The increasing prevalence of diabetes and costly long-term complications associated with poor glycemic control are issues facing health services worldwide. Diabetes self-management, with the support of health care providers, is critical for successful outcomes, however, frequent clinical contact is costly. Text messages via short message service (SMS) have the advantage of instant transmission at low cost and, given the ubiquity of mobile phones, may be the ideal platform for the delivery of diabetes self-management support. A tailored text message-based diabetes support intervention called Self-Management Support for Blood Glucose (SMS4BG) was developed. The intervention incorporates prompts around diabetes education, management, and lifestyle factors , as well as blood glucose monitoring reminders, and is tailored to patient preferences and clinical characteristics.To determine the usability and acceptability of SMS4BG among adults with poorly controlled diabetes.Adults (aged 17 to 69 years) with type 1 (n=12) or type 2 diabetes (n=30), a hemoglobin A1c (HbA1c) over 70 mmol/mol (8.6%), and who owned a mobile phone (n=42) were recruited to take part in a 3-month pilot study of SMS4BG. At registration, participants selected the modules they would like to receive and, where appropriate, the frequency and timing of blood glucose monitoring reminders. Patient satisfaction and perceptions of the usability of the program were obtained via semistructured phone interviews conducted at completion of the pilot study. HbA1c was obtained from patient records at baseline and completion of the pilot study.Participants received on average 109 messages during the 3-month program with 2 participants withdrawing early from the study. Follow-up interviews were completed with 93% of participants with all reporting SMS4BG to be useful and appropriate to their age and culture. Participants reported a range of perceived positive impacts of SMS4BG on their diabetes and health behaviors. HbA1c results indicated a positive impact of the program on glycemic control with a significant decrease in HbA1c from baseline to follow-up.A tailored text message-based intervention is both acceptable and useful in supporting self-management in people with poorly controlled diabetes. A randomized controlled trial of longer duration is needed to assess the efficacy and sustainability of SMS4BG. Globally, diabetes is a significant health issue with increasing incidence worldwide. There is a disproportionate burden of the disease among indigenous peoples internationally  and in NMobile health  is the use of mobile devices, including mobile phones, to deliver health services and information . The fieRecent systematic reviews show that the majority of SMS-based behavior change interventions for disease management have positive short-term impacts on behavioral and clinical outcomes ,12,13. TA 3-month, nonrandomized pilot study was conducted between July and December 2013. All study documents and procedures were approved by the Health and Disability Ethics Committee (13/NTA/55).Eligibility criteria included adults aged 16 to 70 years, a diagnosis of type 1 or type 2 diabetes mellitus, hemoglobin A1c (HbA1c) >70 mmol/mol (8.6%) within the last 12 months, mobile phone ownership, ability to provide informed consent, and ability to read English. An HbA1c result of greater than 70 mmol/mol (8.6%) was utilized as the definition of poorly controlled diabetes in this study, a level associated with increased risk for the development of diabetes complications. Recruitment was carried out across three primary health care practices, two secondary care hospitals, and one community-based organization in Auckland, New Zealand. Clinicians at each site identified potential participants and either enrolled them directly through the study website or referred them to a research assistant to complete registration. Registered participants then received an automated consent text message and were required to reply “Yes” to be enrolled in the program. The program was free to receive but if a participant replied, they were charged NZD $0.20 per message by their network provider. Participants were given a voucher (NZD $20.00) at the conclusion of the study to reimburse them for their time and any costs associated with replying to the messages.At the end of the program all participants (including those that withdrew) were asked to complete questions about their satisfaction with the program, its usefulness and usability, and perceived positive impacts, via a semistructured telephone interview conducted by a research assistant. Engagement with the program was assessed using system-recorded responses to the blood glucose monitoring reminder messages. In addition, participants consented to the research team obtaining their HbA1c test results from their medical records to assess change in HbA1c from baseline to follow-up.t tests were used. Change in HbA1c was calculated using the related-samples Wilcoxon signed-rank test.Descriptive statistics were generated for baseline demographic and clinical characteristics, and measures of engagement with the system. Counts and percentages were reported for categorical variables, and means and standard deviations for continuous variables. To determine whether ratings of usefulness differed between ethnic groups and diabetes type, SMS4BG was developed to provide self-management support for adults with poorly controlled diabetes. The content was developed by a multidisciplinary team, led by a health psychologist (RD) and public health physician (RW). The development followed the mHealth Development and Evaluation framework , which pThe development of the content was informed by a review of the research literature, existing mHealth interventions (targeting diabetes management and related lifestyle behaviors), and current patient resources. The program was informed by two behavior change theories: Social Cognitive Theory  and the To accommodate personal preferences and clinical characteristics, SMS4BG was made up of modules including a core module that all participants received and additional optional modules. Clinician input determined optional module topics. The core module consisted of 2 messages per week on diabetes education, emotional encouragement, and illness perceptions (available in Māori and non-Māori versions). In addition to the core module, if registered as a smoker, the participant received an additional 1 message per month supporting smoking cessation. Participants could also opt to receive additional modules on topics relevant to diabetes management such as insulin, diet, exercise, stress management, and blood glucose monitoring reminders. A summary of the different SMS4BG modules can be seen in The SMS4BG program was designed so that text messages were send-only  with the exception of the blood glucose monitoring reminders, which provided the option for participants to reply with their blood glucose test results. In addition to SMS, there was an accompanying website that patients and clinicians could log onto, allowing them to review a graphical display of the participant’s blood glucose responses sent into the system. The website also provided administrators with the ability to manage the message content and monitor message delivery. To enhance participant engagement, SMS4BG was personalized with the inclusion of each participant’s name in many of the messages. Individuals could also select the frequency and timing of blood glucose monitoring reminder messages—from 1 per week to up to 4 per day.In New Zealand, there is a higher prevalence of diabetes in Māori in comparison with New Zealand Europeans . To ensuOnce developed, messages were reviewed by diabetes specialists and a selection of messages were pretested by people with diabetes. Feedback from this process was incorporated into the messages before they were finalized and entered into the system for testing. Following development, a pilot study was conducted which set out to assess the usability and acceptability of the text message support program in adults with poorly controlled diabetes.A total of 44 potential participants were recruited, with 42 participants consenting to participate. Due to the choice of modules, participants received varying numbers of messages during the 3-month program. Participants received on average 109 (range 8 to 437) text messages from the program during the 3-month period, with an average of 13 messages per week. This included on average 63 (range 8 to 93) send-only text messages per participant over the 3-month program. A total of 34 participants out of 42 (81%) opted to receive blood glucose monitoring reminders, receiving on average 58 (range 9 to 353) reminder messages each over the 3-month period. A total of 827 response messages were received from 26 (76%) of the 34 participants registered to receive reminders. Of those who responded to at least one reminder, participants on average responded to 57% of their reminder messages (range 1 to 99%). For those 8 participants that did not reply , cost was identified as the leading barrier. Only 4 (12%) of the 34 participants reported accessing their graph online to view their blood glucose results. The most frequently reported barriers were no access to computers or Internet and not responding to the messages, and as a result not having a graph to view.not at all useful) to 5 (extremely useful), the mean rating was 3.94 (SD 0.98). Higher mean ratings of usefulness were seen in those with type 2 diabetes  compared to those with type 1 diabetes  (P=.004). Although not statistically significant, higher ratings of usefulness were found for Māori  compared with New Zealand European  (P=.23).A summary of the results of the follow-up interviews is provided in All participants were able to identify at least one positive impact of the program. The majority  of participants reported that the program had a positive impact on their overall blood glucose control. In addition, 49% (19/39) of all participants interviewed reported a positive impact of SMS4BG on their exercise habits, 59% (23/39) on their diet and eating behavior, and 67% (26/39) on their mood. Of the participants interviewed who received the exercise lifestyle module, 83% (10/12) reported a positive impact of SMS4BG on their exercise habits. Of those interviewed who received the healthy eating module, 82% (9/11) reported a positive impact on their diet and eating behavior. Of those interviewed who received the stress and mood module, 67% (6/9) reported a positive impact on their mood. Of those 10 who were registered as smokers, 3 (30%) participants reported that they had quit smoking during the program.Suggestions for improvements in the program included making the