An important use of data obtained from microarray measurements is the classification of tumor types with respect to genes that are either up or down regulated in specific cancer types. A number of algorithms have been proposed to obtain such classifications. These algorithms usually require parameter optimization to obtain accurate results depending on the type of data. Additionally, it is highly critical to find an optimal set of markers among those up or down regulated genes that can be clinically utilized to build assays for the diagnosis or to follow progression of specific cancer types. In this paper, we employ a mixed integer programming based classification algorithm named hyper-box enclosure method (HBE) for the classification of some cancer types with a minimal set of predictor genes. This optimization based method which is a user friendly and efficient classifier may allow the clinicians to diagnose and follow progression of certain cancer types.We apply HBE algorithm to some well known data sets such as leukemia, prostate cancer, diffuse large B-cell lymphoma (DLBCL), small round blue cell tumors (SRBCT) to find some predictor genes that can be utilized for diagnosis and prognosis in a robust manner with a high accuracy. Our approach does not require any modification or parameter optimization for each data set. Additionally, information gain attribute evaluator, relief attribute evaluator and correlation-based feature selection methods are employed for the gene selection. The results are compared with those from other studies and biological roles of selected genes in corresponding cancer type are described.The performance of our algorithm overall was better than the other algorithms reported in the literature and classifiers found in WEKA data-mining package. Since it does not require a parameter optimization and it performs consistently very high prediction rate on different type of data sets, HBE method is an effective and consistent tool for cancer type prediction with a small number of gene markers. Microarray technology provides wealth of information on expression levels of thousand genes that has been used for diagnostic and prognostic purposes for various types of diseases. The data obtained from microarray measurements leads to understanding of genes that are being regulated under the disease conditions including cancer both in biology and clinical medicine at the molecular level An important characteristics of microarray data is the large number of genes relative to number of samples. This high dimensionality in gene space increases the computational complexity while it usually decreases the accuracy of the classification. This fact brings the necessity of gene selection by ranking or gene reduction for the high dimensional gene space. The relevance of genes in cancer occurrence can be categorized into three classes: Strongly relevant, weakly relevant and irrelevant genes The effectiveness of the selected gene subset is measured by its prediction accuracy or error rate in classification. IN microarray experiments, classification of data is a crucial step for the prediction of phenotype of cells. Different machine learning approaches have been employed to analyze microarray data including k-nearest-neighbors In this work, we show that a systematic and efficient algorithm, mixed integer linear programming based hyper-box enclosure (HBE) approach, can be applied to classification of different cancer types efficiently. We first introduce and establish a consistent classification method for different types of microarray data. Second we provide an optimal set of genes as best diagnostic indicators for different cancer types that gives the highest accuracy in classification. Information gain attribute evaluator, relief attribute evaluator and correlation-based feature selection (CFS) methods are used for gene selection. We conduct experiments using 6 well known cancer data sets including leukemia data set http://datam.i2r.a-star.edu.sg/datasets/krbd/) and Artificial Intelligence Laboratory (http://www.ailab.si/supp/bi-cancer/projections/index.htm) databases  with 12,600 genes. Considering the results given in The selected genes by HBE are serine protease hepsin (X07732), nel-related protein 2 (D83018), ao89h09.x1 (AI207842), Cdk-inhibitor p57KIP2 (U22398), DKFZp564I1663-r1 (AL036744), adipsin/complement factor D (M84526), glutathione transferase 4 (GSTM4) (M96233), DKFZp586K1220 (AL050152), aldose reductase (X15414), ADP/ATP translocase (J03592). In fact, many of the genes that have been selected in this study shown to display different expression patterns in prostate cancer tissues. For example hepsin (a cell surface serine protease) is significantly upregulated in human prostate cancer and it promotes cancer progression and metastasis of prostate This data contains 8 patients having relapsed and 13 patients having non-relapse with measurements of 12600 genes.The genes, selected with HBE are shown to play a critical role in prostate cancers, are human cofactor A protein (AF038952), farnesyl-protein transferase beta-subunit (HUMFPTB), glutamine-fructose-6-phosphate amidotransferase (GFAT) (M90516). The function of heterodimeric enzyme farnesyl: protein transferase (FPTase) 3 is to transfer of a 15-carbon isoprenoid moiety to a C-terminal cysteine of many cellular proteins. The inhibition of farnesyl protein transferase affects for the prevention of proper functioning of the Ras protein that leads to oncogenesis or cancer. In fact, elimination of cancer were reported to increase when prostate cancer cell line is treated with farnesyl-protein transferase inhibitor et al. This data set contains 58 samples from DLBCL patients and 19 samples from follicular lymphoma. The gene expression profiles were analyzed using Affymetrix human 6800 oligonucleotide arrays. HBE method is the most accurate classifier among the classifiers with both 10-CV and LOOCV in the DLBCL data set . From thHBE analysis resulted in high classification accuracy with 6 genes which are DNA replication licensing factor CDC47 homolog (D55716-at), gamma-interferon-inducible protein IP-30 precursor (J03909-at), LDHA lactate dehydrogenase A (X02152-at), CD69 antigen (Z30426-at), SLC (AB002409-at), Rad2 (HG4074-HT4344-at). When the function of those proteins and their role in DLBCL were analyzed, we see that these proteins play a significant role in the progression of DLBCL. DNA replication licensing factor CDC47 is a factor that helps the DNA to undergo a single round of replication per cell cycle. CDC47 is not only necessary for DNA replication and cell proliferation, but also for S-phase checkpoint activation upon UV-induced damage The lymphoma data set contains 47 samples, 24 of them are referred to as germinal center B-like group and 23 are activated B-like group. This data set contains expression of 4026 genes.In the lymphoma data set, HBE method is the most accurate classifier using both ten-fold-cross-validation and leave-one-out cross validation with the accuracies of 96.40% and 97.87% respectively . Supportalgorithm selects the following genes: Deoxycytidylate deaminase (19408), lymphoid-restricted membrane protein (JAW1) (16886), PKU-beta = KIAA0137 = protein kinase (20423), T-cell protein-tyrosine phosphatase (17140), TTG-2 Rhombotin-2 (19238), stress-activated protein kinase (JNK3) (19384), and unknown labeled genes with ids 19288, 19274, 13394. Expression of the following genes are currently being used as markers for lymphoma in clinical diagnosis. Deoxycytidylate deaminase (dCMPase) hydrolyzes dCMP into dUMP, and it is suggested that this gene is a marker of the aggression of human lymphoid malignancies There are four different small round blue cell tumors in this data set: Ewing family tumor (EWS), Burkitt lymphoma (BL), neuroblastoma (NB) and rhabdomysarcoma (RMS). The training set contains 63 samples and the test set contains 20 samples. The cDNA microarrays comprise 2308 genes. Small round blue cell tumors (SRBCT) of childhood are diagnosed using single layer neural network et al. et al. et al. et al. The selected genes with their gene ids in SRBCT classification are Fc fragment of IgG, receptor, transporter, alpha (FCGRT) (70394), transmembrane protein (812105), fibroblast growth factor receptor 4 (784224), ESTs (295985), recoverin (383188). FCGRT encodes a receptor binding the Fc region of monomeric immunoglobulin G. This protein both helps to transfer of immunoglobulin G antibodies from mother to fetus across the placenta, and binds to immunoglobulin G to prevent the antibody degradation The contributions of this work are two-fold. The first contribution is that we implement an effective optimization based classifier that gives very high performance and valuable insight into different type of cancer data sets. Previously it has been shown that our approach was successfully applied to protein folding and drug classification problems. HBE approach does not require parameters to optimize in order to obtain high classification accuracies. This method can be used for different types of data without any modifications. The second contribution is finding of optimal predictor genes that give the highest accuracy in classification. This effort can provide to develop antibody assays for the diagnosis of specific types of cancer and to provide accurate diagnostics by only measuring expression of few genes. We have applied our algorithm on publicly available data sets including leukemia data set, two prostate cancer data sets, two lymphoma data sets and SRBCT data set. In conclusion, mixed-integer programming based hyper-box enclosure approach is robust and effective method for microarray analysis.We performed scaling to all data sets by using the expression ofet al. There are generally three types of approaches in feature (gene) selection: Filters, wrappers, and feature weighting. Filter methods eliminate irrelevant features according to some prior knowledge. Wrapper approaches use machine learning algorithms to evaluate the feature subsets; however, they have high computational complexity when they combined with classification algorithms. Feature weighting methods simply weigh features instead of selecting a subset of features that is a combinatorial problem. We employed information gain attribute evaluator, relief attribute evaluator, and correlation-based feature selection (CFS) from Weka machine learning package Information gain evaluates a feature (gene) by measuring the information gain with respect to the class:Relief attribute evaluator is an evaluating algorithm that rates features (genes in our case) due to these facts: (1) how well their values distinguish among samples of different classes (tumor type in our case) (2) how well they cluster instances of the same class Correlation-based feature selection (CFS) is a fast algorithm that reveals a good feature subset that contains features highly correlated with the class, yet uncorrelated with each other The objective in data classification is to assign samples that are described by several attributes into a predefined number of classes. In this study, we propose HBE algorithm for the classification of cancer types. This algorithm consists of integer programming (IP) and mixed integer linear programming (MILP) based components, and the data points belonging to different classes are discriminated by hyper-boxes. The use of hyper-boxes for defining boundaries of the sets that include all or some of the samples in that set can be very accurate on both two-class  and multi-class (more than two tumor types) problems. If it is necessary, more than one hyper-box could be used in order to represent a class. When two classes are both represented with a single hyper-box respectively, the boundaries of these hyper-boxes may overlap. Thus, two boxes could be constructed in order to eliminate this overlapping. The description of the optimization model is given in This step is used to improve the computational efficiency by determining representative seeds for each class . Seed fiAs it is proven in Uney-Yuksektepe Furthermore, different classification models will always develop different models  for different data sets. In classification problems, benchmark data sets are used in order to compare the results of different methods. As the same benchmark data sets for each tumor problem are used to compare distinct models, the comparisons are unbiased and stable for this study. For instance, the most popular data classification method Support Vector Machines (SVM) will produce different hyper-planes for perturbations in the original data set. Hence, differences between the constructed hyper-boxes of different data sets are not an interesting and problematic issue for data classification.The objective of the IP-Seed problem given in Equation (6) is to minimize the distances from each seed to sample of its group (in-class distances) and maximize the average distances from each seed to the samples that belong to other classes (out-class distances). Equation (7) states that every class must have exactly one seed, and integrality of the decision variable Training part studies are performed on a training data set composed of a number of samples Minimization of the number of misclassified samples in the data set with the minimum number of hyper-boxes is the objective of the mixed integer linear programming model. The objective function isThe lower and upper bounds of the hyper-boxes are determined by the samples that are enclosed within the hyper-boxes. Hence, lower and upper bounds of hyper-boxes are calculated by related constraints. Moreover, the bounds of hyper-boxes exist if and only if this hyper-box is assigned to a class. There exist constraints that ensure the assignment of each data point to a single box and single class Since the MILP model is solved for ‘problematic samples’ only, the ‘non-problematic samples’ are assigned to hyper-boxes in a straight forward manner . We defier-boxes .After the initial gene ranking, the optimal gene set is searched by using Supporting Information S1MILP formulation of the hyper-box enclosure approach.(0.05 MB PDF)Click here for additional data file.Supporting Information S2Source codes, scripts and data files.(0.78 MB ZIP)Click here for additional data file.


