Activation of the Wnt signaling in some types of cancer and its relation with chemotherapy resistance is a very interesting issue that has been emphasized in recent years. Although, it is known that increase in the activity of β-catenin is important in blast transformation and drug resistance, the underlying mechanisms are still unclear. In this study, changes in the expression levels of 186 genes that are thought to be important in drug resistance and Wnt signaling pathways were determined by using qPCR method in doxorubicin-sensitive and -resistant HeLa and K562 cell lines. It has been observed that the genes involved in the Wnt signaling pathways are involved in more changes in HeLa/Dox cells (36 genes) than in the K562/Dox cells (17 genes). Genes important for the development of cancer resistance have been found to be significantly different in expression levels of 18 genes in HeLa/Dox cells and 20 genes in K562/Dox cells. In both cell lines, the expression of ABCB1 gene was significantly increased to 160 and 103 fold, respectively. However, despite the resistance to same drug in HeLa and K562 cell lines, it appears that the expression levels of different oncogenes and genes involved in Wnt signaling pathways have been altered. It has been found that although resistance develops to the same drug in both cell lines, the expression levels of different genes have changed. If functional analysis of these genes is performed on patient population groups, these molecules may become candidates for novel therapeutic target molecules. Wnt signaling pathway is involved in both embryonic and adult stages in an organism . This iThe multidrug resistance 1 gene (MDR1) encodes an ABC transporter called P-glycoprotein (P-gp), which is the direct target of the Wnt/β-catenin signaling pathway. Overexpression of P-gp actively pumps out cytotoxic drugs from the cell and by reducing drug concentration in the cell cause drug resistance. The Frizzled 1 protein (FZD1), an important member of the Wnt/β-catenin pathway, and P-gp have been shown to increase in the adriamycin resistant MCF-7 breast cancer cell line. It has been shown that when the FZD1 gene expression is silenced via siRNA, cytoplasmic and nuclear β-catenin levels fall, as well as the level of P-gp decreases and sensitivity to chemotherapeutic agents is regained . Activation of Wnt signaling is known to play a role in the pathogenesis of some solid tumors and leukemia types . IC50 values of both original and drug resistant cells were determined. Doxorubicin resistant variants of original HeLa and K562 cell lines were developed in our laboratory by applying the drug in dose increments. The cells were grown in 25 cmRNA isolation was performed from HeLa and K562 cell lines, which are sensitive and resistant to doxorubicin using TRI Reagent  according to the manufacturer's instructions. RNA samples were assessed qualitatively on an Agilent 2100 Bioanalyzer. RNA samples with sufficient concentration (at least 200 ng/µl) and free of genomic DNA and protein contamination were used for cDNA synthesis. cDNAs were synthesized from total RNA by using random primers of Transcriptor High Fidelity cDNA Synthesis Kit (Roche Life Sciences) according to manufacturer's instructions.PCR-arrays  were performed on 96-well plates using the quantitative RT-PCR method by using Roche Light Cycler 480 instrument. It has been attempted to determine the expression levels of the genes which have a role in Wnt signaling pathways and genes which are thought to be important in resistance development. Genes on the "Wnt Signal Transduction PCR Array" and "Drug Resistance PCR Array" are given in Tables 1RT-PCR analyzes were performed in dublicates using a total of 16 PCR arrays from both the "Wnt Signal Transduction PCR array" and the "Drug Resistance PCR array" from all original and doxorubicin resistant cell lines. Relative amount changes were determined by calculating the Ct values (2-ΔΔCt) obtained for each gene. According to this method; from a Ct value relative to a control gene (beta-actin) in a gene-resistant drug line; the value of ΔΔCt [ΔΔCt = ΔCt resistant-ΔCt original] was obtained by subtracting the Ct value relative to the control gene in the same cell line. The fold change in the amount of gene was obtained by substituting ΔΔCt in the 2-ΔΔCt. The results obtained with the two replicates were evaluated with the SPSS 16.0 program and the differences less than the p-value of 0.05 and those with more or less 2 folds changes in gene expression levels were considered as "statistically significant".50 values at 72 h by XTT assay. In the original HeLa cell line, doxorubicin killed 50 % of the cells at a dose of 2,664 μM  whereas in doxorubicin-resistant HeLa cells this value was found to be 5,470 μM  , while in doxorubicin-resistant K562 cells this value was found to be 0.996 μM (IC50 = 0.996 μM). These results show that doxorubicin-resistant K562 cell line is approximately 32 times more resistant than the original K562 line . The role of Wnt signal transduction in the emergence of many cancer types and its relationship with chemotherapy resistance has been a subject of interest in recent years. There are three types of Wnt signaling pathways; Wnt/β-catenin (canonic), non-canonic and Wnt/calcium. Especially, the mechanisms between Wnt/β-catenin signal activation, blast transformation and drug resistance are being explored . In this study, attempts were made to identify changes in