was carried out at D0 (transplantation time point), D7, and D14.
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10.7150/thno.14064
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All slides were digitally scanned (Digitiser Hamamatsu Photo-nics®, Massy, France) and analyzed with dedicated software ((NDP. view software®, Massy, France).
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10.7150/thno.14064
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Statistics The results are presented as means ± standard deviation for continuous variables, and as percentages for categorical variables. GraphPadPrism (Graphpad Software, La Jolla, CA, USA) software was used for statistical analyses. Fischer's exact test was carried out for comparisons between categorical variables and the nonparametric Mann-Whitney test was used for nonpaired continuous variables. Comparisons between more than two groups were performed with the nonparametric Kruskal-Wallis test. Dunn's multiple comparison test difference in rank sum was used for pairwise comparison. A p-value of less than 0. 05 was considered statistically significant.
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10.7150/thno.14064
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Acknowledgments This work was supported by ENCITE , co-funded by the European Commission under the 7 th Framework Programme. We sincerely thank Sophie Camilleri-Broet ( Department of Pathology, Hôpital Européen Georges Pompidou, Paris, France ) for her assistance in the field of histology; Josette Legagneux ( Microsurgery Training and Research Lab, School of Surgery, Paris, France ) for her great advice in the field of microsurgery, and Nicolas Guegan ( Institut IMAGINE-INSERM 1163, Paris, France ) for his technical assistance.
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10.7150/thno.14064
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Literature search We performed a systematic search on PubMed for articles on breast screening trials. We also searched reference lists in major reviews [17] [18] [19] and in publications on breast screening trials we already had. We retrieved all articles reporting original data of these trials. Three of us (PA, MB, MS) read the articles with looking for cancer-specific fatality data reported by categories of size or lymph-node status or stage. For the sake of finding comparable data on screening for other cancers, we performed a similar literature search for colorectal cancer. Fatality could be reported as a proportion of cancer patients who died because of the cancer or as a survival statistics, which is the reverse of fatality. It could be reported as a measure of the risk (e. g. , the hazard rate) to die from a large (more advanced) cancer compared to the risk to die from a small (less advanced) cancer.
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10.1371/journal.pone.0154113
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Search Strategy We will search the Cochrane Database of Systematic reviews to identify all systematic reviews that included at least 1 RCT involving adult kidney transplant recipients. We will retrieve the full text article of all RCTs included in the systematic review, and also search for all additional articles published from the same trial using key word searches in electronic databases (MEDLINE, Embase) and trial registries. No date or language restrictions will be applied.
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10.1097/txd.0000000000000593
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Types of Studies For feasibility, the Cochrane Database of Systematic reviews will be used as a sampling frame to identify RCTs in kidney transplantation, as has been done in previous analysis of outcomes reporting in RCTs. 22, 50 Based on a preliminary search, we expect that more than 300 RCTs will be included.
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10.1097/txd.0000000000000593
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Data Extraction One reviewer (B. S. ) will extract characteristics of all the trials including: first author, date of publication, country/ies in which the trial was conducted, sample size, participant characteristics (range and mean age, time since transplant, sex), trial duration, name and type of intervention (eg, surgical, pharmacological, psychosocial, lifestyle), and all outcomes as reported in the trial (including definitions, tools for measurement, thresholds, time points or time frames for measurement, change in level or percentage, scores). Two reviewers (N. E. , A. T. ) will cross check the data extraction.
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10.1097/txd.0000000000000593
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Data Analysis and Presentation The data will be entered into Microsoft Excel to assist with data management, tabulation, and analysis. Author B. S. will group all similar outcomes into appropriate outcome domains, which will be reviewed and discussed by the SONG-Tx Steering Group (J. R. C. [Chair], K. B. , J. G. , M. A. J. , L. M. , T. P. P. , D. R. , L. R. , A. W. , G. W. ] and SONG Executive Committee (J. C. C. , S. C. , J. G. , T. H. , B. H. , B. M. , P. T. , W. V. B. , D. C. W. , W. C. W. , A. T. ). The outcome domains will be broadly classified as surrogate, clinical, or patient-reported outcomes. We will identify the number of trials that reported each outcome domain. For each outcome domain, we will assess the number of different outcomes (including measures) and the number of trials that assessed each specific outcome and perform statistical analyses using the software package R (version 3.
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10.1097/txd.0000000000000593
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Purposive sampling to obtain a maximum variation in the demographics, clinical characteristics (patients), and professional experiences and roles (health professionals), and snowballing strategies (where participants can nominate or extend an invitation to other relevant stakeholder members to participate) will be used. Patients/family members will be recruited through participating hospital/university institutions of the SONG Executive, SONG-Tx Steering Group and investigators, patient/consumer organisations, and the SONG Initiative database. Health professionals will be recruited via the collegial networks of the investigators and professional transplantation and nephrology societies.