Vitiligo is characterized by the death of melanocytes in the skin. This is associated with the presence of T cell infiltrates in the lesional borders. However, at present, there is no detailed and systematic characterization on whether additional cellular or molecular changes are present inside vitiligo lesions. Further, it is unknown if the normal appearing non-lesional skin of vitiligo patients is in fact normal. The purpose of this study is to systematically characterize the molecular and cellular characteristics of the lesional and non-lesional skin of vitiligo patients.Paired lesional and non-lesional skin biopsies from twenty-three vitiligo patients and normal skin biopsies from sixteen healthy volunteers were obtained with informed consent. The following aspects were analyzed: (1) transcriptome changes present in vitiligo skin using DNA microarrays and qRT-PCR; (2) abnormal cellular infiltrates in vitiligo skin explant cultures using flow cytometry; and (3) distribution of the abnormal cellular infiltrates in vitiligo skin using immunofluorescence microscopy.Compared with normal skin, vitiligo lesional skin contained 17 genes (mostly melanocyte-specific genes) whose expression was decreased or absent. In contrast, the relative expression of 13 genes was up-regulated. The up-regulated genes point to aberrant activity of the innate immune system, especially natural killer cells in vitiligo. Strikingly, the markers of heightened innate immune responses were also found to be up-regulated in the non-lesional skin of vitiligo patients.As the first systematic transcriptome characterization of the skin in vitiligo patients, this study revealed previously unknown molecular markers that strongly suggest aberrant innate immune activation in the microenvironment of vitiligo skin. Since these changes involve both lesional and non-lesional skin, our results suggest that therapies targeting the entire skin surface may improve treatment outcomes. Finally, this study revealed novel mediators that may facilitate future development of vitiligo therapies. Affecting 0.5%–1% of population worldwide, vitiligo is an acquired pigmentation disorder in which melanocytes are destroyed, resulting in development of porcelain-white patches of skin et alThe pathogenesis of vitiligo is largely unclear. There is evidence that vitiligo is an autoimmune disease Previous vitiligo transcriptome analyses have focused on cultured melanocytes that were isolated from patients with vitiligo, demonstrating possible molecular abnormalities in vitiligo-patient's melanocytes In this study, we performed a transcriptome analysis comparing gene expression profiles of three types of skin biopsies: (1) vitiligo lesional skin (LS); (2) normal appearing non-lesional skin (NLS) of vitiligo patients; and (3) normal skin of healthy volunteers (NS). This was followed by explant skin cultures and immunofluorescence analyses of the cellular infiltrates present in these skin biopsies. The results showed for the first time markers and cells of activated innate immune response not only in the lesional areas, but also in the normal appearing non-lesional areas of vitiligo patients' skin. This conclusion suggests that future therapeutic development needs to consider the role of innate immune activation in the affected as well as the unaffected areas of the skin in vitiligo patients. Therefore, treating the entire skin surface of vitiligo patients may have higher chances of achieving optimal therapeutic results than targeting individual lesions. Further, targeting innate immune activation may represent a promising approach for developing vitiligo therapies in the future.andTwenty-three subjects with vitiligo vulgaris and 16 healthy volunteers were recruited for this study and2. The The study was approved by the Ethical Review Board of the University of British Columbia (Certificate Number C98-0493) in accordance with the contents of the Declaration of Helsinki, and by the Institutional Review Board of the University of British Columbia. Collection of skin biopsies was undertaken after the subjects have signed the informed consent.Skin samples were trimmed to remove visible adipose tissue and homogenized in Trizol  using the tissue homogenizer . RNeasy Fibrous Tissue Mini Kit  was used to extract total cellular RNA according to the manufacturer's protocol. The quality of the RNA was assessed by the Agilent Bioanalyzer 2100 ; and the concentration of the RNA samples was determined by the Nanodrop 1000 spectrophotometer .in vitro transcription in the presence of fluorescent-labeled CTP using the Low RNA Input Linear Amplification Kit, PLUS, Two-Color from Agilent following the manufacturer's instructions. Each microarray was hybridized with 825 ng of amplified cDNA labelled with Cy5  or Cy3  at a specific activity between 8 and 15 pmol/µg. Hybridizations were performed on Whole Human Genome Oligo microarrays , comprising 41,059 60-nt oligonucleotide probes, mostly represented as single spots. Image scanning was performed with the Agilent DNA Microarray Scanner and quantified using Agilent's Feature Extraction software. The results were imported and analyzed with the GeneSpring GX 7.3 software  for statistical computing and visualization. Data normalization was performed within and across the arrays using per gene, per chip normalization according to the Agilent recommendation. To detect the differentially expressed genes between vitiligo LS and NLS and NS, non-parametric Mann-Whitney U tests were used based on group analysis. The genes were ranked according to their false discovery rate-adjusted p-values (with cut off set at <0.05 after Bonferroni corrections). The threshold of expression differences was set at a 2.0-fold increase or decrease in gene expression levels as compared with NS.For the initial screening of gene expression differences in vitiligo skin, RNA from 17 pairs of full-thickness vitiligo skin and 16 normal skin biopsies were used in two-colored DNA microarray analysis following a protocol we previously described Pathway analysis on the differentially expressed genes in vitiligo skin was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.7, which is a standard bioinformatics tool for functional analysis of large gene list derived from high-throughput genomic scanning The candidate differentially-expressed genes that were identified by transcriptome analysis were verified by quantitative real time polymerase chain reaction (qRT-PCR) according to methods we previous reported Immune cells from the skin biopsies of 6 vitiligo patients and 5 healthy individuals were isolated and cultured as previously described 1: SK7), CD56 FITC (Ms IgG2b: NCAM16.2), Ki67 PE (Ms IgG1: B56) and granzyme B APC (Ms IgG1: GB11) , where NK cells were distinguished from the rest of the lymphocytes via positive expression of CD56 and negative expression of CD3. The samples were analyzed on a BD FACSCanto flow cytometer  and data was analyzed with FCS Express Pro Software Version 3 . A total of 10,000 events were collected for each sample, and live cells (positive for the viability stain) were analyzed for the proportion of CD3-CD56+ NK cells. These cells were further gated to obtain the proportion of NK cells expressing granzyme B.Flow cytometry of the immune cells migrating out of the skin biopsies in culture dishes was performed using fluorophore-conjugated antibodies against T cell receptors and natural killer (NK) cell markers. Cells were labeled with Fixable Viability Stain 450 and antibodies against CD3 APC  at 4°C for 20 min. For permeabilization, 0.3% Triton X  was applied to the sections for 10 min at 4°C. After blocking in 10% normal goat serum  for 60 min at room temperature, the sections were incubated overnight in mouse monoclonal anti-human NKG2D , which labels NK cells; rabbit polyclonal anti-human CD3 , which labels T lymphocytes; or rabbit polyclonal anti-human Melan-A/MART1 , which identifies the melanocytes. Then, the slides were treated with Alexa Fluor® 594 goat anti-mouse IgG and Alexa Fluor® 488 goat anti-rabbit IgG . Finally, the sections were counter-stained with DAPI  and mounted in Gold anti-fade medium . The slides were visualized with a Zeiss Axiovert 200M inverted fluorescence microscope . Image processing and quantification were performed with AxioVision Rel. 4.6 software  using the interactive measurement module. Quantification of cells was performed across entire tissue sections, with the resulting data expressed as the mean number of cells per 400× field of view over a range of 20–32 fields of view depending on the size of the specimens.The transcriptome analyses were performed using GeneSpring GX 7.3 software . Additional statistical analyses were performed with GraphPad Prism software . Non-parametric Mann-Whitney U tests were used to compare the difference in gene expression and the quantity of natural killer cells between vitiligo skin and the normal healthy skin.All study subjects had the diagnosis of vitiligo vulgaris, with a mean of 17.8% (range: 1% to 80%) body skin area depigmented Table 1. and We first examined gene expression changes in vitiligo lesional skin (LS) using the skin from healthy subjects as the controls  and 4. BasePathway analysis with Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.7  and Further comparisons were made between the NLS of vitiligo patients and NS from healthy subjects. As shown in in situ immune cell compositions in the tissues Since the gene expression analyses revealed markedly increased NK cell markers in NLS and LS of vitiligo patients, we speculated that the skin of vitiligo subjects contain abnormal infiltration of NK cells. To test this speculation, skin resident immune cells were isolated from cultured vitiligo skin explants To further confirm the increased NK cell infiltration within the skin of vitiligo patients, we next examined the biopsies by immunofluorescence microscopy. As shown in This study examined the gene expression profiles of skin tissues from patients with vitiligo using skin biopsies of healthy individuals as the controls. A potential technical challenge of the current study arises from the fact that melanocytes only account for a small proportion of total cells in the full thickness skin biopsies, which theoretically makes it difficult to detect melanocyte-related gene changes. The fact that the down-regulated genes identified by this non-cell-targeted analysis included the melanocyte markers among the most significantly altered genes demonstrated the robustness of the current approach in picking up gene expression changes even if these changes only account for a small fraction of the total cells present in the skin biopsy tissues.Most of the down-regulation of melanocyte-related genes  in the well-established vitiligo lesional skin (LS) most likely is the result of melanocyte death in the lesional skin. The expression down-regulation of several other genes, including LZTS1, GPR143 and C19orf28 does not have clear explanation at the present. It is possible that these genes are also melanocyte-expressed genes although this remains to be clearly established. The down regulation of SOX10 and PLP1 in vitiligo lesional skin may also be partially explained by the fact that they can be found in melanocytes The significance of the down-regulation of some melanocyte related genes  in vitiligo non-lesional skin as compared with normal skin of healthy individual is not entirely clear, but may represent subclinical melanocyte damage even in the lack of overt death of melanocytes. Alternatively, they may reflect inherent abnormalities present in the vitiligo individuals' melanocytes, as have been suggested to be