the expression levels of 186 genes which have important roles in drug resistance and Wnt signaling pathways in original and doxorubicin resistant HeLa and K562 cells. The selection of the model cell lines to two different types of cancer is to demonstrate possible similar and/or different effects of the same drug in different types of cancer.Resistant variants of HeLa and K562 cell lines were obtained and resistance development were determined by XTT cytotoxicity analysis. According to the results, the doxorubicin resistance in the HeLa cell line was about 2-folds higher than that of the original HeLa line; and the doxorubicin-resistant K562 cell line developed about 32 times more resistance than the original K562 line. It is seen that the amount of resistance developed to the same drug is significantly different in different cancer cell lines. The expression of the ABCB1 gene encoding MDR1 protein in both cell lines was significantly increased to 160 and 103 folds, respectively, in doxorubicin resistant HeLa and K562 cell lines. In many studies, it has been shown that overexpression of MDR1 in cancer cells is responsible for resistance to chemotherapy drugs Haber, 1992); the in92; the iIt is known that oncogen activation is related to the development of cancer formation and drug resistance. For this reason, many cancer drugs have been developed to target oncoproteins . It hasThere are also significant differences between the doxorubicin-resistant HeLa and K562 cells in the expression of the genes involved in the Wnt signaling pathway. Among 93 genes included in this study; expression levels of 36 genes in HeLa/Dox and 17 genes in K562/Dox were observed to change with respect to drug-sensitive variants. Activation of Wnt signaling pathways begins with the binding of the Wnt protein to the FZD and LRP5/6 receptors in the target cell membrane. Some of the beta-catenin that accumulates in the cytoplasm enters the nucleus, causing transcription of target genes . In HeLal., 2017). In anoal., 2017). Three In conclusion; this study provides important information on the level of gene expressions in terms of determining the relationship between doxorubicin resistance and changes in the Wnt signaling pathway in HeLa and K562 cell lines. However, in order to investigate these relations in more detail, functional analyzes at the protein level are required in the following studies.This work was supported by TÜBİTAK 3001 project (Project No: 214S634).The authors declare that they have no conflict of interest.


Although it has been recognized for a long time that the predisposition to cardiovascular diseases (CVD) is determined by many risk factors and despite the common use of algorithms incorporating several of these factors to predict the overall risk, there has yet been no global description of the complex way in which CVD risk factors interact with each other. This is the aim of the present study which investigated all existing relationships between the main CVD risk factors in a well-characterized occupational cohort. Prospective associations between 12 behavioural and clinical risk factors  were systematically tested using Cox regression in 10,736 middle-aged individuals free of CVD at baseline and followed over 20 years. In addition to independently predicting CVD risk (HRs from 1.18 to 1.97 in multivariable models), these factors form a vast network of associations where each factor predicts, and/or is predicted by, several other factors . Both the number of factors associated with a given factor (1 to 9) and the strength of the associations (HRs from 1.10 to 6.12 in multivariable models) are very variable, suggesting that all the factors do not have the same influence within this network. These results show that there is a remarkably extensive network of relationships between the main CVD risk factors which may have not been sufficiently taken into account, notably in preventive strategies aiming to lower CVD risk. Over the last decades, many risk factors have been independently associated with cardiovascular diseases (CVD) through prospective observational studies , 2. ThisDespite the recognition that these factors do not act separately but jointly to determine CVD risk  and the The interest of taking into account the relationships between the risk factors is potentially high, not so much for the identification of individuals with an increased CVD risk that is usually quite good  but for As a first step to take into account this interaction network, the present study aims to provide a global view of all the prospective relationships between the main behavioural and clinical CVD risk factors in a single cohort followed over a 20-year period.The analyses were performed in a cohort of middle-aged volunteers working at the French National Gas and Electricity Company, who were all recruited in 1989 and followed since then (GAZEL cohort) . The recAll the data were obtained from self-administered questionnaires, completed annually by volunteers during follow-up, which contained a variety of inquiries into their lifestyle and the occurrence of health events. Among the risk factors that were considered in the analyses, gender and age were reported as such. Parental history of CVD referred to the occurrence of coronary heart disease before the age of 60 in the mother or the father. The inquiry into the occurrence of hypertension, dyslipidemia, diabetes, sleep disorder and nonfatal CVD events  was composed of