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10.1097/txd.0000000000000593
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ACKNOWLEDGMENT The SONG-Tx initiative is endorsed by The Transplantation Society. The authors also want to acknowledge the following organizations for their partnerships and support: Cochrane Kidney and Transplant, Kidney Disease Improving Global Outcomes (KDIGO), PKD International, World Transplant Games Federation, Australasian Kidney Trials Network (AKTN) , Christchurch Kidney Society, Kidney Health Australia , Transplant Australia , The Kidney Foundation of Canada , British Kidney Patient Association , European Kidney Transplant Association (EKITA) of the European Society of Transplantation, European Renal Best Practice (ERBP) , and the UK National Kidney Federation. All SONG Collaborators are listed here: songinitiative. org/who-we-are/partnersand-supporters/.
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10.1097/txd.0000000000000593
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CMV QNAT was performed once a week for the first 3 mo, once a month between months 3 and 6, and then every 2 months up to 1 year and if CMV disease was suspected clinically. During the virological monitoring of CMV disease, the assay was performed once a week until two consecutive negative CMV DNAemia QNATs occurred. Antiviral drug resistance was suspected when persistent viral replication was observed after >2 weeks of appropriate antiviral therapy and was confirmed by fulllength sequencing of the UL97 and UL54 genes (25) , performed at the French National Cytomegalovirus Reference Center (Limoges, France). Sequences were compared with the AD169 reference sequence using the Gene Librarian 3. 2 software (Visible Genetics Inc. , Siemens, France) (26, 27).
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10.1111/ajt.13781
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Flow cytometry analysis and monitoring of Vd2 neg cd T cells Blood samples were analyzed at the Bordeaux University Hospital immunology laboratory by flow cytometry using a FC500 flow cytometer from Beckman Coulter. All blood samples were withdrawn on EDTA anticoagulant in Vacutainer 5-mL tubes (BD Biosciences, Mountain View, CA) and kept at room temperature until processed. Following the manufacturer's recommendations, labeling was carried out on whole blood and red blood cells lysed at room temperature with a Versalyse (Beckman Coulter France, Villepinte, France) lysing solution added to Iotest fixative solution (Beckman Coulter, Macon, France). Events were acquired with the dedicated CXP-1 software. Vd2 neg cd T cells were detected with anti-Vd2 and anti-PAN-d, purchased from Beckman Coulter France.
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10.1111/ajt.13781
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Acknowledgments We thank Catherine Rio as a nurse coordinator. We also acknowledge the technicians from the Laboratories of Virology and Immunology in Bordeaux University Hospital and the Centre National de R ef erence des Cytom egalovirus (Limoges) for their significant contribution to this study.
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The GWAS meta-analysis results included three associations in novel loci (rs17603856 (6p23), rs1887428 (9p24) and rs669763 (16q13)) with genome-wide levels of significance (P < 5 × 10 -8 ; Fig. 1b ). In addition, the major histocompatibility complex (MHC) and, to a lesser extent, the IRF5 locus on chromosome 7 showed significant transancestral heterogeneity (Fig. 1b ). We then carried out a two-stage replication study incorporating rs17603856, rs1887428 and rs669763. We scanned the 1KG imputed data for association at loci independent of those previously published and excluding the MHC. We successfully genotyped a total of 66 SNPs at 56 loci (SNP selection is described in the Online Methods) in an additional 3,043 cases and 5,074 controls of Chinese ancestry recruited from Anhui Province. Eighteen of these SNPs (at 17 independent loci) showed association in this replication study, passing a false discovery rate (FDR) of 0.
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10.1038/ng.3603
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01. These included rs17603856 and rs1887428 but not rs669763, which failed quality control. We then genotyped these 18 SNPs in a European replication cohort comprising 1,478 cases and 6,925 controls3. Data from an additional European-American GWAS (1,165 independent cases and 2,107 controls) were also included in this final analysis15 (Supplementary Table 2a ). Of the 18 candidate SNPs, 11 showed a standard genome-wide level of significance (P < 5 × 10 -8 ) in the combined meta-analysis (11, 381 cases and 24,463 controls) of all three main GWASs and the three replication studies (Table 1 , Supplementary Fig. 1 ). The strongest association signal after this meta-analysis was that for rs1887428 (9p24; P = 2. 19 × 10 -17 ). Other statistically significant associations were found at rs34889541 (1q31.
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We then carried out replication using a second European GWAS15 independent of our main European GWAS and de novo genotyping in a new data cohort of European ancestry.
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Statistical analysis Association testing-After imputation, we analyzed each GWAS data set for association (SNPTEST36), fitting an additive model. We used the inverse variance method for metaanalysis, combining data from the three studies for SNPs with an imputation INFO score of >0. 7 in all three studies. Testing for heterogeneity. We tested for heterogeneity between the association signals in the Chinese and European data using Cochran's Q statistic (1 degree of freedom in this case). The P values on the -log 10 scale are plotted in Figure 1b. Q-Q plots (one per chromosome) for the heterogeneity P values can be seen in Supplementary Figure 9a , and Bland-Altman plots for differences in genetic effect (log odds ratio) estimates are in Supplementary Figure 9b. Assessment of shared association between ancestries-To assess the extent to which genetic association with SLE was shared between the Chinese and European populations, we compared association results in the European GWAS3 with a meta-analysis of both Chinese GWASs, for SNPs published as associated in European3 and/or Chinese studies4,6-9.