present by the observation of gene expression abnormalities in purified melanocytes from vitiligo individuals bright NK cells are found in the blood and target lesions of patients with systemic lupus erythematosus and rheumatoid arthritis The unique findings from the current study include notably the discovery of the up-regulation of multiple genes of the killer cell lectin-like receptor (KLR) family including multiple NK cell activation markers such as KLRK1 , KLRC2 and KLRC4 Previous studies have shown an increase in the number of circulating NK cells in the blood of vitiligo patients The fact that increased NK cell infiltration was observed in the normal-appearing NLS of vitiligo patients may indicate an unfavorable generalized melanocyte-microenvironment in the skin compartment for the melanocyte to survive. However, what gives rise to the NK cell/innate immunity activation in vitiligo skin is not clear. It has been documented that NK cells respond to cellular signals released by cells under stress In addition to increased NK cells, this study revealed the presence of cytotoxic T cells in both vitiligo LS and NLS Figure 3To our knowledge, this is the first genomic expression analysis on the skin of patients with vitiligo. Our results strengthened the notion that immunity and inflammation play important roles in vitiligo pathogenesis. Moreover, our study specifically highlights the potential pathogenic role of the aberrantly heightened innate immunity, especially NK cells, in the local microenvironment of melanocytes in vitiligo skin. Further studies in the future are needed to verify if increased NK cells and heightened innate immunity are the direct cause of melanocyte death in vitiligo. Finally, the observation of activated innate immunity in the normal appearing non-lesional skin far away from the lesional border suggests that therapeutically targeting the activated innate immune responses in both lesional and non-lesional vitligo skin may represent a viable approach for developing vitiligo therapies in the future.


To the Editor: Multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type 104 has emerged during the 1980s and 1990s as a world health problem because of its implications in animal and human disease   have recently been reported for serovars Typhimurium DT104, Agona, and Albany to confer different multidrug resistance phenotypes (dfrA10 gene coding for trimethoprim (Tm) resistance was found downstream of the pse-1 integron in a third unusual integron structure involving orf513 in the variant antibiotic resistance gene cluster called Salmonella genomic island 1-A (ApCmFfSmSpSuTcTm)  isolated from a human case in Belgium.Multidrug-resistant serovar Typhimurium definitive phage type 104 was disseminated globally with several outbreaks in humans and animals. At present, in contrast to the world health problem of multidrug-resistant serovar Typhimurium definitive phage type 104, human cases of infections with other Salmonella genomic island 1-A   were performed using primers corresponding to the left and the right (with or without retron) Salmonella genomic island 1 junctions to the chromosome as described previously   . This hypothesis is also supported by macrorestriction analysis, which showed that the strains from poultry and the human case-patient had identical XbaI and BlnI pulsed-field gel electrophoresis patterns and thus indicate that they are clonally related. Moreover, the patient was not in contact with poultry during his stay in Turkey and, to date, very little is known about the epidemiology of multidrug-resistant serovar Agona strains in this country. Further investigations on the epidemiology of multidrug-resistant serovar Agona strains harboring Salmonella genomic island 1 are warranted to avoid such strains becoming a worldwide epidemic, as was the case for multidrug-resistant serovar Typhimurium definitive phage type 104.To our knowledge, this is the first report describing a human infected by a serovar Agona strain harboring


Studies of bimanual actions similar to activities of daily living (ADLs) are currently lacking in evaluating fine motor control in Parkinson’s disease patients implanted with bilateral subthalamic deep brain stimulators. We investigated basic time and force characteristics of a bimanual task that resembles performance of ADLs in a group of bilateral subthalamic deep brain stimulation (DBS) patients.Patients were evaluated in three different DBS parameter conditions off stimulation, on clinically derived stimulation parameters, and on settings derived from a patient-specific computational model. Model-based parameters were computed as a means to minimize spread of current to non-motor regions of the subthalamic nucleus via Cicerone Deep Brain Stimulation software. Patients were evaluated off parkinsonian medications in each stimulation condition.The data indicate that DBS parameter state does not affect most aspects of fine motor control in ADL-like tasks; however, features such as increased grip force and grip symmetry varied with the stimulation state. In the absence of DBS parameters, patients exhibited significant grip force asymmetry. Overall UPDRS-III and UPDRS-III scores associated with hand function were lower while patients were experiencing clinically-derived or model-based parameters, as compared to the off-stimulation condition.While bilateral subthalamic DBS has been shown to alleviate gross motor dysfunction, our results indicate that DBS may not provide the same magnitude of benefit to fine motor coordination. Bilateral deep brain stimulation (DBS) of the subthalamic region has been identified as an effective procedure for the treatment of cardinal motor signs in advanced Parkinson’s disease (PD) Despite such reports, few have ventured beyond evaluation via the UPDRS-III and basic features of unimanual prehension Given that motor symptoms, medical side effects, and general quality of life worsen with PD progression While clinically determined stimulation parameters have been effective in improving overall motor function in uni- and bilaterally implanted PD patients To investigate the effects of bilateral subthalamic DBS on everyday motor function, we have chosen to evaluate impairments in fine motor control using an ADL-like task to assess upper extremity function in three different stimulation conditions: clinically determined DBS parameters, patient-specific model-derived DBS parameters, and off DBS stimulation. Expanding upon the methods used in previous studies A priori, differences between the two stimulation parameter conditions are not expected, as both conditions are programmed with the intention of alleviating motor symptoms of PD. Given that improvements in manual coordination and grip force control have been reported previously with subthalamic DBS in comparison to off stimulation states Ten participants with advanced PD participated in this study (mean ± sd: age 61±8 years), Ethics and procedures for this study were evaluated and approved the Institutional Review Board of the Cleveland Clinic. All participants gave written informed consent according to the procedures approved by the Cleveland Clinic Institutional Review Board, in accordance with the Declaration of Helsinki. All data were collected onsite at the Cleveland Clinic, in Cleveland, Ohio, USA. None of the patients evaluated in this study exhibited cognitive issues that were significant enough to have next of kin provide informed consent. Capacity was determined via clinical cognitive evaluation using the Mini-Mental State Examination (MMSE).Non-Rotation and Rotation tasks, respectively) between the two sensors were used throughout each testing session  and lower (static) objects were freely moveable; however, the location and orientation of the static object was prescribed. Subjects were not permitted to move the static object during testing. Any trial in which movement of the static object occurred was omitted.Overall, eight different bimanual configurations were performed in each testing session. Five trials were collected in each of the eight tested configurations . Details regarding each of the tested conditions can be found explicitly described in No contact of either transducer was permitted prior to trial onset; subjects were instructed to begin each trial with both hands placed palm down on the surface of the table. Up to three practice trials were offered to each subject prior to the onset of data collection for each condition. On average, each subject performed one practice trial. In each of the three sessions, the presentation of conditions was block randomized for each subject; articulation type was used as the blocking factor.Data were collected during three visits to the Neural Control Laboratory at the Cleveland Clinic. Each visit was separated by a minimum of 72 hours. Each participant completed the experiment under three different stimulation conditions: clinically-determined DBS parameters , patient-specific model-derived DBS parameters (Model), and off DBS stimulation (Off).The clinically-determined DBS stimulation parameters used in this study were the settings derived for each patient via standard clinical care. These settings were stable for at least 6 months prior to study participation. The clinical programming of stimulators was overseen by an experienced DBS programming team consisting of a programming nurse and a movement disorders neurologist specializing in Parkinson’s disease.Model derived stimulation parameters were defined from a patient-specific DBS computer model of each side of the patient’s brain created in Cicerone v1.2, a freely available academic DBS research tool Based on previous experience developing patient-specific models of therapeutic subthalamic DBS Model settings were defined using theoretical predictions of the VTA. The VTA provides an electrical prediction of the volume of axonal tissue directly activated by DBS for a given stimulation parameter setting. The software provided the ability to interactively visualize and evaluate a wide range of stimulation parameter settings and calculate the overlap of the VTA with the brain anatomy and target volume. Following completion of the experimental testing we used Cicerone to quantify the VTAs for each patient under both the Model and Clinical settings, along with their respective overlap with the subthalamic nucleus atlas volume available in the software.G/dtdF) in each of the tested conditions. Total grip and load forces were considered twice the measured value as only two force-torque sensors were used in the experimental setup. Correlation between grip and load forces within each hand was computed to evaluate within-limb control during task performance. Data analysis algorithms related to the analysis of raw force-torque data signals, data filtering techniques, and movement onset and termination identification were based on previous work Grip and load forces were analyzed several different ways in this experiment. At the most basic level of analysis, the maximum magnitudes of the exerted forces were evaluated, as well as the rate of grip force production , and Task , Stimulation , and Transducer . Evaluation of conditions appropriate for parametric analyses were performed prior to statistical analyses. For all ANOVAs, the assumption of sphericity was verified using Mauchly’s sphericity test. If sphericity was violated, the degrees of freedom were adjusted as necessary using Greenhouse-Geisser corrections. Correlation coefficient (r) values from the regression analysis were subjected to Fisher z-transformation to mitigate the ceiling effects inherent to these variables. Non-transformed data are presented