two consecutive questions: first, “in the list below, indicate the diseases you are suffering or have suffered from during the past twelve months” and secondly, “among the diseases you have cited, what are the new ones, i.e., those you were not suffering from over a year ago”? Body mass index was calculated from weight and height values drawn from the questionnaires. Depressive status was assessed with the Centre of Epidemiologic Studies Depression scale and defined as scores ≥ 17 in men and ≥ 23 in women, which includes both moderate and severe depression . The inqAs the validity of self-reporting for assessing the incidence of risk factors and CVD events is always questionable, a quality control study has been performed in the GAZEL cohort by comparing CVD disorders that were reported in the questionnaire in 1992 with physician’s diagnoses obtained from the sick-leave database of the company . It show2), overweight (25 kg/m2 ≤ BMI < 30 kg/m2) or obese (BMI ≥ 30 kg/m2). Smoking was classified into 3 groups  and alcohol consumption into 4 patterns using a slightly modified version of the World Health Organisation classification to take into account the distribution of the consumption in the French population: non-drinker, light drinker , moderate drinker  or heavy drinker . Except for gender, age and parental CVD, the incidence of each factor during follow-up was also considered as an outcome that can be predicted by the other factors at baseline. In this case, physical inactivity, hypertension, dyslipidemia, diabetes, sleep disorder and depression were used as such. Body mass index, smoking and alcohol consumption were transformed into binary variables as follows: obese versus optimal/overweight, smoker versus non-smoker/ex-smoker and non-moderate drinker (non-drinker/light drinker/heavy drinker) versus moderate drinker.Twelve clinical or behavioural factors were used to predict the risk of CVD events during follow-up. The rationale behind this choice was that these traditional risk factors are commonly used by physicians in their daily practice where the prevention may take place and they are likely to be available in cohorts in which the present analyses could be reproduced. Gender, parental cardiovascular disease, physical activity, hypertension, dyslipidemia, diabetes, sleep disorder and depression were coded as binary variables . Age (range 39–54 years) was divided into tertiles and body mass index (BMI) into the usual 3 categories: optimal ) versus log(time) transformation) that the assumption holds for the 47 significant associations reported in the results . Note that the assumption was not verified for the 52 non-significant associations as the identification of those violating the assumption would not allow to include them in the network of significant associations but only to suspect them to be false negatives. Despite the fact that annual questionnaires produced interval censored data in the unit of one year, standard Cox regressions were used in place of proportional hazard regressions for interval censored data because it was assumed that the failure to report incident CVD events or risk factors or the delay with which they are reported in the following questionnaires do not greatly modify the estimation of hazard ratios as there is no evidence in the GAZEL cohort that the failure frequency or delay length vary to a large extent between the volunteers exposed and those not exposed to the risk factors.Cox proportional hazard regression models ) were used to compute hazard ratios and 95% confidence intervals for incident CVD events and for the incidence of each risk factor during a 20-year follow-up running from 1993 to 2013. The baseline year was 1993 because it was the first year in which all the risk factors were simultaneously assessed. Time to event was measured from baseline to the first occurrence of CVD events or the first detection of the risk factors as provided by the annual questionnaires. For each factor, the time-dependent number of volunteers at risk analysed in the corresponding Cox regression is shown in S3 Fig), it is not negligible when compared to the incident rates of CVD events or of the risk factors, especially towards the end of the follow-up. Censoring deaths as the events of interest might therefore bias the observed associations to some extent. Nevertheless, Cox regressions were used in place of competing risk models for two reasons. The first one is that the main purpose of the study is to explore the presence or absence of relationships between the risk factors rather than to determine accurately their incidence. It has been shown that censoring competing events is acceptable in this case as the HRs are relatively similar in standard Cox regressions and competing risk methods [Censored events include all deaths (n = 707) among which some were of cardiovascular origin (n = 112). This certainly leads to an underestimation of the magnitude of the associations with nonfatal CVD events as fatal and nonfatal events are supposed to have the same risk factors. However, this underestimation remains minimal given that the proportion of fatal events in comparison to non-fatal events is very low (7.8%). The reason of this choice is that the cause of death was not sufficiently documented to allow an accurate assessment of fatal events. Note that when analysing the incidence of risk factors, volunteers who have had a nonfatal CVD event before the first detection of