program longer, allowing for two-way communication with health care professionals through the program, making it free to reply to the messages, allowing for greater choice in the timing of the messages, and greater personalization. Few technical issues were reported—of the 39 participants interviewed, 2 (5%) reported issues accessing their graph, and 8 (21%) participants reported that not having credit/money on the phone account meant they could not reply with their blood glucose test results.P=.001) for the 26 participants out of 42 (62%) for whom complete data was available.Baseline HbA1c values were obtained for all participants, but follow-up results were only available for 26 (62%) of the 42 participants. A significant improvement in HbA1c was found from baseline (median 89.50 mmol/mol) to follow-up , Pacific  or Asian decent . This indicates that this type of technology is not limited by demographic characteristics and the text message content was relevant to a wide range of people with poorly controlled type 1 and type 2 diabetes.Most participants were satisfied with the number and frequency of the messages they received, which may be due to participants being involved in the selection of the modules they received and, therefore, having some degree of control over the number of messages they received.Although visual feedback was provided in the form of a graph of submitted blood glucose results, this feature was not utilized by the majority of participants. The leading barrier for not accessing the graph was lack of Internet access either at home or on their mobile phones. Previous studies have reported greater improvements in HbA1c with combined mobile and Internet-based interventions compared to studies utilizing mobile intervention alone . Our finStrengths of the SMS4BG program included that it was theoretically informed, system initiated (ongoing intervention not dependent on participant behaviors), personally tailored, and provided participant choice. Many previous diabetes text messaging programs have had limited reach or were designed specifically for one diabetes type, age group, or single diabetes management behavior. SMS4BG was designed for adults of all ages with both poorly controlled type 1 and type 2 diabetes and provided support for self-management and encouragement in people’s everyday lives rather than focusing on specific diabetes-related tasks. In addition, SMS4BG utilized simple technology and, therefore, had less potential for technical issues that have been a limitation in previous mHealth studies.Another strength of the current study was the inclusion of an indigenous version. With a higher prevalence of diabetes seen in Māori compared to NZ Europeans , diabeteThis study had several limitations, including the absence of a control group and a small sample size. Although positive change in glycemic control was seen without a control group or adequate sample size, this difference must be interpreted with caution. The lack of complete follow-up HbA1c results limits the generalizability of the improved glycemic control results. The target population (poorly controlled) were likely not attending medical appointments as regularly as guidelines state and, therefore, the lack of clinical results could be expected. Future studies could include text messages around the importance of HbA1c tests and reminders to go for tests as a way of potentially overcoming this issue. The pilot study was of short duration and as diabetes is a condition requiring long-term management, longer interventions may be more appropriate. Another limitation was the lack of follow-up to assess whether any effects of SMS4BG were maintained beyond the program itself. A larger and longer-term randomized controlled trial will need to be carried out to establish the efficacy of SMS4BG on self-management behaviors, self-efficacy and clinical outcomes, and its sustainability and cost-effectiveness.The current study adds to the evidence for the use of mHealth in delivering personally tailored diabetes self-management support and, particularly, the use of text messaging as a medium of delivery. The positive pilot study results indicate that this type of broad reaching EMI could be successful in engaging adults with poorly controlled type 1 or type 2 diabetes and assisting with improved diabetes self-management. Further refinement of SMS4BG is needed based on the pilot study feedback, followed by a larger randomized control trial to determine its efficacy.


Traditional cancer treatments have centered on cytotoxic drugs and general purpose chemotherapy that may not be tailored to treat specific cancers. Identification of molecular markers that are related to different types of cancers might lead to discovery of drugs that are patient and disease specific. This study aims to use microarray gene expression cancer data to identify biomarkers that are indicative of different types of cancers. Our aim is to provide a multi-class cancer classifier that can simultaneously differentiate between cancers and identify type-specific biomarkers, through the application of the Binary Coded Genetic Algorithm (BCGA) and a neural network based Extreme Learning Machine (ELM) algorithm.BCGA and ELM are combined and used to select a subset of genes that are present in the Global Cancer Mapping (GCM) data set. This set of candidate genes contains over 52 biomarkers that are related to multiple cancers, according to the literature. They include APOA1, VEGFC, YWHAZ, B2M, EIF2S1, CCR9 and many other genes that have been associated with the hallmarks of cancer. BCGA-ELM is tested on several cancer data sets and the results are compared to other classification methods. BCGA-ELM compares or exceeds other algorithms in terms of accuracy. We were also able to show that over 50% of genes selected by BCGA-ELM on GCM data are cancer related biomarkers.We were able to simultaneously differentiate between 14 different types of cancers, using only 92 genes, to achieve a multi-class classification accuracy of 95.4% which is between 21.6% and 38% higher than other results in the literature for multi-class cancer classification. Our findings suggest that computational algorithms such as BCGA-ELM can facilitate biomarker-driven integrated cancer research that can lead to a detailed understanding of the complexities of cancer.The online version of this article (doi:10.1186/s12859-015-0565-5) contains supplementary material, which is available to authorized users. Somatic or genetic mutations in key regulatory genes may cause the molecular machinery to lose control over the regulation of cell proliferation, differentiation and death that can in turn lead to clonal proliferation, causing cancer. Identification of cancer through morphological features of tumor cells has serious limitations since similar histopathological appearances can imply various clinical and risk conditions. Recent studies in cancer genomics have created a body of knowledge that has facilitated better understanding of the complexities of cancer. Advances in molecular diagnostics have helped to make cancer classification that is more objective and precise. The complexity of cancer can be coded in terms of underlying principles that determine the transformation of normal cells to cancer cells ,2.Biomarkers are measured characteristics of biological conditions that can indicate favourable or adverse conditions present in cells. Advances in cancer research have revealed that mutational oncogenes and tumor suppressor genes are molecular markers characteristic of cancer. The application of computational methods to identify biomarkers that encode these cancer causing changes can provide clinicians with a valuable tool that could lead to advances in the understanding, treatment and prognosis for cancer.Microarray data typically consists of thousands of gene features with only a few hundreds of samples. Computational biologists have applied Genome wide association studies using advanced statistical and bioinformatics techniques to better understand the etiology of cancer. Several studies in gene selection and classification methods have used the frequently used Global Cancer Mapping (GCM)  microarrIn our previous publication, an integer coded genetic algorithm and Extreme Learning Machine (ICGA-ELM)  multiclasimultaneously classify different types of cancers accurately and to help identify biomarkers. The Binary Coded Genetic Algorithm (BCGA) combined with the neural network based Extreme Learning Machine (ELM) is used to obtain high classification accuracy. BCGA-ELM is tested primarily on the GCM data set along with several other cancer data sets. These results are compared to popular classification methods using the Weka software [The objective of this study is to select the best set of features (genes) that can software . BCGA-ELsoftware ,2.Several multi-class and binary class microarray data sets are used in this study. Global Cancer Map (GCM) is primarily used in this study to illustrate the capabilities of the BCGA-ELM algorithm in selecting cancer related biomarkers and in obtaining high classification accuracy. Other cancer data sets are included in this study to show the robustness and generalization capabilities of BCGA-ELM in selecting meaningful biomarkers that can achieve high accuracy, irrespective of the algorithms that are used for classification.GCM is an oligonucleotide microarray data obtained from solid tumors of epithelial origin . GCM datIngenuity Pathway Analysis (IPA®) is used to identify biomarkers among the selected candidate genes for four data sets  and the fast learning Extreme Learning Machine (ELM) ,22. The Genetic algorithm (GA) was developed  to desigThe solution for our gene selection problem is coded as a binary string of length 16063, representing the total number of genes. A '0' in the string indicates exclusion of the gene in that position and a '1' represents inclusion of the gene  is computed using Equation 1. The final number of genes selected (92) is determined by the number of genes present in the solution set with the highest accuracy. A 20-fold cross-validation of the chosen gene set (represented by binary string) may guarantee a stable and robust solution for gene selection.The data are divided into training (75%) and testing (25%) sets randomly. ELM classifier is used to compute training and testing accuracies. Random splitting and classification is processed 20 times on each of the 200 binary strings. Fitness value f = 1 and d = 0.98 in Equation 1. The process of selecting the best genes continues during successive generations until the termination (convergence) criterion (maximum number of generations or maximum accuracy) is satisfied. In our experiments we use the following