in the figures to avoid confusion. Post-hoc pairwise comparisons to elucidate differences among Stimulation  state were performed with Bonferroni corrections were used to evaluate significant effects of the ANOVA analyses. Data regarding VTA measurements were found to obey normal distributions and were therefore evaluated via paired t-tests between on-stimulation states . Non-parametric analyses were used in the evaluation of UPDRS-III scores. Kruskal-Wallis analyses were used to determine if differences existed in UPDRS scores across the three stimulation states. Mann-Whitney tests were then used to determine differences in UPRS-III scores between stimulation states . All ANOVAs were performed using SPSS; all non-parametric analyses were performed using Minitab, due to ease of usability of the interface for non-parametric analyses.The data are presented in the text and figures as means and standard errors. Analyses of variance (ANOVAs) were performed on the data with the factors of: UPDRS-III scores for each of the DBS states revealed differences between the on-DBS states versus the off-DBS state . Both clinically-determined and model-derived parameters were associated with lower overall UPDRS-III scores as compared to off-stimulation , see Task and Method. Task time was greater when the two objects were being connected to each other  and during rotational actions , illustrated in The overall time to perform the bimanual tasks (task time) was not different across DBS conditions. Similar to analysis of healthy young individuals Stimulation: F1.3,134.6 = 7.4, p<0.005), confirmed by post-hoc analysis. Average grip force during testing with Model DBS parameters was not significantly different from Clinical or Off DBS conditions. Average grip forces were also found to be greater when exerted on the dynamic transducer . The disproportion of applied grip forces between the two transducers was most notable during the Off DBS stimulation condition. This finding was confirmed by the presence of a Stimulation x Transducer interaction , illustrated in Across all conditions, the average grip force produced was 11.6±0.9 N and 15.7±1.0 N for the static and dynamic transducers, respectively. Average grip force was affected by DBS stimulation state and transducer, as shown in Transducer: F1.0,122.9 = 61.6, p<0.001) and during the Clinical DBS condition . Maximal grip force was largest during the Clinical DBS condition as compared to the Off DBS condition, whereas the Model DBS condition did not differ from the other two stimulation states, confirmed via post-hoc. An imbalance in applied maximum applied grip force between the two transducers was most notable during the Off DBS state, such that larger maximal grip forces were applied to the dynamic transducer, confirmed by the Stimulation x Transducer interaction , illustrated in Similar to the average grip force findings, maximum grip force was significantly larger on the dynamic transducer  and when participants were experiencing the Clinical DBS condition ; however, no interaction effects were found, as shown in The rate of grip force production (dFG/dt) exhibited similar trends to average and maximal grip force production, such that dFG/dt was significantly larger on the dynamic transducer  and in non-rotation tasks , shown in Transducer x Method interaction .In this analysis of within-hand coordination, the kinetic coupling measures did not show effects of stimulation state. Despite lack of a stimulation effect, within-hand grip-load force correlation was larger for the static transducer .The VTAs for the Clinical and Model DBS are located in In this investigation of the effects of DBS on fine motor control, we hypothesized DBS would improve basic features of bimanual dexterity. Specifically, we hypothesized that testing clinically-determined and patient-specific model-derived DBS parameters would be associated with reduced bradykinesia and improved grip force production . With respect to these hypotheses, analysis of the current data set did not reveal significant DBS-related improvements in bradykinesia during bimanual tasks; however, stimulation condition did affect the magnitude of grip forces exerted. Consistent with our third hypothesis, grip-load coupling was found to be intact in PD patients independent of treatment state. As per our fourth hypothesis, we expected both clinically-determined and model-derived DBS stimulation conditions to be associated with lower overall UPDRS-III motor scores as compared to the off-stimulation condition. Despite the limited stimulation effects on bimanual actions, significant differences in UPDRS-III scores were found. In the following paragraphs, we address the differential effects of DBS condition on motor function as well as the physiological and programming implications of DBS.Regarding our hypotheses, a prominent feature of PD movement, bradykinesia (measured by task time), was not different across stimulation conditions, contrary to earlier reports Despite these findings, differences in grip force production due to stimulation state were exhibited. Abnormal grip force production has been reported with PD In the current study, clinically-determined and patient-specific model-derived DBS parameters both produced improved clinical rating scores. However, patient-specific model-derived DBS parameters do not appear to be as effective in improving motor performance in ADL-like tasks compared to clinically-determined DBS parameters. While previous work has shown equivalence in motor performance between the two parameter groups under dual-task conditions (i.e. simultaneous performance of a cognitive and motor task)


These assemblies can be programmed to serve as structural scaffolds for secondary chromophore molecules with light-harvesting properties. Like in natural systems, the local and global spatial organization of these synthetic scaffolded chromophore systems plays a crucial role in their emergent excitonic and optical properties. Previously, we introduced a computational model to predict the large-scale 3D solution structure and flexibility of nucleic acid nanostructures programmed using the principle of scaffolded DNA origami. Here, we use Förster resonance energy transfer theory to simulate the temporal dynamics of dye excitation and energy transfer accounting both for overall DNA nanostructure architecture as well as atomic-level DNA and dye chemical structure and composition. Results are used to calculate emergent optical properties including effective absorption cross-section, absorption and emission spectra and total power transferred to a biomimetic reaction center in an existing seven-helix double stranded DNA-based antenna. This structure-based computational framework enables the efficient  Programmed self-assembly of nucleic acids offers the unique ability to scaffold chromophore molecules at precise locations and orientations in 3D space to construct DNA-based light-harvesting antennas and wires with custom optical properties . Proof-oImportantly, the 3D spatial organization and conformational flexibility of chromophore molecules functionalized to nucleic acid scaffolds plays a critical role in their exciton transport and light-harvesting properties due to the sensitive nature of FRET interactions to both nanometer-scale inter-chromophore distance and transition dipole moment orientation . Coherenin silico design and evaluation of light-harvesting and photonic nucleic acid nanostructures functionalized with chromophores via chemical conjugation to specific DNA bases . Subsequent energy minimization and molecular dynamics of the atomic structure that could in principle be performed to refine the atomic model are beyond the scope of the present work.dsDNA atomic coordinates are generated from the finite element model by positioning standard atomic coordinates of the four base pairs A-T, T-A, G-C and C-G at finite element nodes using the topology and sequence information of the DNA nanostructure, the chain identifiers, residue sequence numbers and residue names. The atomic structure of each reference base pair is translated and rotated to the corresponding position and orientation of the pertinent finite element node (Supplementary Note S2). Although most of the FRET modeling results presented here are for the rigid mechanical ground-state of the nucleic acid nanostructure, the effects of conformational flexibility are examined for the dsDNA-based wire with intercalating dyes. When dsDNA flexibility is accounted for, it is assumed that FRET occurs on timescales that are significantly shorter than the shortest relevant fluctuation timescale of dsDNA, so that FRET may be evaluated for the Boltzmann-weighted DNA conformational ensemble computed from normal mode analysis. Relative dye conformational flexibility with respect to the DNA nanostructure itself that can in principle be incorporated for intercalating and base-substituted dyes, as previously performed by FRET-matrix . In this approach, the linker length and dye radius are used to compute the sterically accessible conformational space of the dye, as previously performed for Cy5 and AF488 dyes conjugated to DNA . The positions and orientations of intercalating and base-substituted dyes are modeled structurally using 3DNA (Supplementary Note S1). Additional PDB structures may be incorporated into the present modeling framework as they become available. The transition dipole moment vector position and orientation of YO-PRO-1 relative to the dye chemical structure is given by wiThe master equation  is solveThe overall FRET efficiency, which is a measure of the effectiveness of exciton transport through the multi-dye system, is calculated from the total integrated fraction of the initial exciton population recombined both radiatively and non-radiatively at the defined acceptor sites,The conformational fluctuations of the positions and orientations of scaffolded dyes are in general due to (i) the conformational flexibility of the dsDNA scaffold and (ii) the relative conformational flexibility of the dye with respect to the DNA scaffold, which may include relatively small fluctuations if the dye is intercalated, tightly bound or base-substituted/conjugated  or signiBecause FRET efficiency (and any other FRET-related quantity) is highly non-linear with respect to dye position and orientation, it may not be justified to use the mean position and orientation of dyes to compute FRET between dyes with positions and/or orientations that fluctuate significantly in time. In general, when the FRET timescale is much faster than the dye translational and rotational timescales (veraging .Supplementary Note S4). Molecular dynamics simulations or polarized anisotropy measurements are needed to more precisely model For dyes that are tethered to DNA via long flexible linkers, the characteristic translational diffusion timescale for the dye to sample the entire AV, ntire AV . On the 0.1−1 ns . For typA similar argument for base-substituted and intercalating dyes justifies the use of static averaging for both position and orientation when the conformational dynamics of DNA are considerably slower than 0–100 ns . Althoug0–100 ns ,51.Supplementary Note S5). Decay-Associated Spectra (DAS) are calculated from the rate matrix Supplementary Note S5). In cases where conformational fluctuations are considered, interactions between dyes are computed based on their mean positions.The overall steady-state absorption and emission spectra of the light-harvesting or photonic device may be calculated from the exciton population of each dye and their individual absorption and emission spectra, quantum yield, molar absorptivity and