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Although this did not identify any additional shared loci (Supplementary Table 1b ), there was suggestive evidence for two loci (P < 0. 05 after multiple testing adjustment within loci but not after adjusting across loci). Consistency of association between ancestries-We tested the hypothesis that the genome-wide association signals were consistent between the two populations. Post-1KG imputed association data were used for SNPs with INFO > 0. 7. These genome-wide association signals were separated into 1-Mb regions (moving 1-Mb windows across the genome, 2,698 in total). We removed the extended MHC with a conservative buffer zone (chr. 6, from 20 Mb to 40 Mb), leaving 2,678 regions. We also removed regions that had an excessively (more than 2 s. d. from the average) low (N < 1,000) or high (N > 3,000) density of SNPs.
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This removed only 10% of the regions, leaving 2,338 regions. The lowest P value within each window was taken as the strength of association for that particular window. Each P value within each region was adjusted for multiple testing using a Bonferroni adjustment, to avoid bias in ranking agreement owing to the lowest P value being correlated with the number of statistical tests. The 1-Mb regions within each population's data were then ranked according to the P value (lowest P value having rank 1). We tested agreement in ranking using Kendall's τ statistic. Supplementary Figure 7c shows heat maps of the ranks for all 2,338 regions, the top 250 regions and the top 50 regions. The order in the heat maps was determined by the sum of the ranks. For comparison, we also included a simulated ranked data set; we permuted the numbers 1-2,338 in two separate data sets and produced a heat map ordered by the sum of the ranks.
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Testing for independent effects within loci-We tested for independent effects of the two SNPs (rs17321999 and rs7579944) within the 2p23. 1 locus by fitting a multiple regression model with both SNPs as explanatory variables (results for each SNP in this analysis are conditional on the other SNP as a covariate). We checked LD between the two SNPs in all data sets. We combined the conditional results in meta-analysis in the same way as in the single-marker analysis. Selection of SNPs for replication study-We used a number of criteria to select SNPs for replication in the Chinese samples. We chose only SNPs that were not within a 1-Mb window of loci that had previously been published as associated with SLE. We selected SNPs that had P value significance levels at meta-analysis of <10 -4. Three SNPs in loci not previously reported as associated with SLE had a genome-wide level of significance (P < 5 × 10 -8 ) after meta-analysis.
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SNPs spanning a 1-Mb window were considered as one region, and we selected only independent SNPs within this region, using LD as a measure of independence. We carried out a gene-based test on the meta-analyzed data, using only SNPs with INFO scores > 0. 9, with the software KGG39-41. One SNP from each of the loci that passed a gene-based test at the level of P < 10 -5 was chosen; some of these had already been selected as having P < 10 -4 in the meta-analysis as single markers. In total, 105 SNPs were selected for replication in the Chinese replication cohort. Of these, 66 passed QC, and 18 SNPs with FDR < 1% were taken forward to the European replication.
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Genotyping of replication data Genotyping of 130 SNPs was carried out for the 3,614 cases and 5,924 controls forming the Chinese replication set, using the Sequenom platform. This set of 130 SNPs included 105 SNPs in loci not previously reported as associated with SLE and 25 SNPs in loci that had previously been published as associated with SLE. The 105 potential new SLE SNPs included, in some cases, multiple SNPs in the same loci where we had some evidence of independence. We carried out several QC steps: we removed SNPs with >10% missing data (25 SNPs), and then subjects with >5% missing data. Two SNPs were monomorphic. Of the remaining 103 SNPs, 77 were in regions of the genome with potential new SLE associations. We removed 13 SNPs after we checked the genotyping allele intensity plots closely for clustering quality and tested for Hardy-Weinberg equilibrium (HWE).
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SNPs were removed if HWE P < 1. 00 × 10 -4. After QC, the Chinese replication consisted of 3,043 cases and 5,074 controls with genotyping on 64 SNPs. The European replication data comprised 1,478 cases and 6,925 controls genotyped for 18 SNPs with an FDR of 1% in the Chinese replication study. The cases were of European ancestry and were a subset of those used in the replication study in the European GWAS3; in the current study we carried out new genotyping on these 18 SNPs, and the controls were the same as used in that study (these samples were checked for European ancestry using a principal component analysis spiked with HapMap samples; see the original paper3). One of the 18 SNPs typed in the European replication cohort for this study (rs2297550) failed genotyping, and the remaining 17 SNPs passed QC (<3% missing data, HWE P > 1.
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Gene expression data Gene expression data came from two sources. We obtained data from Fairfax et al. 17 and unpublished data from B. Fairfax and J. Knight for NK cells, naive monocytes, monocytes stimulated by lipopolysaccharide (harvested after 2 h and 24 h), monocytes stimulated by interferon, and B cells. We obtained CD4 (CD4 + T cells) and CD14 (CD14/16 + monocytes) data from a previous study of gene expression in immune-related cells16. We made an adjustment for multiple testing using FDR = 0. 01. To test whether observed associations between SNPs and expression levels of cis-acting genes were due to chance, we calculated the RTC score18.
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