settings for GA: crossover probability 80%, mutation probability 20%, selection probability for normalized geometric ranking method is q = 1% over 50 generations. Through many iterations and evaluations, we arrive at a smaller set of 92 genes that satisfies our objective to obtain high accuracy.In our experiments we use ωc : xt → yt as accurately as possible. A comprehensive description of the ELM algorithm is given in [i,Yi), select the appropriate activation function and number of hidden neurons.Given training samples and class labels  for estimating the class label in the test set.Use the calculated weights . This is shown to influence the generalization performance ,15 of thY is the observed class value and T is the calculated output value of the class, for a given set of hidden neurons H and input parameters V and b. The best weights and bias values for the ELM can be found using search techniques and optimization methods that are not very computationally intensive. These parameters are stored and used later on to determine the class of new samples.where In this paper we display an overall accuracy as a general measure of method performance. Overall accuracy is a ratio of number of correctly classified samples to total number of available samples.The BCGA-ELM algorithm selects the minimum set of 92 candidate features (from GCM data) that have the best discriminatory power to differentiate between 14 types of cancers, with 95.4% accuracy . Figure An in-depth, insilico analysis of this data using IPA® and iReport® show some interesting results. This analysis indicates that over 52 of the 92 genes are determined to be significantly differentially expressed genes (DEGs). Figure simultaneous multi-class classification of the data and gives high classification accuracy. The minimum number of genes required by each method to achieve maximum generalization performance is also given.Table From Table The accuracy for multiclass data sets Breast and Leukemia, with 30 and 11 features respectively, are 100% for BCGA-ELM and for the Weka algorithms (with a single exception for Leukemia which is 97.1% under Naive Bayes). The results are lower by 33.3% and 6.7% for HC-k-TSP and mul-PAM respectively (between 5 and 27 features) for Breast cancer while they are lesser by 2.9% for Leukemia. For Lymphoma (using 27 features), BCGA-ELM achieves 100% while the Weka packages yield between 72.6% and 93.64%. The lowest results are for Naive Bayes, which seems to be the general pattern for all data sets. We have given comparative results for other methods in the literature only when they are clearly stated as multi-class computations.For the six binary data sets  BCGA-ELM achieves 100% classification accuracy for all these sets, with reduced features ranging between 11 and 92 genes  to hallmarks of cancer (HC). To our knowledge, we have not seen such a large selection of biomarkers present in the candidate set of genes selected from the GCM dataset features . The remaining 40 genes, other than the 52 biomarkers that were identified by IPA® and iReport®, may be investigated further to determine if they are related closely to the pathogenesis of cancer. Similarly Table Clinical and histopathological data are generally used to establish the diagnosis and treatment of cancer patients. Under difficult or advanced disease conditions, these data are not sufficient to make clear diagnosis or propose treatments. According to Hanahan and Weinberg ,2, thereIngenuity Pathway Analysis® (IPA) and iReport® have identified 52 differentially expressed genes (DEGs), out of the 92 genes selected by BCGA-ELM, as known biomarkers that are closely related to the six hallmarks of cancer. This type of information can be used for the diagnosis and treatment of cancer. The expression changes were interpreted in the context of pathways, biological processes, disease phenotypes and molecular interactions. These hallmarks include cell processes such as proliferative signalling (HC1), developing resistance to cell death (HC2), immortalizing cells through replication (HC3), promoting growth of new blood vessels (vasculogenesis) to sustain tumors (HC4), invading healthier tissues (HC5) and promoting spread of cancer to other parts of the body (HC6). These activities include self-sufficiency in growth signal, insensitivity to anti-growth signals, tissue invasion and metastasis, limitless replicative potential, sustained angiogenesis and evading apoptosis.Figure Figure For the longest time traditional treatments for cancer centered on cytotoxic drugs and adjuvant therapies lacking precision to treating particular cancers; however, there is a tremendous shift towards creating therapies focusing on molecular targets that are rationally designed, aimed to bring greater efficacy with less harmful side effects.The proposed BCGA-ELM selects a minimum of 92 target genes (GCM) with a testing accuracy of 95.4%, which is between 21.6% and 38% higher than other results in literature for multi-class cancer classification. The molecular targets as identified in this study by the BCGA-ELM based multi-class algorithm has been shown to be reflective of the hallmarks of cancer [We hope that the BCGA-ELM algorithm can facilitate biomarker-driven integrated cancer research that can lead to a detailed understanding of the complexities of cancer. This understanding can lead to the development of drugs that are specific to each type of cancer that might be tailored to the needs of individual patients, leading to personalized medicine.


Retroviral DNAs are profoundly silenced at the transcriptional level in embryonic cell types. The transcriptional profile of pluripotent stem cells has been demonstrated to be extremely heterogeneous from cell to cell, and how the silencing of retroviral DNAs is achieved is not yet well characterized.In the current study, we investigated the transcriptional silencing dynamics in stem cells by independently monitoring the expression of two Moloney murine leukemia virus (MMLV) retroviral vectors newly introduced into embryonic carcinoma (EC) cells. Although MMLV is efficiently silenced by epigenetic mechanisms in most such cells, a small number of the doubly-transduced EC cells transiently show double-positive proviral expression. These cells were sorted and their expression patterns were studied over time as silencing is established.Our data suggest that retroviral silencing occurs stochastically, in an individual locus-specific fashion, and often without synchronous silencing of both viruses in the same cells. Surprisingly, the chromatin modifications that mark the silenced proviruses are unchanged even in cells that temporarily escape silencing. This local silencing effect is a feature of stem cell epigenomic regulation that has not previously been revealed. Developmental programs are executed by the tightly controlled and temporally coordinated transcriptional regulation of large sets of genes. As cells move through developmental stages during embryogenesis, groups of genes are often synchronously turned on and off to induce specific changes in cell phenotype and in the capabilities needed for tissue and organ formation. This gene regulation is mediated by trans-acting transcription factors, and is accompanied by long-lasting alterations in chromatin folding, histone modifications, and DNA methylation at the genomic regions of the genes being regulated. While the sets of coordinately regulated genes may sometimes be genetically linked at a single chromosomal region, they are most often unlinked and dispersed at many disparate locations on many chromosomes. In these cases the trans-acting machinery must find and act on the multiple loci in a coordinated, synchronous manner. The timing of these regulatory events can be tightly synchronous, but there is some inherent noise or variability in the regulatory machinery, and thus there can be considerable cell-to-cell fluctuation in gene expression . As a reRetroviral DNAs provide a unique tool for the analysis of gene expression because they can be introduced into the genome at will by infection, and because they are introduced as “naked”, unmodified, newly synthesized DNA that is assembled into chromatin by the addition of new nucleosomes. Vector genomes that express reporter genes are powerful tools to monitor regulation of transcription in infected cells . RegulatIn previous studies we showed that although most ES cells silence the incoming retroviral DNA, a small subpopulation of infected cells (‘escapees’) evade silencing . When soTo monitor the stability of retroviral restriction in embryonic cells, two MMLV viral vectors, one containing a GFP gene and one an mCherry reporter gene, were separately packaged into virus particles, and the viruses were then mixed and used to co-infect F9 embryonic carcinoma (EC) cells at a multiplicity of infection (MOI) of approximately 2. Cells of the F9 line, originally isolated from a murine teratocarcinoma ,23, are Fluctuations in retroviral silencing have been reported before for endogenous  and exogper se may have been lost in these cells.In an attempt to isolate a minor population of cells that displayed a global and stable escape from silencing, we sorted the initial double positive population from infection with PBSpro virus a second time, 22 days after the initial sort Figure A. This tTo determine the chromatin state of the active loci, we analyzed the genomic regions of the five twice-sorted cell populations by chromatin immunoprecipitation (ChIP) for active H3K4me4) and suppressive  histone modifications. In F9 cells sorted as double positive in the first sort and double negative in the second sort, the proviral sequences were highly enriched for H3K9me3 Figure A and H3KK4me4 andTo determine whether the stochastic changes in viral expression were correlated with changes in the chromatin state, the one-color expressing cell populations were analyzed by chromatin immunoprecipitation (ChIP). As these cells were sorted as double-positive in the first sort, each cell contains at least one of each provirus, which was at least transiently expressed. After 22 days in culture, many of these cells had silenced one proviral copy but not the other one. Expression of the positively-sorted allele was not stable and was silenced after several weeks Figure D and E. cis-acting nature of the suppression seen in these cells is reminiscent of the cis-acting control of expression seen in the course of silencing of MLV vectors introduced in successive infections [The experiments above show that embryonic cell populations that have silenced most proviruses still contain some cells that transiently transcribe the viral DNAs at low frequencies, and then quickly shut them off. We can recover such expressing