fluorescence lifetime . I. I13). ISupplementary Note S5). Model results (Supplementary Note S6). Although the FRET transfer rate between those pairs varies significantly due to the conformational fluctuations, it remains much higher than the relaxation rate (∼108 s−1) and its average value is similar to the rigid wire case, so that the overall transfer efficiency is only weakly affected.To model FRET in this wire, 1000 dye conformations are simulated for each mean number density of intercalated dyes to account for the variable numbers and positions of the dyes that intercalate randomly along the dsDNA helix . Variati results b are in  results . Intereset al.  is considerably less than the Py-Cy3 Förster distance (3.7 nm) b. StrongIsotropic averaging is assumed for the calculation of the exciton population and steady-state spectra. Exciton population dynamics for these antenna designs illustrate multiple exponential decays for the 1:1:1 and 6:6:1 constructs a. SteadyDAS spectra also quantify energy transfer from Py to Cy3 and from Cy3 to AF b. Due toFor light-harvesting antennas designed to maximally absorb sunlight, the solar irradiance spectrum may be considered in computing the total power transferred to the reaction center (RC) from the antenna absorption cross-section c. For exSupplementary Note S7).High transfer rates might reduce the accuracy of isotropic averaging. For example, high calculated transfer efficiency from Py to Cy3 using isotropic averaging is∼96% for the 1:1:1 antenna and range from 96 to 99% for the 6:6:1 antenna, which implies FRET lifetimes in the range of 0.8 ps to 12.5 ns and 0.5 ps to 33 ns, respectively, that can get significantly shorter than typical rotational lifetime 0.1−1 ns . Hence, Supplementary Note S8. In future antenna designs, intercalating and base-substituted or -conjugated dyes and porphyrins may be employed instead of tethered dyes due to the ability to more precisely control their positions and orientations, and pairwise single-molecule FRET measurements may be required to resolve differences between theoretical and experimental light-harvesting and optical properties.Interestingly, the preceding theoretical results suggest a considerably higher Cy3-to-AF energy transfer than that measured experimentally . Thus, it is feasible using the present computational modeling framework to efficiently explore both DNA nanostructure solution shape as well as multi-dye composition in the future design and optimization of high molecular weight biomimetic DNA-based light-harvesting constructs. However, quantum mechanical modeling will be required for the consideration of coherent inter-dye energy transfer.To demonstrate the potential of the proposed structure-based procedure to model and compute FRET transfer for antenna constructs of this molecular weight, we applied our framework to a megadalton DNA origami FRET cascade . In thisin silico distinct DNA-based light harvesting and photonic device architectures prior to experimental synthesis and validation. Further, the atomic-level models that are generated here may be used as input to higher resolution computational modeling approaches including molecular and Brownian dynamics to account for dynamical structural and quantum properties that are not considered in this work. The latter are likely to play an important role in the design of highly efficient light-harvesting antennas such as those found in nature . Atomic structure generation, dye AV generation and optical property calculation software is available upon request.Atomic models of DNA nanostructures are available as part of the CanDo webserver (Supplementary Data are available at NAR Online.Office of Naval Research [ONR N000141210621 to K.P. and M.B.]; and the Army Research Office ; are gratefully acknowledged. E.B. additionally acknowledges support from the Natural Sciences and Engineering Research Council of Canada. Funding for open access charge: Office of Naval Research [ONR N000141210621].Conflict of interest statement. None declared.


Cardiac metabolic imaging based on hyperpolarized 13C-labeled pyruvate shows great potential for assessing the metabolic changes that the heart undergoes during ischemia  of the iIn this work, we explore multi-echo measurements using single-shot echo-planar imaging (EPI) readouts for thin slice dynamic cardiac metabolic imaging of small animals.Healthy female Wistar rats (200g) were scanned in a 9.4T horizontal bore Bruker MRI system. A home-built multi-sample dissolution DNP system was used to hyperpolarize pyruvate . During Images for the metabolites are shown in Figure By using the multi-echo technique, the requirements on the gradient system performance are relaxed. In contrast to using a spectral-spatial excitation, a simple slice excitation can be used that allows for thinner slices. This is especially important in small animal imaging. Due to the thin slices, the dephasing and partial volume effects are significantly reduced, and the effective in-plane resolution is improved. This is especially important in separating the signal from pyruvate between myocardium and blood pool when using kinetic modeling. The high in-plane resolution is of importance for metabolic imaging after selective occlusion of one coronary artery, and for studies of the metabolic changes that the heart undergoes under controlled experiments in small animals.Marie Curie Intra European FellowshipSwiss National Science Foundation


Stipiturus mallee is an endangered passerine whose global distribution is confined to fire-prone, semi-arid mallee shrublands in south-eastern Australia. This species, with poor capacity for dispersal, has undergone a precipitous reduction in distribution and numbers in recent decades. We used genetic analyses of 11 length-variable, nuclear loci to examine population structure and processes within this species, across its global range. Populations of the Mallee Emu-wren exhibited a low to moderate level of genetic diversity, and evidence of bottlenecks and genetic drift. Bayesian clustering methods revealed weak genetic population structure across the species' range. The direct effects of large fires, together with associated changes in the spatial and temporal patterns of suitable habitat, have the potential to cause population bottlenecks, serial local extinctions and subsequent recolonisation, all of which may interact to erode and homogenise genetic diversity in this species. Movement among temporally and spatially shifting habitat, appears to maintain long-term genetic connectivity. A plausible explanation for the observed genetic patterns is that, following extensive fires, recolonisation exceeds in-situ survival as the primary driver of population recovery in this species. These findings suggest that dynamic, fire-dominated landscapes can drive genetic homogenisation of populations of species with low-mobility and specialised habitat that otherwise would be expected to show strongly structured populations. Such effects must be considered when formulating management actions to conserve species in fire-prone systems.Fire is a major disturbance process in many ecosystems world-wide, resulting in spatially and temporally dynamic landscapes. For populations occupying such environments, fire-induced landscape change is likely to influence population processes, and genetic patterns and structure among populations. The Mallee Emu-wren  Fire is a major disturbance process that changes landscape structure in many ecosystems worldwide The effects of fire-induced landscape change on ecological and population processes are complex, and major gaps remain in our knowledge Following disturbance by fire, subsequent population recovery will be influenced by many factors including the number and demographic parameters of survivors Stipiturus mallee. The Mallee Emu-wren is one of the tiniest members (4–6.5 g) of the family Maluridae, endemic to the semi-arid zone of south-east Australia Eucalyptus spp. ‘Mallee’ refers to the growth form of Eucalyptus spp. characterised by a large underground lignotuber from which multiple stems sprout. The Mallee Emu-wren is a resident habitat specialist and has a patchy distribution. Multi-scale studies of the habitat requirements of this species show that its distribution is strongly influenced by the post-fire age of vegetation, as a consequence of seral changes in the structure of ground-storey spinifex grass (Triodia scariosa) on which it depends Triodia-mallee vegetation older than 15 years since last burnt, with a slight preference for vegetation 15–29 years of age This study examines the genetic diversity and structure of a globally endangered bird species, the Mallee Emu-wren In recent decades, the Mallee Emu-wren has undergone a precipitous reduction in numbers and distribution. Large wildfires (>10 000 ha), exacerbated by drought, have caused the extinction of populations in parts of the south-west of its range. Large fires and inappropriate fire regimes that reduce the amount of habitat of suitable age are considered a significant threat to remaining populations. Geographic range contraction has occurred such that the Murray-Sunset and Hattah-Kulkyne National Parks in northern Victoria now support an estimated 92% of the global population. Small, scattered and isolated populations occur in South Australia in Billiatt and Ngarkat Conservation Parks and only one pair was recorded in the Big Desert/Wyperfeld reserve complex in Victoria in 2006 This study examines the genetic diversity and structure of the Mallee Emu-wren across its global range. The primary objective was to examine spatial genetic patterns among populations across the species' range to draw inferences about population structure and processes. Its occurence in a fire-prone environment, coupled with the intrinsic characteristics of the Mallee Emu-wren (e.g. poor flight capability) strongly suggest that it may exhibit a metapopulation structure at the landscape-scale, with relatively strong patterns of genetic divergence (i.e. population genetic structure) expected among geographically-dispersed sampling units The Mallee Emu-wren is inconspicuous and rare, has patchy distribution and occur in low density Long-term ecological and genetic studies on other species of the family Maluridae show males are philopatric, and related males (e.g. brothers) have a tendency to occupy neighbouring territories. Dispersal tends to be female biased, although the pattern and distance varies within populations and among species Mallee Emu-wrens were captured either by trapping in monofilament mist nets, or by throw nets after being lured by playback recordings of calls (©David Stewart/Nature Sound). The species is sexually dichromatic and males are easily distinguished from females by the presence of brilliant sky-blue throat and breast feathers All work was conducted under Deakin University Animal Ethics Committee approval (A35/2005) and research permits issued by the Department of Sustainability and Environment, Victoria (10003389) and the Department of Environment and Heritage, South Australia .Malurus splendensM. cyaneusStipiturus malachurusDNA was extracted from samples using a standard ethanol/chloroform extraction method Taq DNA polyermase, 5× buffer, 0.25 mM dNTPs, 0.5–4.5 mM MgCl2 (Promega/MBI Fementas) and 5–10 pmol of each primer pair in a total volume of 20 µl. Microsatellite products were run on an ABI® Capillary Analyser (Perkin Elmer) or a Li-Cor 4200 and 4300 Global IR2 two-dye DNA sequencer for separation and sizing. Putative homozygotes were amplified and genotyped twice to confirm their status. Positive and negative controls were used in all reactions.PCR reactions