the factors were also censored. Although the mortality rate is relatively low . The comparison with multi-adjusted Cox regressions excluding these volunteers with the 20-year follow-up indicates that the bias is very small as all the associations with incident CVD events and the vast majority of those with incident risk factors are conserved .The risk of CVD events was assessed in 10,736 of the 20,625 volunteers originally included in the cohort after exclusion of those who have had a nonfatal CVD event before the beginning of the follow-up period (n = 247) or those who had missing data in one or more of the risk factors included in the regression models (n = 9778). Additionally, when the outcome was the incidence of CVD risk factors during follow-up, volunteers who were exposed to the risk factors before the follow-up period or at baseline were also excluded before performing the analyses (n = 163 to 8282 depending on the factor). Note that as volunteers lost to follow-up were among those who were excluded due to missing values, all the volunteers included in the analyses were followed over the whole period of 20 years except when they died during this time period. These analyses can therefore be seen as a retrospective study conditional on the follow-up period or death within this period. Although the number of volunteers lost to follow-up is very low (n = 619), a sensitivity analysis was made to test whether the exclusion of these volunteers may bias the results of the analyses. For this purpose, multi-adjusted Cox regressions including volunteers lost to follow-up were performed with a follow-up running up to the date of the last available questionnaire  would require to use multivariate imputation by chained equations which is time-consuming and not always devoid of biases [S2 Table, the rates of hypertension, dyslipidemia, diabetes and depression, as well as those of smoking and physical inactivity, are significantly lower in the selected volunteers. To test to which extent this selection may affect the results presented in this study, two types of sensitivity analyses were performed. The first one was to run Cox regressions weighted by the inverse probability of being selected; this probability was calculated by using a multivariate logistic regression model including five risk factors, averaged over the 4-year period (1989–1992) preceding the baseline year in order to minimize the number of missing values, which were significantly associated with the selection (OR (95%CI): gender W/M 0.78 (0.71–0.87), hypertension Y/N 0.84 (0.75–0.94), dyslipidemia Y/N 0.81 (0.68–0.98), depression Y/N 0.71 (0.64–0.78) and smoking Y/N 0.77 (0.71–0.84). The comparison of the outputs of weighted (S3 Table) and unweighted multi-adjusted Cox regressions shows that all the associations with incident CVD events and most of those with incident risk factors are conserved . The second sensitivity analysis consisted of comparing the outputs of gender-age adjusted Cox regressions in volunteers with complete data either for all the risk factors  or only for the factor analysed in each model ; the number of volunteers excluded is large in the first case while it is much smaller and varies from one model to another depending on the risk factor in the second case. S4 Table shows that all the associations with incident CVD events and most of those with incident risk factors are conserved ; note that the number of gender-age adjusted associations in volunteers with complete data in all the risk factors is greater than the number reported in the Results where only the associations observed in multi-adjusted models are shown. In both sensitivity analyses, it appears that the variations in the HRs (95% CI) explaining the shift of some associations above or below statistical thresholds are small in the vast majority of the cases.The choice of using only volunteers with a complete set of data was made by considering that a multiple imputation approach in a cohort where the distribution of missing values is not monotonous , all but one  of the associations with incident CVD events and most of those with incident risk factors are conserved . Most importantly, this does not modify the conceptual framework summarizing the main findings of the study  were therefore performed to estimate the bias that may be introduced by considering these factors as time-independent. As shown in he study  whether An issue related to multiple testing is the risk of having false positives which increases with the number of tests performed (n = 17 × 9 × 3 in the present case). To take this problem into account, a Bonferroni correction could be applied in order to keep only the tests with p<0.0001 (0.05/(17 × 9 × 3)). However, this correction comes at the cost of increasing the probability of having false negatives, which would be clearly the case in this study where several well-known dose-dependent associations are observed at 0.0001<p<0.05. Thus, rather than choosing between keeping potential false positives or excluding potential false negatives, we decided to show all the associations with p<0.05 leaving open the possibility of narrowing the significance threshold to 0.01, 0.001 or 0.0001, depending on the priority of the reader. In fact, this issue is not critical as the main conclusions that can be drawn from the analyses, i.e., the existence of an extensive network of relationships between the main CVD risk factors, its conceptual framework and