cells by sorting, and we can watch the kinetics of the shutoff on a cell-by-cell basis. The results suggest that the silencing of retroviruses in these cells is most often a stochastic event mediated through local changes occurring independently at each provirus, and is not mediated by a global change in the state of the cell that acts simultaneously in a coordinated way on all the proviruses in a cell. This characteristic seems to be true whether the cells utilize the highly efficient PBSpro-directed machinery, or the less efficient PBS-independent machinery. The local, fections ,27.EC cells have long been known for their ability to silence incoming viral DNAs, first documented for the silencing of the papova viruses . This beIt was previously shown that genes expressed at low levels tend to be bound by fewer transcriptional regulators, both activators and repressors, which results in high cell-to-cell variability . This vaThe status of the histone modifications and DNA methylation at the proviruses suggests that our EC cells typically mark the incoming viral DNAs for silencing soon after infection. Even those cells selected as transiently double-positive and single-positive, as well as double-negative, contain proviral DNAs exhibiting the chromatin and DNA marks of silent genes. Thus, the brief expression of the proviruses in these cells occurs in spite of these marks, and relatively quickly responds to the marks by shutting down. It is also possible that because most cells contain more than one copy of each virus, and because only one copy in the double-positive population may be expressing at any given moment, the silencing chromatin marks that we observe are present on the silent alleles. The expressed alleles thus may be at least partially depleted of the silencing marks, and this loss may be obscured by the silent alleles.trans, and that the expression of both GFP and mCherry vectors is not solely a consequence of their integration sites. We surmise that the cell sorting regimen has selected for a cell population that has stably lost some components of the silencing system. Further analysis of the repertoire of expressed genes in these cells may ultimately allow for identification of the basis of their permissivity.The rare population of cells that are stable expressors of both GFP and mCherry, recovered as high expressers after two sorts separated by many days in culture, are unusual escapees of the silencing process. These cells may have been selected to have proviral integrations at special loci. They may be similar to an EC line described by the Jaenisch lab, isolated after selection for expression of a marker gene delivered by a vector with a proline PBS . It is nThe silencing of newly introduced retroviral genomes in embryonic cells is achieved by the recognition of the viral DNAs by transcription factors and chromatin modifying machinery. This machinery acts on the independently integrated proviruses in a temporally asynchronous manner, such that each provirus can be silenced without strict coordination with another provirus in the same cell. The findings suggest that there is a stochastic aspect to the determination of the chromatin modifications that are imposed on each provirus. Rare clones in which more profound escape from silencing arise by more dramatic global changes in the embryonic cell state.F9 and NIH3T3 cells were cultured in DMEM with 10% FBS, 2 mM glutamine, 1000 U/mL penicillin, 100 mg/mL Streptomycin. F9 cells were cultured on gelatinized tissue culture plates. All cells were cultured at 37°C in 5% CO2. Viruses were prepared as previously described using pNCA-GFP/mCherry vectors  pseudoty-DDCT. The copy number values given were obtained by averaging results from three PCR reactions. Uninfected NIH 3 T3 cells were used as negative control. In the flow analysis results presented, all numbers were normalized to the infection efficiency as seen by the % of NIH3T3 expressing cells. The numbers are given as GFP-positive F9 cells/GFP-positive NIH3T3 cells x100; this is to correct for variation in multiplicity with different viruses.The difference in threshold cycle (CT) values (DCT) between Q-PCR assays using two of the primers on the pro-virus, 40NT and PBS, was used to monitor proviral DNA copy number. The Gapdh primer set for the genomic DNA was used to normalize the amount of genomic DNA. A one copy number standard was established by infecting NIH 3 T3 cells at a very low multiplicity of infection (MOI) with the MLV-GFP vector and sorting the GFP(+) cell population , ensuring that a single copy of GFP virus was present . The ratGFP-positive cells were isolated on a cell sorter . Data were acquired on an automated cell analyzer  and analyzed with FlowJo software (Treestar).https://www2.appliedbiosystems.com). See Additional file Bisulfite conversion of genomic DNA was carried out using the Zymo EZ DNA Methylation-Gold™ Kit. PCR primers were designed using Methyl Primer Express software version 1.0  and then purified using QIAquick PCR purification kit (Qiagen). Antibodies used (about 5 μg per 10–30 μg of DNA) were: Anti YY1 , Anti Trim28 , Anti-trimethyl-Histone H3 (Lys9) , Anti-trimethyl-Histone H3 (Lys27)  and Anti-trimethyl-Histone H3 (Lys4) . IP with IgG antibody  resulted in enrichment level < 1. Amplification was carried out by real-time PCR, and the bound/input values were then normalized by setting the negative control gene results to 1. Multiple assays of the same sample or the same gene sequence were analyzed in separate immunoprecipitations. All immunoprecipitations were repeated at least 3 times. Primer sequences used for qPCR are listed in Additional file 10The authors declare that they have no competing interests.SS designed the experiments, carried out the work and drafted the manuscript. EM provided advice and help with interpretation of the data and reviewed the findings. SPG designed the program, supervised the work and drafted and edited the manuscript. All authors read and approved the final manuscript.Proviral DNA copy number. (A) After infection of F9 cells, the initial unsorted and various sorted populations were analyzed for infection efficiency by qPCR. Results are shown in arbitrary units after normalization to Ctrl = cells with single viral copies. (B and C) NIH3T3 cells were infected with 1/10 of the concentration that was used to infect the F9 cells in panel A, with (B) PBSpro – GFP and mCherry viruses or with (C) PBSgln – GFP and mCherry viruses, and followed for expression of the markers by flow analysis. On the right is an example of the flow analysis dot plot recorded 27 days after infection (white) and in comparison to an uninfected control (black).Click here for fileExpression of reporter genes in subclones over time after sorting for double-positive populations. Panels A-E: Double-positive cells sorted after infection with PBSpro virus were subcloned and analyzed by flow cytometry for 40 days after sorting. Results show variability in expression levels in five individual clones that is possibly affected by the various integration sites of the proviruses in the clones.Click here for fileCpG methylation in sorted populations is similar whether selected for single or medium double-positive expression. Clones from the indicated populations (A-C) were scored for %mGC at either the GFP locus (left) or mCherry locus (right). Values are average of 10 clones. Results for examples from each population are shown.Click here for filePrimer and Probe List.Click here for file


Spinal fusion surgery techniques develop together with technologic advancements. New complications are seen as the result of new techniques and these may be very severe due to spinal cord and vascular structures in the lumbar region. The posterior lumbar interbody fusion cage (PLIFC) was shown to enhance spinal fusion and to prevent pseudoarthrosis due to its basic dynamic characteristics. PLIFC migrations are usually observed during the postoperative period, just after the mobilization of the patient and usually toward spinal canal. Migration to the retroperitoneal region is a extremely rare condition in the literature. In this article we discussed three cases of PLIFC antepulsion into the retroperitoneal region during the intraoperative period. Spinal fusion surgery techniques develop together with technologic advancements. New complications are seen as the result of new techniques and these may be very severe due to spinal cord and vascular structures in the lumbar region. Consequently, related mortality and morbidity may increase , 2. The In this paper, we discuss the one-year outcomes of PLIFCs that showed antepulsion into the abdominal cavity during the intraoperative period.Case 1: A 60-year-old female patient was admitted to our clinic with complaints of low back pain and leg pain that increased with walking, as well as neurogenic claudication. She underwent an operation for spinal stenosis and degenerative disc disease between L3-S1. A bilateral transpedicular screw system (BTSS) was placed at the L3-L4-L5-S1 levels. Total laminectomy and bilateral foraminotomy were performed on the L3-L4-L5 spine. A PLIFC was placed without problems through fluoroscopy control after the dura and roots were eliminated and L5-S1 discectomy had been performed; fluoroscopy showed the cage to be located in retroperitoneal region as anterior resistance was not felt during placement of the left PLIFC. The CT images obtained in the early postoperative period confirmed that the cage was in the retroperitoneal region at L5-S1 level  and PLIFC has gradually increased for treatment of spinal instability and/or spinal stenosis and related complications have also increased , 2, 5–7.Lumbar disc prosthesis (LDP) cases dislocated to anterior have been reported in literature and some of these cases were dislocated in the early period and some in the late period. In all these cases, removal is recommended in the early period prior to adhesion development in the operative field. Disc prosthesis developing in the late period may lead to injuries of the small intestine, colon, and ureter, in addition to the complications related to great vessel compression or rupture . SurgicaExcessive curettage could lead to tearing of the anterior longitudinal ligament (ALL) and annulus fibrosus when applying PLIFC. The cage should be hammered carefully under scope control. Excessive power should be avoided during tightening of the screw. The L5-S1 region should be closely monitored for acute injury of organs and vascular structures in case of anterior migration of PLIFC although it is a less risky region for injury. We consider that early retroperitoneal explorative surgery is not required unless intra-abdominal organ and vascular injury develop.