for microsatellites and product separation were performed in two different laboratories, with cross-referencing quality control. PCR reactions for each marker were optimised using the following: 20–40 ng of sample DNA, 0.5 Units Go ISF) were calculated in FSTAT 2.9.3.2 Standard measures of genetic diversity for length-variable markers were obtained in various programs, treating the six geographic locations as separate populations. GENALEX V6.0 FST calculated in GENEPOP V4.0 D, adjusted for small sample sizes (estD)FST and estD as measures of genetic differentiation each have different strengths and shortcomings, so we present both to infer population differentiation FST and estD values theoretically range from zero to one, with zero indicating no differentiation  FST is a widely used estimator of population genetic structure, with which many researchers are familiar D may be more appropriate for highly-variable markers (e.g. microsatellites) ST, .Enhanced effects of genetic drift in small, isolated populations are expected to lead to increased genetic differentiation among sites. The extent of genetic differentiation between each pair of sites was estimated using two allele-frequency-based measures: FIS - which is an expression of excess of heterozygotes relative to proportions expected under Hardy-Weinberg equilibrium The heterozygosity excess test in the program BOTTLENECK V1.2.03 To assess the extent of genetic population structure across the species' global range, individual genotype-based Bayesian clustering algorithms were implemented in both STRUCTURE 2.3.3  tructure was run using the admixture model with correlated allele frequencies, and K values 1 to 10. Twenty replicate runs were performed for each K value. Each run was 3×106 Markov Chain Monte Carlo (MCMC) repetitions following a burn-in period of 106 repetitions.SK≈5 populations K values 2 to 9. The Deviance Information Criterion (DIC) was used to select the model that best fit the genetic data K, and the most likely value of K was selected by visually assessing the point at which DIC first reached a plateau and the number of clusters to which individuals were proportionally assigned. The 10 runs with the lowest DIC values for the selected K-value were retained and their admixture estimates were averaged using CLUMPP V 1.1.2 TESS incorporates information on individual geographic coordinates and has been shown to be more powerful than non-spatial algorithms, especially in weakly differentiated populations Analyses of the nuclear loci show overall moderate to low levels of genetic diversity across the global range of the Mallee Emu-wren, with signatures of recent population bottlenecks in two locations, and local effects of genetic drift in others such that only a single sampled population showed neither phenomenon , 3 & 4.FIS values were positive and significant (p = 0.05) for Hattah-Kulkyne National Park, with two loci  showing significant homozygote excess FIS, none were significant. Linkage disequilibria were not detected for any loci pairs.e excess . One locA) was variable among loci, ranging from 2 to 32 alleles . As was expected based on the theoretical and empirical behaviour of the metrics, significant estD values were higher than their respective FST values  heterozygosity excess was detected for the Murray-Sunset National Park (East) and Ngarkat Conservation Park samples under the two-phase mutation model, indicating evidence of recent bottlenecks in these two populations (Significant (ulations .K = 1). Increased power resulting from the incorporation of spatial information in TESS, revealed the presence of two weak spatial genetic clusters across the study region (o. 6) were assigned to one cluster (cluster 2 in o. 2), the remaining populations within the Murray-Sunset and Hattah-Kulkyne reserve complex  were more strongly assigned to the alternative cluster  (consistent with reported demographic declines) and some evidence of genetic drift in other locations, indicate these localities may have experienced recent declines in effective population size. Bottlenecked and strongly inbreeding populations are important to identify for conservation management because of genetic threats to individual fitness and population viability The prevalence of several monomorphic loci and evidence of a bottleneck in the Ngarkat Conservation Park population  & 4 is cFingilla teydea polatzeki, a critically endangered passerine endemic to the island of Gran Canaria. Despite a 50% decline in the global population of this sub-species , temporal sampling found no genetic signature of a bottleneck. Furthermore, the post-fire population retained a high level of genetic diversity Our results contrast with those from a study of the genetic effects of a forest fire on the Blue Chaffinch A second genetic threat to the long-term viability of the Mallee Emu-wren can presumed to be the erosion of quantitative genetic variation necessary for adaptive evolution FST values ; that is, populations have similar allelic frequencies indicating substantial genetic connectivity  from within the fire footprint in-situ survivors is less likely to be accompanied by loss of much original genetic diversity, except in the presence of very strong, sustained or repeated bottlenecks Collectively, the genetic patterns and population structure found in this study can be attributed to the spatial and temporal patterns of fire in mallee ecosystems. Most fires are small (<100 ha in size), but intense landscape-scale wildfires exceeding 10,000 ha occur within the region every 10–20 years in-situ survival as the primary means of population recovery of the Mallee Emu-wren. These large fires  create vast homogenous areas in which the ground layer , shrub and canopy strata are all consumed Amytornis spp. of the arid-zones of Australia Ningaui yvonneaeGiven large, severe wildfires dominate the mallee landscape in-situ survival may occur in environments where fires leave numerous unburnt refuges, as in the case of the Blue Chaffinch of Gran Canaria discussed earlier. Unburnt refuges were prevalent throughout the fire area and were thought to enable a sufficient proportion of individuals to survive and persist, thereby mitigating the loss of genetic variability in the post-bottleneck population In-situ survivorship in unburnt refuges has been attributed to the rapid demographic recovery in birds , and weak genetic structure (STRUCTURE/TESS) is an optimistic message for the conservation of this species. The lack of marked population differentiation across its global range means that for management purposes  this species can be treated as a single genetic unit. Nevertheless, the finding of disrupted fine-scale population processes, as illustrated by the demographic and genetic impoverishment in the Ngarkat Conservation Park population, supports implementation of actions to assist population recovery. Reintroduction programs or the genetic restoration The apparent genetic connectivity of the Mallee Emu-wren inferred from the low population differentiation Click here for additional data file.Text S1Summary of CERVUS parentage analysis.(DOCX)Click here for additional data file.


The success of fat grafting in breast reconstruction depends on fat retention. The use of stem-cells-enriched fat graft is an alternative method for graft stability.A case of nipple-areola sparing mastectomy double stage reconstruction with the use of stem cells enhanced fat graft is reported.Fat grafting is growing as a new and promising tool in reconstruction following nipple and areola sparing mastectomies as a way to restore a sufficient and reliable subcutaneous space in the mastectomy flap. This combined with an anatomical gel implant offers an integrated system of achieving a natural shaped breast. Through preservation of the areola–nipple (A/N) complex, the nipple–areola sparing mastectomy (NASM) serves to reduce the negative psychological impact on women without increasing the incidence of local relapse ,2. For minvolvement of the skin above the neoplastic area;evidence of neoplastic infiltration of the A/N complex (nipple retraction or haematic secretion);distance between the neoplasia and the A/N complex of less than 10 mm;tumour multiplicity;relapse in case of previous breast tumour;suspicious axillary adenopathy;primary chemotherapeutic treatment.Actually, NASM is contraindicated in cases of the following:With NASM, the mammary gland is removed as well as the skin above the neoplasia when necessary. The incision can be made either inside the A/N complex (reverse omega), in the superior-outer quadrant with a radial course, or in the infra-mammary fold. An immediate reconstruction is usually performed with a tissue expander, which is replaced after six months by a definitive prosthesis with eventual symmetrization of the contralateral breast.The patient is a 37-year-old woman with a positive genetic test for BRCA1 and a nodular neoformation in the superior-outer/superior-inner quadrant of the right breast  She underwent bilateral NASMs with a right sentinel lymph node biopsy on 20 June 2007.Final histological examination documented infiltrating ductal carcinoma on the right side  with a minimum distance between the tumour and the nipple margin greater than 10 mm.The sentinel lymph node biopsy was negative pT1c, psN0(i-).The biological characterization of the neoplasm was the following: ER neg-PGr neg-ki67 40%, c-erb-B2 neg, score 0. Histological examination of the tissue from the left breast demonstrated micro-fibroadenoma intracanalicolare (QSE) in the background of fibrocystic mastopathy.Immediately following the mastectomies, a bilateral 450 cc tissue expanders were positioned in a submuscular pocket Figure 2The post-operative course was free of complication. In August 2007, the patient underwent prophylactic bilateral oophorectomies. On 28 January 2008, the patient underwent the second stage of the breast reconstruction. A partial bilateral capsulotomy was followed by a careful injection of cell-enhanced autologous fat into the subcutaneous areas. A total of 85 cc of cell-enhanced fat graft was injected on the left breast and 90 cc on the right. The tissue was obtained through a syringe-based liposuction of 355 cc of adipose tissue from abdomen and external thighs, which was performed at the beginning of the case. The tissue was then processed using an automated device (Celution 800/CRS-Cytori) to produce a cell-enhanced fat graft.The surgical procedure was completed with placement of 370-cc bilateral silicone gel implants Figure 3The patient was discharged on post-operative day 1 and had no complications in the post-operative course. The aesthetic result was evaluated during a ten month follow-up after the second procedure. The patient evaluated the result as excellent, the surgeon as good. Graft retention in the mastectomy flap was evaluated with MRI and ultrasound at three months The oncological follow-up was negative at 17 months for local relapses and systemic metastasis.Autologous fat grafting has been used to treat soft tissue defects for decades with varying degrees of success. Fat would be an ideal filler given the easy accessibility, lack of immunogenicity and low cost of adipose tissue, but survival of fat grafts has not been predictable ,5. SeverIn this case report, a new technique is used to enrich a patient's fat with her own ADRCs to create a natural soft tissue filler. Cell-enhanced fat along with a silicone implant is used for breast reconstruction after NASM. The follow-up demonstrates that ADRC-enhanced fat grafting is a feasible option for breast reconstruction, which may lead to a more natural appearance than implant alone.