its potential interest in prevention, are fundamentally the same whatever the significance threshold which is selected.All analyses were performed with the statistical discovery software JMP Pro 12 .Table 1, a total of 1694 nonfatal CVD events occurred during follow-up  corresponding to 7.9 events per 1000 person-years. The distribution of these events according to the risk factors at baseline is shown in Table 2with the hazard ratios. All the factors independently predict CVD risk with multi-adjusted HRs varying from 1.18 to 1.97.Among the 10,736 volunteers retained for the analyses, whose baseline characteristics are shown in .Only one risk factor independently predicts the incidence of non-moderate alcohol consumption  or 46.0 cases per 1000 person-years) with multi-adjusted HRs ranging from 1.13 to 1.14 (Table 4).The incidence of smoking  or 6.4 cases per 1000 person-years) is independently predicted by two factors with multi-adjusted HRs varying from 1.18 to 1.29 (Table 5).Six factors independently predict the incidence of physical inactivity  or 22.6 cases per 1000 person-years) with multi-adjusted HRs ranging from 1.14 to 1.73  or 8.8 cases per 1000 person-years) is independently predicted by 5 factors with multi-adjusted HRs varying from 1.20 to 1.90.Table 7).The incidence of hypertension  or 19.9 cases per 1000 person-years) is independently predicted by 8 factors with multi-adjusted HRs ranging from 1.09 to 2.78 (Table 8).Nine factors independently predict the incidence of dyslipidemia  or 23.9 cases per 1000 person-years) with multi-adjusted HRs varying from 1.09 to 1.51  or 5.4 cases per 1000 person-years). with multi-adjusted HRs ranging from 1.15 to 6.12.Table 10).The incidence of sleep disorder  or 29.6 cases per 1000 person-years) is independently predicted by 5 factors with multi-adjusted HRs ranging from 1.13 to 1.88 (Table 11).Four factors independently predict the incidence of depression  or 15.1 cases per 1000 person-years) with multi-adjusted HRs varying from 1.11 to 1.78  with a number of predictive or predicted factors ranging respectively from 0 to 9 and from 0 to 7 at p<0.05 (from 0 to 5 and from 0 to 4 at p<0.0001) as reported in Table 13.Fig 1). Non-modifiable factors  obviously only predict and are not predicted by other factors. Behavioural factors  form very few associations with each other, they predict several clinical factors and are predicted by a small number of non-modifiable or clinical factors. Upstream clinical factors  form a few associations with each other, they predict many downstream clinical factors and are predicted by many non-modifiable or behavioural factors. Downstream clinical factors  form many associations with each other, they predict very few factors but are predicted by a large number of non-modifiable, behavioural factors or upstream clinical factors.The present study systematically reports prospective associations between the main behavioural or clinical CVD risk factors in a cohort of middle-aged volunteers free of CVD at baseline and followed over 20 years. The 12 factors retained for the analyses are all independently associated with the risk of CVD events in a dose-dependent manner for non-binary factors and in agreement with the literature , 36–42. In theory, the extent of this network of relationships between the risk factors underlines the necessity of considering all the factors together to evaluate CVD risk. Approaches such as structural analyses should allow to determine the relative contribution of each factor to the overall CVD risk and to explore what type of relationships  exists between the different factors . An adjaIn contrast, integrating this network in the design and implementation of primary or secondary preventive strategies aiming to lower CVD risk, whose efficiency has remained very disappointing , 27, mayThe present study has several limitations. A first one concerns the very limited set of CVD risk factors that have been tested among all those which are available . A seconThe present study assessed in a single cohort all prospective associations between the main behavioural and clinical CVD risk factors. It provides a global view of the extensive network of relationships formed by these factors in parallel with their independent associations with CVD risk, highlighting the inadequacy of considering a factor or even a few without taking into account the interactions with the other factors. The extent of this network is probably underestimated and not sufficiently taken into account in the preventive strategies aiming to lower CVD risk, explaining perhaps in part their lack of efficiency.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Exergames are interactive video games used for exercise and may have therapeutic value in people with degenerative ataxia. The purpose of this study was to investigate potential effects of exergaming training on cerebellar ataxia in people with spinocerebellar ataxia type 3 (SCA3). Nine individuals with SCA3 were recruited and randomized to either exergaming or conventional group for a 4-week training period. The severity of ataxia was measured as the primary outcome by the Scale for the Assessment and Rating of Ataxia (SARA) and by the directional control of the limit of stability test. The secondary outcomes included upper-limb function and gait performance. After training, participants in the exergaming group had a significant decrease in the total SARA score and the gait-posture