Stargardt disease is the most common hereditary macular degeneration in juveniles. It is characterized by macular dystrophy associated with loss of central vision in the first or second decade of life, a “beaten-metal” appearance in the fovea or parafoveal region, yellowish flecks around the macula or in posterior area of the retina, progressive atrophy of the bilateral foveal retinal pigment epithelium, and the “dark choroid” sign on fundus fluorescein angiography in most cases. We report a case of Stargardt disease in a 26-year-old Caucasian female submitted to rehabilitative training with microperimetry MP-1 to find a new preferred retinal locus (PRL) and to train her to better her quality of life. Best corrected visual acuity, mean retinal sensitivity, fixation, bivariate contour ellipse area, and speed reading were evaluated before and after the training and results were discussed.  Stargardt disease is an autosomal recessive condition commonly seen in early adulthood characterized by the presence of classical yellow subretinal flecks at the posterior pole. The macular changes can range from fine granularity to beaten bronze metal appearance to geographic atrophy. Fishman et al. analysed ninety-five patients with Stargardt macular dystrophy for visual loss with age and they showed that the probability of maintaining a visual acuity of 20/40 or better in at least one eye was 52% by age 19, 32% by age 29, and 22% by age 39; furthermore in the population studied by Fishman, once a patient's visual acuity dropped below 20/40, it tended to decrease rapidly and stabilize at 20/200 [We report a case of Stargardt disease in a 26-year-old Caucasian female that presented to us complaining of visual impairment in both eyes for 3 years.Presentation, clinical findings, and morphological changes are discussed. After ascertaining the diagnosis, the patient underwent a rehabilitation protocol by biofeedback training with MP-1 trying to find a new preferred retinal locus (PRL) and improve her quality of life. The rehabilitation protocol to which the patient was subjected was showed and results were discussed.A Caucasian female aged 26 years complained of a decrease in visual acuity progressive for 3 years. There was no previous ophthalmic history and her general health was good. The patient denied having eye pain, redness, photophobia, or irritation. She was not taking any medications and denied any medications allergies. The best corrected visual acuity (BCVA) was 20/200 in the right eye (RE) and 20/100 in the left eye (LE). The examination of the anterior segment in both eyes was unremarkable: the bulbar conjunctiva was white and transparent, anterior chamber clear, and cornea transparent. Furthermore, pupils were normal in size or shape and reactive to light, and no lens opacification was seen. The fundus examination revealed the existence of bilateral atrophy of the foveal retinal pigment epithelium (RPE) and photoreceptors and yellow-orange flecks distributed around the macula and the midretinal periphery. The vitreous is transparent; the examination of the extreme peripheral retina is normal.The photographs in autofluorescence by Fundus Autofluorescence  described a granular appearance of macula with alternating of hypoautofluorescent  and hyperautofluorescent (lipofuscin deposition) dots. These lesions are accompanied by flavimaculatus spots. The fluorescein angiography  confirmed the presence of macular atrophy accompanied by a reorganization of the pigment epithelium. There is an oval shaped window defect centered on the fovea. Optical coherence tomography  showed a bilateral macular atrophy, with disappearance of the ellipsoid line. The electroretinogram and electrooculogram were normal. All these diagnostic tests concluded the diagnosis of Stargardt disease type 1.We decided to submit the patient to rehabilitation program to improve her quality of life. Then a written informed consent was obtained by her. The rehabilitation protocol consisted of a 25-item questionnaire  to test her quality of life, reading speed test (words/minute) calculated by trial lenses in addition to reading appropriate for the age, on a text in Times New Roman 18 printing body, calculating the number of words per minute, then microperimetry and fixation test with microperimeter MP-1, and then 10 training sessions with MP-1 acoustic target biofeedback.Microperimetry and fixation test were performed with MP-1 microperimeter  using the automated program, the threshold test of 4-2 strategy, and a 1° single cross fixation target. However, at the beginning of the study the size was enlarged to a 2° single cross fixation target when the patient was not able to see the 1° single cross fixation target.Fixation stability was quantified according to Fujii classification and also by calculating the bivariate contour ellipse area (BCEA) encompassing 68% of fixation points based on collected fixation data after 30 seconds. The patient had a lot of eccentric and unstable loci of fixation around the fovea and in fact the initial BCEA was very large because the eccentricity of the PRL from the fovea ranged from 1.8° to 8.6°. We have also calculated retinal threshold sensitivity measured in all eyes using the Goldmann III target (round shape with a white background) with stimulus intensity ranging from 0 to 20 dB. Stimulus presentation time was 200 ms. After microperimetry we have chosen the preferred retinal locus (PRL) more suitable to train in an area 3° above the fovea in both eyes, which is an eccentric point compared to the fovea but in which there was greater retinal sensitivity, and we trained it: the patient was asked to move their eyes according to audio feedback which advised her whether she was getting closer to the desired final fixation position.2 to 1.87 deg2 in the RE while in LE it became from 3.27 deg2 to 1.54 deg2 and this result demonstrated an objective improvement of fixation , the assessment of distant and near visual acuity, reading speed test, and fixation and microperimetry tests were repeated. BCVA was maintained till the follow-up after a year. The mean retinal sensitivities before and after 1 year in the RE were 2.9 dB and 3.7 dB, respectively, while in the LE they were 3.5 dB and 4.8 dB, respectively. She has demonstrated an improved fixation that increased from being unstable to relatively unstable according to Fujii classification in both eyes; BCEA became from 3.52 degfixation . The reaABCA4) gene A photoreceptor cell-specific ATP-binding transporter gene (ABCR) that is mutated in recessive Stargardt macular dystrophy [ ABCA4 also result in retinitis pigmentosa and cone-rod dystrophy and have been linked to age-related macular degeneration (AMD) [Stargardt disease, first described in 1909, is an autosomal recessive macular dystrophy. The majority of people affected by the disease present with uncorrectable, decreased visual acuity during their teenage years, which most often progresses to legal blindness. To date, there is no approved intervention. Stargardt disease is marked by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE), degeneration of the neuroretina, and subsequent loss of vision. The condition results from mutations in the ATP-binding cassette, subfamily A, member 4  , 6. Biofon (AMD) –10 have on (AMD) .Visual rehabilitation with biofeedback is a therapeutic approach that has been applied in different ocular pathologies characterized by visual deterioration and loss of fixation stability: nystagmus, AMD, glaucoma, anisometropia, amblyopia, retinitis pigmentosa, oculocutaneous albinism, myopic maculopathy, vitelliform dystrophy, posttraumatic macular scar, and cone dystrophy.Olivo et al. demonstrated that Stargardt disease patients showed a significant gray matter (GM) loss bilaterally in the occipital cortices, extending into the right precuneus, and in the frontoorbital cortices. At tract-based spatial statistics (TBSS), significant reductions in fractional anisotropy were detected throughout large regions in the supratentorial white matter (WM), more pronounced in the posterior areas. Gray matter volume correlated directly with mean visual sensitivity in the right middle frontal and left calcarine gyri and inversely with retinal thickness in the left supramarginal gyrus .In this case report we wanted to find a new fovea (PRL) and turn it into a trained retinal locus (TRL) . Our resImprovement through biofeedback training in patients who suffer from macular disease either remaining stable or worsening, where the traditional treatment cannot offer further results, is of interest and well worthy of attention. The reasons for this improvement are probably due to the fact that we trained a “retinal motor” PRL, with appropriate retinal sensitivity, so as to increase the number of correct fixation saccades and rereference the oculomotor system.Andrade has shown that patients are usually unaware of their scotoma because when the retina is damaged by a local lesion (induced scotoma), the cortical neurons driven by stimuli originating in this region do not remain inactive but become selective to stimuli originating in other parts of the retina. This process occurs in two distinct steps, each with its own time scale: (a) a fast redistribution of receptive fields (RFs) in the area of the lesion and (b) a long-term reorganization that leads to the final RF configuration. Cortical neurons located in the retinotopic position corresponding to the scotoma receive some degree of activity from the unimpaired neurons in the area surrounding the lesion . This is