Apis mellifera) from apparently healthy colonies. Although infections, toxins, and other stressors have been associated with the onset of CCD, the pathogenesis of this disorder remains obscure. Recently, a proteomics study implicated a double-stranded DNA virus, invertebrate iridescent virus (Family Iridoviridae) along with a microsporidium (Nosema sp.) as the cause of CCD. We tested the validity of this relationship using two independent methods: (i) we surveyed healthy and CCD colonies from the United States and Israel for the presence of members of the Iridovirus genus and (ii) we reanalyzed metagenomics data previously generated from RNA pools of CCD colonies for the presence of Iridovirus-like sequences. Neither analysis revealed any evidence to suggest the presence of an Iridovirus in healthy or CCD colonies.Colony collapse disorder (CCD) is characterized by the unexplained losses of large numbers of adult worker bees ( Apis mellifera) in hives, while both resources and brood remain. Since the initial description of CCD in 2006, annual colony losses in the United States have exceeded 30%, a number significantly greater than previous average losses Honey bee decline has become an increasingly important problem worldwide, and has been attributed to multiple underlying causes Nosema ceranaePicornavirales such as Israeli acute paralysis virus Although CCD continues to be associated with an annual colony loss of approximately 10% in the United States Iridovirus genus (Family Iridoviridae) has been proposed to act synergistically with N. ceranae to cause the development of CCD A. cerana) A. mellifera protein sequences. To reconcile this discrepancy and test the intriguing hypothesis proposed by Bromenshenk et al., we surveyed CCD and healthy A. mellifera colonies from the United States and Israel for the presence of several invertebrate iridescent viruses, and re-analyzed previously generated metagenomic data from CCD colonies for Iridovirus sequences.Recently, a double-stranded DNA virus (dsDNA) belonging to the Iridovirus genus. These were collected in 2007–2009 from 35 colonies distributed across nine commercial apiaries, seven of which have been previously described One hundred and sixty-three bees were surveyed for the presence of members of the Iridovirus genus, a second nested assay was developed based on an alignment of IIV-6 and IIV-31 (GenBank accession number AJ279821), the only members of the Iridovirus genus with available DNA polymerase sequence data. Finally, an assay targeting the only sequence available for IIV-24 (GenBank accession number AF042340) was developed in a highly conserved region of the capsid. PCR assays were performed in 20 µl reactions containing 0.25 µM of each primer, 0.125 mM dNTPs, and 1 mM MgCl2. The primer annealing temperatures for each assay are given in Armadillidium vulgare) were used as positive controls. The IIV-24-specific assay was also able to detect the IIV-6 positive control. The sensitivity of each assay was evaluated by serial dilution of the IIV-6 plasmid standard. To verify the quality of our extracted nucleic acid, we also determined the copy number of the bee nuclear genome present using a β-actin quantitative assay Invertebrate iridescent viruses have been shown to replicate primarily in the gut walls, Malpighian tubules and fat-bodies of bees Iridovirus genus (GenBank Tax ID: 10487) and a homology search with blastx Following the report of an IIV-6-like virus present in samples from the same colonies surveyed by Cox-Foster et al. in 2007 Iridovirus in healthy and CCD colonies. All four assays were optimized to detect 1–10 DNA copies. We found no evidence of Iridovirus in any of the 163 bees, despite detecting an average of 105–106 DNA copies of β-actin per microliter in these samples. During the initial characterization of IIV-24 in diseased bees (A. cerana), it was estimated that each bee contained an average of 1010–1011 viral particles Iridovirus sequences publicly available and the absence of any nucleic acid or amino acid sequences linked to the Iridovirus identified by mass spectrometry Iridovirus previously associated with disease in bees PCR assays were used to test for the presence of members of Iridovirus in CCD bees. Using an e-value cut-off set at 0.0001, two of the four RNA pools (representing pools of CCD positive samples) did not reveal any sequences with homology to any Iridovirus. The third and fourth pools, comprised of samples from healthy colonies and royal jelly, contained seven reads with low similarity to IIV-6 . Blasting these individual reads against the entire non-redundant protein-coding database revealed a higher similarity to bacterial or bee genomic sequence in all cases . Although these results do not exclude the possibility that Iridovirus sequences may have been present at extremely low concentrations in the 2007 sample set, they do not lend support to a linkage between invertebrate iridescent viruses and CCD.The results of our reanalysis of the metagenomic data from earlier studies of CCD colonies N. ceranae is also required) would also be present at levels high enough for the detection of iridescence. However, during the examination of bee tissues from a large number of CCD colonies, no iridescent qualities were observed (data not shown).Typical signs of infection with invertebrate iridescent viruses include lethargy, early and rapid mortality and in bees, the loss of flight A. mellifera proteins when the host proteome is included in the analysis Admittedly, the possibility exists that the invertebrate iridescent virus detected by mass spectrometry is unrelated to those targeted here, although we consider this to be unlikely. Indeed, it has been suggested that the peptides identified as IIV-6-like by Bromenshenk et al.


During passive motion, the human tibiotalar (ankle) joint behaves as a single degree-of-freedom 1DOF) system DOF syste. In thesA 1DOF Spherical Parallel Mechanism is analyzed, based both on joint anatomy and kinematics: the calcaneal-fibular and tibio-calcaneal ligaments are modelled as binary links of constant length, and relevant bones are connected by a spherical pair centred at the pivot point . GeometrIn all specimens, the model replicated passive motion with a very good precision Figure .The passive motion of the ankle joints can be approximated well by a 1DOF spherical mechanism, despite the simple structure of this model. Replication of the original experimental motion can be a little worse than using previous mechanisms  (Figure


The Anti-Clot Treatment Scale (ACTS) is a 15-item patient-reported instrument of satisfaction with anticoagulant treatment. It includes a 12-item ACTS Burdens scale and a 3-item ACTS Benefits scale. Its role in clinical trials and other settings should be supported by evidence that it is both clinically meaningful and scientifically sound. The aim of the study was to evaluate the measurement performance of the ACTS  in patients with venous thromboembolism based on traditional psychometric methods.ACTS Burdens and Benefits scale data from a large clinical trial (EINSTEIN DVT) involving 1336 people with venous thromboembolism were analysed at both the scale and item level. Five key psychometric properties were examined using traditional psychometric methods: acceptability, scaling assumptions, reliability ; validity ; and responsiveness. These methods of examination underpin the US Food and Drug Administration recommendations for patient-reported outcome instrument evaluation.Overall, the 12-item ACTS Burdens scale and 3-item ACTS Benefits scale met the psychometric criteria evaluated at both item and scale levels, with the exception of some relatively minor issues in the Dutch language version, which were just below reliability criteria . A consistent finding from item-level evaluations of aggregate endorsement frequencies and skewness suggested that response scales may be improved by reducing the number of response options from five to four.Both the ACTS Burdens and ACTS Benefits scales consistently satisfied traditional reliability and validity criteria across multiple language datasets, supporting it as a clinically useful patient-reported instrument of satisfaction with anticoagulant treatment in clinical trials.NCT00440193 Patient-reported outcome (PRO) instruments are rapidly becoming the primary or secondary outcome measures of choice in pivotal clinical trials, research and practice , which mVenous thromboembolism (VTE) encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE) occurs with an incidence rate of 1 to 2 per 1000 persons per annum in Western countries, with two-thirds of cases presenting with DVT . VTE canThe Anti-Clot Treatment Scale (ACTS) is a 15-item patient-reported instrument of satisfaction with anticoagulant treatment. It includes a 12-item ACTS Burdens scale and a 3-item ACTS Benefits scale. The ACTS also includes two additional global questions (see Appendix A). The ACTS was developed based on the original conceptual model of the Duke Anticoagulation Satisfaction Scale (DASS) following a literature review, interviews with experts and patients, and qualitative cognitive debriefing interviews -12. The If the ACTS is to be considered suitable for future measurement of the burdens and benefits of anticoagulation therapy in patients with VTE, it should satisfy stringent criteria as a reliable and valid instrument. This study provides clinical researchers with a comprehensive evaluation of the reliability and validity of the ACTS using traditional psychometric methods in line with current guidelines.Bayer Pharma AG provided anonymised, blinded ACTS Burdens and Benefits scale datasets from EINSTEIN DVT, a large clinical trial involving patients with acute symptomatic DVT treated with rivaroxaban or enoxaparin/VKAs . The incPatients were asked to complete a questionnaire booklet containing the ACTS and Treatment Satisfaction Questionnaire for Medication version 2 (TSQM II) during follow-up visits. The measurement performance of the ACTS Burdens and Benefits scales was evaluated in the following languages: Dutch, Italian, French, German and English, and then a pooled dataset of all language versions. In this paper, the scale-level analyses for the separate study/language versions  and both item and scale-level analysis for the pooled language versions datasets  are presented. Further information is available from the authors.The ACTS is a 15-item, patient-reported measure of satisfaction with anticoagulant treatment. It includes 12 items that assess the burdens of anticoagulant treatment and three items that assess the benefits of anticoagulant treatment. Patients are asked to rate their experiences of anticoagulant treatment during the past 4 weeks on a 5-point scale of intensity . The ACTS Burdens total score ranges from 12 to 60, and the ACTS Benefits total score ranges from 3 to 15. When used in clinical research, it is recommended that the ACTS Burdens scores are reverse-scored so that higher ACTS Burdens and Benefits scores indicate greater satisfaction with treatment. For the purposes of this psychometric