Number representations change through education, although it is currently unclear whether and how language could impact the magnitude representation that we share with other species. The most prominent view is that language does not play any role in modulating the core numeric representation involved in the contrast of quantities. Nevertheless, possible cultural hints on the numerical magnitude representation are currently on discussion focus. In fact, the acquisition of number words provides linguistic input that the quantity system may not ignore. Bilingualism offers a window to the study of this question, especially in bilinguals where the two number wording systems imply also two different numerical systems, such as in Basque-Spanish bilinguals. The present study evidences linguistic prints in the core number representational system through the analysis of EEG oscillatory activity during a simple number comparison task. Gamma band synchronization appears when Basque-Spanish bilinguals compare pairs of Arabic numbers linked through the Basque base-20 wording system, but it does not if the pairs are related through the base-10 system. Crucially, this gamma activity, originated in a left fronto-parietal network, only appears in bilinguals who learned math in Basque and not in equivalent proficiency bilinguals who learned math in Spanish. Thus, this neural index reflected in gamma band synchrony appears to be triggered by early learning experience with the base-20 numerical associations in Basque number words. Does language learning modify our numerical system? Basic magnitude processing implies the specialization of the quantity code with an essential role of the intraparietal sulcus, but also the orchestration of temporo-occipital, left inferior parietal or frontal cortex in the acquisition of numerical symbols and for arithmetic learning –5 VerbalBmath group) or in Spanish (LLSmath group). In the case of Basque-Spanish bilingualism, number words from the two languages are not just two codes for naming a certain quantity, but imply two different systems for numeric organization: vigesimal (base 20) vs. decimal (base 10) systems. Here, we measured the implicit effects of these two different numerical systems on brain oscillations during Arabic digit comparison. Number comparison is generally taken as a task that taps into an abstract analogue magnitude representation [A test for possible long-lasting associations between particular number wording systems and quantity was possible through the selection of proficient, balanced Spanish-Basque bilinguals, who had learned math either in Basque .This neurodynamic relationship between language and the representation of numerical magnitude was evaluated here by measuring the electroencephalography (EEG) response while bilingual participants performed a numeric size comparison task between pairs of Arabic digits. A subsequent analysis of large-scale neural synchronization was carried out by computing the phase coupling ,9 betweeSmath group) and nine reported learning math in Basque (LLBmath group). Aside from LLmath, the groups were equivalent in proficiency: for the LLSmath group, proficiency in Spanish, as measured by the Boston Naming Test (BNT) [Bmath group, BNT scores were 53.4 (2) and 50.2 (3.63) respectively . Thus, both groups were slightly more proficient in Spanish and were equivalent in relative proficiency . On average, the LLBmath group self-reported using Basque 39% of the time and Spanish 61% of the time. The LLSmath group used Basque 44% of the time and Spanish 56% of the time.Eighteen equally proficient Spanish-Basque bilinguals participated in this study  The avest (BNT)  was 54.8This study was approved by the Ethical Committee for Clinical Research within the Guipuzcoan public health sector (CEIC), which is the pertinent external committee for the assessment of studies involving human subjects in our region. The study was approved attending to the provided information: procedures to recruit participants , informed consent procedures that were implemented, copies of the information sheets and consent forms. Participants gave written informed consent. The application of all relevant European and national  was assessed (i.e. Declaration of Helsinki). This assessment was requested by the FP7-PEOPLE-2010-IEF funding body, which also assessed all the relevant ethical documentation and procedures.A subset of the stimuli included in Salillas and Carreiras (2014)  ERP studThe hypothesis and relevant contrasts were thus based on a comparison of responses to the two types of pairs across groups, with identical items used for each group of participants and identical target numbers for both Basque and Common pairs.For all the 72 experimental pairs, the largest number  was presented first. In order to avoid the inference of any rule, 36 fillers were presented in the same order , and a pool of 108 fillers were presented in the opposite order . These filler pairs did not follow any verbal form relationship and had random numerical distance between the first and second number.Each trial consisted of a fixation point that appeared for 1000 ms followed by a first number that remained on the screen for 300 ms. This first number was followed by an ISI of 350 ms in which a blank screen was presented. The second number then appeared for 300 ms. The appearance of the second number was the onset trigger for EEG analyses. After a blank screen had been shown for 700 ms, a question mark signaled the request of a delayed response (see Smath 30.5 (sd 3.7) for Common pairs and 29.8 (sd 3.4) for Basque pairs; LLBmath 31.3 (sd 3.8) for Common pairs and 28.4 (sd 3.8) for Basque pairs.EEG was recorded from 27 scalp electrodes embedded in an Easy-Cap in a 10–10 system array, and was referenced online to the left mastoid. Six additional electrodes were used to record blinks (from below the eye) and horizontal eye movements (placed at the outer canthi), using the left mastoid processes as a reference. Electrode impedances were maintained below 5 kOhms. The EEG was amplified with Brain Amp amplifiers, with the band pass set from 0.01 to 100 Hz, and sampled at a rate of 500 Hz. The output of the bioamplifiers was fed into a 32-channel, 12-bit analogue-to-digital converter, which was located inside a PC computer. Presentation software was used to present the visual stimuli and record the behavioral responses. Brain Vision Recorder software was used to deliver the event and timing codes to the data acquisition PC, so that the epochs of EEG activity corresponded precisely to the events of interest. Trials with artifacts due to eye movements, excessive muscle activity, or amplifier blockage were eliminated semi-automatically offline, in order to restrict them to the analyzed segment. Maximal allowed absolute difference after time = 0 ms. in each segment was kept to 150 μV. Maximal and minimal allowed amplitude was +90 μV and -90 μV respectively. And lowest activity (max—min) was kept to 0.5 μV. The average number of trials by condition was: LLThe raw EEG signal was first segmented into a series of epochs lasting 2350 ms including 1350 ms preceding the second digit. Electrodes placed near the eyes were excluded from the analysis in order to avoid biological artifacts. The continuous 50 Hz (AC) component was filtered in each epoch using a zero-phase filter that keeps the biological 50 Hz signal. Then, the filtered signal was analyzed with a sliding-window fast Fourier transform , in all trials and subjects for each condition. Using this process, we obtained the signal phase for frequencies between 1 and 70 Hz, with 1 Hz frequency resolution. Phase information was then used to compute a time-varying phase-locking value (PLV), which is an index of neural synchrony ,12 This The PLV is the length of the vector resulting from the vector sum of difference vectors for the trials , where N is the number of trials:N) was obtained by subtracting the average activity of the baseline (μ) from the filtered signal (S) divided by the standard deviation of the baseline (σ), in a frequency-by-frequency manner:The PLV index ranges from 0 to 1, with a value of 1 indicating perfect synchronization , and a value of 0 representing the total absence of synchrony . Phase synchronization across an entire trial for each frequency bin was normalized to a 400 ms baseline preceding TN second digit onset. The normalized signal  in search of time-frequency windows showing significant effects . SubsequFor the topographical analysis of phase synchrony we controlled for the statistical effects of multiple comparisons by choosing a very conservative significance threshold (p < 0.00006). This threshold was set on the basis of the distribution of synchrony values during the baseline. The threshold was chosen such that the number of cases larger than the threshold divided by the total number of cases was equal to p = 0.00006. By choosing this significance level, one line per analysis window could be explained by chance, given the fact that there were 27 electrodes with 351 possible combinations (27x26/2 = 351) .We performed the analysis of EEG phase synchrony with Matlab 7.0.4  using algorithms developed by Dr. Eugenio Rodriguez and others .Bmath group than LLSmath group from 150 ms to 250 ms  and from 350 ms to 450 ms  after TN second digit onset, whereas Beta phase synchrony was significantly higher for LLSmath group than LLBmath group from 140 ms to 280 ms  after TN second digit onset. For the Common pairs condition, we not observed significant differences in these time-frequency windows between the LLBmath group and LLSmath group.Results are shown in Bmath group, we observed two significant increase of Gamma phase synchronization during Basque pairs condition. The first one among fronto-parietal sites mostly over the left side of the scalp, from 150 ms to 250 ms after TN second digit onset, and the second between bilateral frontal and parietal sites, from 350 ms to 450 ms after TN second digit onset. In the same condition, the LLSmath group showed a strong increase of Beta phase synchronization mainly among occipital-parietal site, from 140 ms to 280 ms after TN second digit onset. During Common pairs condition, we observed an increase in Beta phase synchrony, but not Gamma, in both groups, among posterior sites mostly over the left side of the scalp, from 140 ms to 280 ms after TN second digit onset.The spatial distribution of phase synchrony over the scalp for each experimental condition is depicted in The results reveal that language shapes the magnitude representation that we share with other species. It has previously been shown that early math learning shapes memory networks for arithmetic . We haveBmath group. The early synchronized fronto-parietal network may reflect the maintenance of verbal to quantity links, as suggested by recent proposals [The data show clear differences between the two groups in terms of the frequency bands and phase synchrony patterns associated with comparisons related to the base 20 system. The differences, which appear around 200 ms after participants start the comparison process, suggest gamma band activation likely based on a left fronto-parietal network. It is the activity of that fronto-parietal network in gamma phase synchrony what seems to be specific to the Basque pairs for the LLroposals , 5, 17. Smath and for Common pairs in LLBmath), and is thus posited to reflect non-linguistic, quantity-related processes. Note that the base 10 system is not only a naming system, but a system in which math is generally structured . Therefore, the neural integration of the base 10 system does not need to be linguistic in origin. In fact, the base 10 system has a route in the widespread use of fingers for counting. Base 10 is used for the place coding in multi-digit numbers. In turn, base 10 structure is a feature of a particular representational system rather than a fundamental mathematical fact. Nevertheless, it is a feature that is incorporated into many of the algorithms that children learn for arithmetic performance [All the rest of conditions showed similar beta activity in bilateral central-posterior networks. This latter network was common to both groups when early acquired verbal links were not used (i.e. for both pairs in the LLformance .It is the claim of all theoretical approaches that a verbal code is not needed when magnitude is contrasted using Arabic digits –7, 20. EIn neurophysiological terms, the reciprocal relationship between the number system and number words could imply the integration—dynamic and transient—of neural networks widely distributed across the brain. It has been proposed that the integration of brain activity is the result of transient synchronization of neural activity . Neural


Extraskeletal Ewing's sarcoma (EES) is a rare presentation, representing only 15% of all primary Ewing's sarcoma cases. Even more uncommon is EES presenting as a primary focus in the spinal canal. These rapidly growing tumors often present with focal neurological symptoms of myelopathy or radiculopathy. There are no classic characteristic imaging findings and thus the physician must keep a high index of clinical suspicion. Diagnosis can only be definitively made by histopathological studies. In this report, we discuss a primary cervical spine EES in a 53-year-old man who presented with a two-month history of left upper extremity pain and acute onset of weakness. Imaging revealed a cervical spinal canal mass. After undergoing cervical decompression, histopathological examination confirmed a diagnosis of Ewing's sarcoma. A literature search revealed fewer than 25 reported cases of primary cervical spine EES published in the past 15 years and only one report demonstrating this pathology in a patient older than 30 years of age (age = 38). Given the low incidence of this pathology presenting in this age group and the lack of treatment guidelines, each patient's plan should be considered on a case-by-case basis until further studies are performed to determine optimal evidence based treatment. Ewing's sarcoma is a malignant primitive neuroectodermal tumor (PNET) that primarily presents during the first two decades of life. Approximately 85% of those cases present primarily in the skeleton. With an annual incidence of 1–3 per million, it is generally a rare diagnosis . There hA 53-year-old male first presented to his primary care provider with a 2-month history of progressive complaints that started as a stiff neck and subjective right shoulder blade mass that was never objectively noted by any physician with some right upper extremity pain. This pain reportedly then spread to the left upper extremity with pain radiating down the posterior side to the left proximal wrist. Due to these complaints, he was evaluated by his primary care provider and started on ibuprofen. Upon awakening the following morning, the patient reported the sudden onset of left arm weakness that was not antigravity. Conversely however, the left upper extremity pain had remitted. With this sudden change in neurological function, the patient's primary care provider obtained an MRI of the cervical spine. The patient was told that the MRI showed a bony lesion and he was referred to our institution's orthopedics clinic. One month later he presented to the orthopedic clinic where the patient noted that his left upper extremity pain had returned. The patient and his wife also noted that, over the previous two-month period, he was losing muscle mass most profoundly in the left biceps and deltoid. Furthermore, he had also unintentionally lost 10 pounds over the previous three months. Due to his suspicious presentation and cervical spine lesion noted on the MRI, the patient was referred to our neurosurgical clinic.On examination, the patient appeared emaciated and easily fatigable. Muscle strength testing revealed 2/5 in the left upper extremity with exception of the biceps at 3/5 and handgrip of 4-/5. His right upper extremity was 3/5 in the deltoid and 4-/5 in all other muscle groups. The bilateral lower extremities were 5/5 for strength. There were no myelopathic signs. A repeat MRI performed at our institution showed an ill-defined predominantly extradural enhancement along the left aspect of the cervical spinal canal, most apparent at C6-C7, but also extending between C2 and C7 . A CT chThe patient underwent a C6-C7 laminotomy with decompression of the left C7 nerve root and biopsy of the extradural lesion, accomplishing the goals of operation including decompression and definitive diagnosis. Intraoperative baseline SSEPs were attempted; however no electrophysiological recordings could be obtained apart from minimal recordings in the right upper extremity.Histopathological studies established the diagnosis of Ewing's sarcoma. Our pathologists noted round, hyperchromatic nuclei with little cytoplasm and multiple mitosis suggestive of a primitive neuroectodermal tumor in surrounding necrotic tissue . FurtherUpon awakening postoperatively, the patient was noted to be severally quadriparetic and had suffered a myocardial infarction. He was transferred to the surgical intensive care unit where he was kept on flat bed-rest and high mean arterial pressure control. He recovered remarkably well. First, he regained movement in his lower extremities and subsequently regained antigravity ability in his right upper extremity. He continued to improve with the help of physical and occupational therapy. At his 6-month follow-up appointment his muscle strength was slowly improving with 3/5 in the left upper extremity with exception of handgrip, which was 4-/5. His right upper extremity was 4-/5 throughout. The bilateral lower extremities were 4/5 in strength. He was started on a course of chemotherapy and radiation therapy prior to discharge. To date, he has completed radiation therapy with a total treatment of 55.8 Gy. He is currently undergoing infusion treatment sessions of vincristine, actinomycin, and cyclophosphamide alternating with ifosfamide and etoposide.Extraskeletal Ewing's sarcoma of the spinal canal remains a rare diagnosis. It has been a subject of interest since its first published description in 1969 . EES affIn reviewing previously published case reports of primary EES of the cervical spine there were some patterns of presentation. The average age at presentation was 19.7 years and there were no cases identified that presented in a patient past 38 years of age. There was a slight propensity for the pathology to present in males (42%) compared to females (29%) in cases where gender was specified. However, one must take into account that 29% of the cases identified did not report the patient's gender. A further breakdown of these previous cases is summarized in Due to the rarity of this pathology, there are few large retrospective studies available for review. However, one such study performed by Ilaslan at the Mayo Clinic was able to identify 125 patients with primary spinal EES out of 1,277 patients with primary Ewing's sarcoma treated at the clinic from 1926 to 2001. The average age of the patients was 19.3 years (range 4–54 years). When the EES was organized by level in the spinal column, there were only a total of 4 patients identified (3%), further showing the infrequency of this pathology [In terms of imaging, reports vary and there appears to be no classical findings of an EES mass. On CT, there tends to be a mass that enhances heterogeneously . On MRI Definitive diagnosis is made by histopathological studies. Findings include uniform small round blue cells with scant possibly slightly eosinophilic cytoplasm notable on light microscopy. Two cellular markers frequently employed by pathologists are CD-99, which is expressed in both Ewing's sarcoma and lymphoma, and CD-45, which is only expressed in lymphoblastic lymphoma . TypicalConcerning treatment, there is strong evidence that early and aggressive treatment leads to the most favorable outcomes , 17, 23.Survivorship of primary EES of the spine post treatment was fairly consistent across different studies. A study by Venkateswaran from St. Jude's Children's Research Hospital of 33 patients showed an estimated 5-year survival rate of 48.1% (±8.9%) . This waThe occurrence of an extraskeletal Ewing's sarcoma remains rare, which typically affects adolescents with a male preponderance. It is especially rare to be found in the spine, especially the cervical spine. We have shown with this case report and in a review of prior literature that it is not unheard of for EES to present in the spine, regardless of age, and in our case, even in late adulthood. While it remains rare for an EES to present in the spine, it should remain a viable differential diagnosis of an ill-defined spinal mass noted on imaging, independent of the patient's age. Standard treatment often involves a multidisciplinary approach of surgery, chemotherapy, and radiation therapy with the particular preferential order determined on a case-by-case basis and has a survivorship of approximately 45% at 5 years after treatment.


Nearly 50% of the horses participating in endurance events are eliminated at a veterinary examination (a vet gate). Detecting unfit horses before a health problem occurs and treatment is required is a challenge for veterinarians but is essential for improving equine welfare. We hypothesized that it would be possible to detect unfit horses earlier in the event by measuring heart rate recovery variables. Hence, the objective of the present study was to compute logistic regressions of heart rate, cardiac recovery time and average speed data recorded at the previous vet gate (n-1) and thus predict the probability of elimination during successive phases (n and following) in endurance events. Speed and heart rate data were extracted from an electronic database of endurance events (80–160 km in length) organized in four countries. Overall, 39% of the horses that started an event were eliminated—mostly due to lameness (64%) or metabolic disorders (15%). For each vet gate, logistic regressions of explanatory variables  and categorical variables (age and/or event distance) were computed to estimate the probability of elimination. The predictive logistic regressions for vet gates 2 to 5 correctly classified between 62% and 86% of the eliminated horses. The robustness of these results was confirmed by high areas under the receiving operating characteristic curves (0.68–0.84). Overall, a horse has a 70% chance of being eliminated at the next gate if its cardiac recovery time is longer than 11 min at vet gate 1 or 2, or longer than 13 min at vet gates 3 or 4. Heart rate recovery and average speed variables measured at the previous vet gate(s) enabled us to predict elimination at the following vet gate. These variables should be checked at each veterinary examination, in order to detect unfit horses as early as possible. Our predictive method may help to improve equine welfare and ethical considerations in endurance events. Fédération Equestre Internationale (FEI) since 1982. The first endurance world championships were held in 1998 in the United Arab Emirates (UAE). Since then, the number of entries in endurance events worldwide has increased more than four-fold [Endurance riding is an international, long-distance equestrian sport that has been regulated by the Elimination rates appear to have increased over recent years . This a The HR is a good indicator of stress and health status in horses ,14,15. IHence, we hypothesized that (i) qualified and eliminated horses differ in terms of their speed profiles and cardiac recovery variables, and (ii) this difference can be used to detect unfit horses and prevent health incidents during endurance events. The objective of the present study was to compute logistic regression models using the HR, CRT and AS recorded at the previous vet gate (n-1) and thus predict elimination at subsequent phases (vet gate n and following) of the event.Endurance events  are split into four, five or six successive phases of approximately 30–40 km. At the end of each phase, each horse undergoes a veterinary inspection  was approved by the local Animal Care and Use Committee . However, the present study did not involve any experiments on animals. All the horses having provided data for the present study had competed in official events organized according to the FEI rules ). In tot-the type of event, according to the distance ridden in one day: 1-star events (80 to 119 km: 27.2%), 2-star events (120 to 139 km: 54.9%) and 3-star events (140 to 160 km: 17.9%).-a posteriori from a meteorological database and are given in the country in which the event was organized: FRA, SPA+POR and UAE. Climate data (temperature and humidity) for these zones were extracted At each vet gate, the HR is measured by a veterinarian using a stethoscope. The CRT was calculated as the difference between the arrival time (at the end of the phase) and the time of veterinary inspection (referred to as the "time in" by the FEI endurance rules ). When t-the AS for each phase.-the CRT and HR at each vet gate.-if appropriate, the number of the vet gate at which the horse was eliminated and the reason for elimination. Horses can be elimination at any vet gate if the horse is considered to be unfit.--Lameness (LA), for horses consistently observed to be lame by a panel of veterinarians. Lameness is “an irregularity of gait which must be consistently observable at trot, or an equivalent gait; and is observable through evaluation by trotting the horse on a loose lead in hand straight out and back, without prior flexion or deep palpation; which must be observed to cause pain, or threaten the immediate ability of the horse to safely perform athletically".-Metabolic reasons (ME), for horses that failed to achieve the required heart rate threshold  required by the event’s distance and number of stars) or did not meet the criteria for over biological parameters . Metabolic status is assessed by “the examination and recording of those parameters that indicate the horse’s fitness to continue including (but not exclusive to) mucous membranes, capillary refill time, hydration, intestinal activity, behavior and cardiac recovery index”.-Retirement (RET), for horses voluntarily withdrawn from event by their rider despite having passed all required veterinary inspections up to that point.-Elimination for other reasons (OR), for horses eliminated from the event for reasons other than those listed above.event outcome: at the end of the event, the horses were divided in two groups: qualified (Q) horses had finished the event and passed all veterinary examinations, whereas eliminated (EL) horses had been eliminated by the judges for one of the four following reasons as defined in the following FEI endurance rules :-LamenLastly, in order to check the validity of the predictive logistic regressions, we computed the probability of elimination for an independent dataset of 80 horses having competed in 1, 2 and 3-star endurance events organized in 2014. The 80 horses were randomly selected so that the dataset contained 10 qualified and 10 eliminated horses for each phase .We used predictive logistic regression analysis to estimate probability of elimination from vet gates 2 to 5 and at the finish. The input variables were AS, CRT and HR measured at the previous vet gate (n-1). Given the absence of pre-ride data (CRT and HR at rest), horses eliminated at vet gate 1 (i.e. after the first phase of the event) were not included in the study. For each vet gate, an equal number of Q and EL horses were selected at random from the total set of available horses. Logistic regression consisted in computing the probability of be classified as qualified (Q) or eliminated (EL), using three explanatory variables  and two categorical variables (distance and country) as input variables. Briefly, the odds ratio was log-transformed  in order to iteratively optimize the best model of the categorical and quantitative explanatory variables . In Of the 7032 starting horses, 38.94% (2738) were eliminated during an event; 64.46% of these eliminations were due to LA, with 15.23% due to ME, 14.35% due to RET and 5.96% due to OR . Most el2 value derived from logistic regression is not a good indicator of validity because it estimates the percentage of variation in the binary dependent variable (Q or EL) accounted for by the explanatory variables AS, CRT and HR at the previous vet gate (n-1). In the present study, R2 values were between 0.12 and 0.37; however these values did not accurately reflect the logistic regressions’ predictive power.The optimum logistic regression was computed for each vet gate (gates 2 to 5). The best indicators of validity are the percentage of well-classified observations and the AUROC, which characterize the logistic regression model’s ability to predict a horse’s classification as EL or Q . The perUsing the same dataset, we also computed logistic regressions to predict the reason for elimination  at each vet gate . The corThe ROC curves enabled us to determine the false positive rate and false negative rate with regard to the probability of elimination. When considering the horse’s health, it is better to have false positives than false negatives. A probability of 50% would have generated too many false negatives. Hence, we chose a probability of elimination of 70% as a threshold for classifying the predictions based on an independent dataset from 2014. Using this threshold, 75% of the 80 horses were classified correctly . The errn due to an HR of 65 or more. Using the logistic regression equations with a HR value set to 64, we computed all the combinations of CRT and AS that gave a probability of elimination of between 20% and 100%. The results are presented using a colour scale in order to show the probability of elimination when the CRT and AS were above the computed thresholds at the previous vet gate (n-1)  . Brieflyte (n-1) , respectte (n-1) . Althougte (n-1) .By analyzing a large dataset from equine endurance events (from 80 to 160 km in distance) organized in four different countries, we were able to compute logistic regressions and estimate the probability of elimination at each vet gate. On average, between 64.8 to 86.6% of all eliminations at vet gates 2 to 5 could be predicted from data collected at the previous vet gate. Furthermore, the predictive models’ robustness was confirmed with a new, independent dataset; 75% of the eliminations were correctly predicted. These results confirm the relationship between CRT, HR and AS measured at phase n-1 and elimination at phase n. Changes in CRT, HR and AS during the event are clearly associated with an increased probability of elimination at the next vet gate. The higher the speed, the higher the probability of elimination. This result is in line with previous studies ,8. The AThe data collected in the present study corresponded to a quarter of the FEI-registered endurance events organized between 2007 and 2011. These data were more representative of the major events, which have a high enough budget to finance electronic time-keeping. Hence, our results might have been biased by the high-level events. Most of the events in the database took place in three European countries (SPA-PORT and FRA), with few events in the UAE. Other countries with major endurance events  were not included in the database. Strictly, the results presented here can be applied in FRA, ESP, PORT and UAE—the countries on which our regression models were based—and thus should be evaluated with caution in other countries  the horse’s fitness and the prevailing environmental conditions  ,8.Although several studies ,8 have hEliminations for LA or MET are intended to prevent tired horses from continuing the event and then developing conditions associated with exhaustion and illness ,25. Desp2 obtained from a logistic regression analysis is not an appropriate measure of the quality of the binary model response because it is only an approximate value. This was confirmed in the present study because the low but acceptable R2 values (0.12–0.37) were associated with rather high AUROC values (0.68–0.84), which confirmed the model’s ability to distinguish between two groups. In the present study, we chose to favor a higher false positive rate over a high false negative rate, so that an unfit and potentially ill horse could be identified as early as possible. Hence, we chose a probability of elimination of 70% for the validation test, this corresponded to a false positive rate of 16.3% and a false negative rate of only 8.7%. It was not possible to include more explanatory variables (such as weather conditions or the horse’s age) in the models because the small sample size and unequal number of Q and EL horses in each subgroup would have prevented high-quality prediction. Including a putative effect of double starts in the statistical models was not possible because it would have generated too many the degrees of freedom for robust calculations. We also computed also logistic regressions to predict the reason for elimination at each vet gate, however the correct prediction rate was too low for most of the vet gates .Multiple regression, discriminant analysis and logistic regression can potentially all be used to classify horses as Q or EL as a function of the input variables available in event records. When discriminant analysis and logistic regression were compared, the latter method appeared to be the best for a two-group context (as in the present study) . FurtherLastly, the present study made it possible to calculate CRT threshold values that could be usefully applied to examinations at the vet gate: horses requiring more than 11 min to achieve an HR below 65 at vet gates 1 and 2 or more than 13 min at vet gates 3 and 4 have a 70% chance of being eliminated at the next vet gates . This information has great practical value and suggests that it would be possible to detect tired horses earlier in the event than veterinarians currently do. It has been demonstrated that the current vet gate examination enables veterinarians to detect physiologically compromised horses . ApplicaOur results show that it is possible to compute predictive logistic regressions for each vet gate (2 to 5) and correctly detect between 61.8 and 86.6% of horses subsequently judged to be unfit, by using AS, CRT and HR measured at the previous vet gate). The CRT appears to be a particularly relevant predictive variable for detecting unfit horses during the event. We suggest that the CRT should be checked at each vet gate, in order to identify unfit horses earlier and thus improve their welfare. In practice, CRT threshold values of 11 min (at vet gates 1 and 2) and 13 min (at vet gates 3 and 4) are associated with a 70% probability of elimination at the next vet gate. We encourage the veterinarians involved in endurance events and the FEI to apply this new knowledge, in order to improve the welfare of endurance horses and prevent health problems during or after endurance events.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.