evaluation, however, the original raw score data were analysed. French, Dutch, Italian, German and English language versions of the ACTS were created previously in accordance with a standard protocol to achieve conceptual equivalence in the translation, including: forward/backward translation, reconciliation, review and pilot testing . FurtherFor validation purposes, the TSQM II was also included. This is an 11-item PRO instrument that assesses patient satisfaction with treatment. It includes four scales: two items that assess the effectiveness of treatment (TSQM II Effectiveness), three items that assess side-effects (TSQM II Side-effects), three items that assess convenience of treatment (TSQM II Convenience) and two items that assess global satisfaction  . PatientPsychometrics is a well-established scientific field that is concerned with the measurement of subjective judgements using numerical scales and the evaluation of the measurement properties of such scales . The most widely used methods for evaluating measurement performance are known as ‘traditional’ psychometric methods . TraditiBased on data collected, the following psychometric properties of the ACTS were examined: acceptability ; scaling assumptions; internal consistency reliability and test-retest reproducibility; aspects of validity ; and responsiveness. Table The EINSTEIN DVT ACTS validation dataset included 1336 patients (96% response rate) at day 15 (average age was 57 years [standard deviation (SD) 16] and 42% were female). Across each of the questionnaire language versions, this comprised: Dutch ; Italian ; French ; German ; and English . The pooled EINSTEIN DVT dataset, including all language versions, comprised 1336 patients . Overall, patients participating in the treatment satisfaction sub-study had similar baseline characteristics to the full EINSTEIN DVT trial population (a full description of patient demographics is presented elsewhere).For each language version, there was a low level of missing data for all item and scale scores for both the ACTS Burdens and ACTS Benefits . This means that scale scores could be computed for >95% of patients. There was a reasonable distribution of ACTS Burdens scores , and an excellent distribution of ACTS Benefits scores (mean 100%). Floor and ceiling effects were generally low for both scale scores  and data skewness was slightly higher for the ACTS Burdens scores than for the ACTS Benefits scores  , with the exception of the Dutch version . The homogeneity coefficient mean ranged from 0.24 to 0.75 . Therefore, scale scores could be computed for >96% of patients, which was slightly higher than the individual country analysis owing to the effect of pooling the datasets. At the scale level, there was a good distribution of ACTS Burdens scores (77%) and an excellent distribution of ACTS Benefits scores (100%). Scale-level floor and ceiling effects were generally low for both scale scores (range 0–11%). Data skewness was slightly higher for the ACTS Burdens scale scores than for the ACTS Benefits scale scores . At the item level, the ACTS Burdens scale ceiling effects ranged from 37% to 77%. In both datasets, in relation to aggregate endorsement frequencies, for all items, three of five response categories met the >10% criterion, but two of five response categories were <10% (between response categories 4 and 5) and 10 of 12 items fell outside the skewness criterion . The ACTS Benefits scale had much lower ceiling effects, which ranged from 15% to 19%. In relation to aggregate endorsement frequencies, for all items, three of five response categories met the >10% criterion, but two of five response categories were <10% (between response categories 4 and 5) and all items passed the skewness criterion . This indicated that items in each scale measured a common underlying construct and contained a similar proportion of information. In addition, principal components analysis factor loadings >0.48) and tests of item convergent/discriminant validity (>0.39) supported this finding, thus further indicating that all items in each of the scales passed the criteria , Cronbach’s alpha (>0.85) and test-retest intraclass correlations (>0.86) for both scales in both datasets passed the criteria, supporting their reliability . Responsiveness statistics comparing day 15 scores with all the other time points individually supported a trend of increasingly higher ACTS Burdens and Benefits scales scores over time, with low but increasing effect size statistics   . ScalingFirst, construct validity analyses in the form of testing hypothesised correlations between the ACTS Burdens scale and four scales of the TSQM II were supported in the pooled dataset but were slightly lower than expected. This issue may reflect the fact that, although both measures focus on treatment satisfaction, the constructs captured by the ACTS and TSQM II are more distinct than would be first expected. On closer inspection, the TSQM II items that capture ‘Effectiveness’, ‘Side-effects’, ‘Convenience’ and ‘Global satisfaction’ are significantly different from the ACTS Burdens and Benefits items. Thus, despite some overlap between the two instruments, there are key differences – for example, the TSQM II Effectiveness scale has one of two items addressing symptom alleviation, not directly impacted by anti-coagulation or measured by ACTS. Furthermore, the TSQM II Side-effects items do not directly address the important anticoagulation-specific side-effects of bleeding or bruising, which are measured in ACTS Burdens items. In addition, the TSQM II is more narrowly targeted at the medicine, the ACTS being more inclusive of the services and difficulties of undergoing anticoagulation therapy. Thus, the findings from the analyses should be interpreted with these facts in mind.The second issue that requires further exploration is that, across all language versions, findings from item-level tests of aggregate endorsement frequencies and skewness suggested that response scales may be improved by reducing the number of response options from five to four. This is also reflected in the findings from the scale-level targeting, which also revealed slightly skewed distributions across the board. One potential cause for this is that there may simply have been too many response options for respondents to discriminate between, especially at the more satisfied extreme of the choices. This is not uncommon in PRO instruments  and it hThe responsiveness analyses, which can be considered to be preliminary, revealed a modest but stepwise increase in ACTS Burdens and Benefits scale scores over the six time points. The associated responsiveness statistics were moderate but were in the range that would be expected clinically. This is because scale responsiveness and treatment effectiveness are inseparably linked ,30. The a priori clinically anchored hypotheses. The second limitation is that the small size of the English-language versions  required that these be combined prior to psychometric analyses. Further testing using adequate samples in each country would provide useful additional evidence for the relative measurement performance of the ACTS scales.Our study has two key limitations. First, although the scope of our psychometric evaluation of the ACTS in patients being treated for acute DVT was relatively comprehensive, there are further analyses that would aid our understanding of the measurement performance of the ACTS Burdens and ACTS Benefits scales. These would include further known groups and discriminant validity tests based on clinically sensible sub-grouping (based on predefined hypothesis-driven selection) and responsiveness analyses assessed against Overall, across the different language versions, evidence from the psychometric evaluation of the ACTS in patients treated for acute DVT supports the use of the 12-item ACTS Burdens scale and the 3-item ACTS Benefits scale. The ACTS can provide essential information about VTE-related treatment satisfaction from the patient’s perspective. This new instrument will complement current clinical outcome measures and facilitate multicentre studies for comparison of treatments and patient populations. Thus, the ACTS has the potential to support treatment trials, cost-effectiveness analysis and patient education, providing crucial data for clinical researchers, clinicians and patients. Further potential psychometric research on the ACTS includes exploring reducing the number of response options from five to four, providing evidence that both ACTS Burdens and Benefits scales are able to detect a clinically meaningful change over time and exploring the potential of using the scales in clinical practice settings.During the past 4 weeks…bleeding as a result of anti-clot treatment limit you from taking part in vigorous physical activities? .1 How much does the possibility of 2 How much does the possibility of bleeding as a result of anti-clot treatment limit you from taking part in your usual activities? .bruising as a result of anti-clot treatment?3 How bothered are you by the possibility of avoid other medicines (e.g. aspirin) as a result of anti-clot treatment?4 How bothered are you by having to limit your diet? .5 How much does anti-clot treatment daily aspects of anti-clot treatment? .6 How much of a hassle (inconvenience) are the occasional aspects of anti-clot treatment? .7 How much of a hassle (inconvenience) are the and occasional aspects of your anticoagulation therapy during the past 4 weeksNow I want to ask you about daily difficult is it to follow your anti-clot treatment?8 How time-consuming is your anti-clot treatment?9 How worry about your anti-clot treatment?10 How much do you frustrating is your anti-clot treatment?11 How burden is your anti-clot treatment?12 How much of a negative impact has your anti-clot treatment had on your life?13 Overall, how much of a confident are you that your anti-clot treatment will protect your health? 14 How reassured do you feel because of your anti-clot treatment?15 How satisfied are you with your anti-clot treatment?16 How positive impact has your anti-clot treatment had on your life?17 Overall, how much of a ACTS: Anti-Clot Treatment Scale; DASS: Duke Anticoagulation Satisfaction Scale; DVT: Deep vein thrombosis; FDA: US Food and Drug Administration; PE: Pulmonary embolism; PRO: Patient-reported outcome; SD: Standard deviation; TSQM II: Treatment Satisfaction Questionnaire for Medication version 2; VKA: Vitamin K antagonist; VTE: Venous thromboembolism.LB is an employee of Bayer Pharma AG. SC, DL and SS were supported in part through a grant from Bayer Pharma AG.SC conducted, analysed and interpreted the data and wrote the manuscript. DL was involved in guiding the study, including design and analysis of data. LB and SS were involved in providing additional input and reviewing drafts of this manuscript. All authors read and approved the final manuscript.Copyright of the ACTS instrument is held by Bayer AG, Germany (2006). All rights reserved. For information on or permission to use, please contact Mapi Research Trust; http://www.mapi-trust.org.