SARA subscore. Participants in the conventional training group did not show a significant improvement in selected outcome measures after the 4-week training period. No significant difference was found between groups for any of these measures. Our results suggested that the exergaming training program significantly decreased ataxia. These results support implementation of exergaming training for people with SCA3. SCA3 is characterized by slowly progressive gait ataxia and is often associated with truncal ataxia, limb ataxia, and dysarthria3. There is no effective pharmacologic treatment for decreasing the ataxia or disease progression, although, physical therapy plays an important role in controlling ataxia and improving or maintaining function through exercise training4. In general, physical therapy programs for degenerative cerebellar ataxia are based on intensive static and dynamic balance and coordination exercises5. There is some evidence that such therapeutic exercise training alleviates ataxic symptoms and improves functional activities in people with cerebellar ataxia10.Spinocerebellar ataxia (SCA) is an autosomal, dominantly inherited neurodegenerative disease with multiple subtypes. More than 30 genetic subtypes have been described. The most common subtypes are SCA type 3 (SCA3) in many countries5. Exergames are video games that incorporate virtual reality and serve as an exercise tool. These video games usually involve balance and coordination challenges and help facilitate the participants’ adherence to the intervention. Exergames could present a novel, advantageous treatment tool for training individuals with SCA10. A previous study evaluated the effects of an 8-week balance and coordination training program  using Microsoft Xbox Kinect video games in children with progressive SCA11. This single-group study showed that the video game-based coordinative training alleviated several signs of ataxia in adolescents with progressive ataxia11. Recently, we developed a balance-based exergaming program and demonstrated that it had a positive effect on postural stability in people with Parkinson’s disease12. In the present study, we modified that program, adding elements for coordinative training. We conducted a randomized controlled pilot trial and evaluated the effects of a 4-week program with this system, compared with a 4-week period of conventional training (12 training sessions), on ataxia in adults with SCA3. We hypothesized that those who underwent a 4-week exergaming intervention would demonstrate equal or superior performance on outcome measures compared with those who underwent conventional training.A recent systematic review suggested that the use of virtual reality tools appears to have therapeutic value in people with degenerative ataxiaA total of 16 individuals were screened and 9 enrolled between 2014 and 2015. Among them, 5 were assigned to the exergaming group and 4 to the control training group. All participants were assessed before and after the 4-week training period. All 9 attended all training sessions. None of the participants reported adverse events. A flow diagram of the study protocol is shown in Fig. The demographic and clinical characteristics of participants in both groups are presented in Table P = 0.038) and total SARA score (P = 0.042). A near-significant trend for an improvement in the limb-kinetic SARA subscore (P = 0.059) was also observed after exergaming training. Control training showed a marginal trend to improve the limb-kinetic SARA subscore (P = 0.059) and total SARA score (P = 0.068). There were no significant differences between the two groups for any of these measures.The results of the interventions are presented in Table This study showed that the exergaming training program significantly improved the SARA scores in people with SCA3. The exergaming training, however, did not provide better results than conventional training with respect to decreasing ataxia or improving functional performance. To our knowledge, this is the first randomized controlled trial to show that exergaming training could be adopted to improve ataxia in adults with SCA3.8. The natural disease progression of SCA3 includes an annual increase in SARA score of 1.1–1.6 points15. These findings imply that exercise intervention may be able to postpone disease progression, especially the ataxia. Also, based on the results from those related studies, it seems that the more intensive the training, the greater is the reduction in the SARA score11.The current study tested two training programs with similar training principles and protocols. Both programs consisted of balance and coordination training. We recorded average decreases in SARA scores of 1.5 and 2.0 points after conventional training and exergaming training, respectively. These results are in line with previous findings, which reported that a 4-week course of intensive coordinative training or rehabilitative training decreased SARA scores by 2.8–5.2 points10. It is reasonable to speculate that the Kinect sensor provides specific motor practice using motion capture, which offers precise real-time information for monitoring limb movement and guiding performance. Our findings showed that exergaming training yields a specific effect on ataxia that goes beyond mere improvement in subjects’ game scores. Therefore, people with cerebellar ataxia have enough learning capacity to benefit from exercise or motor training programs. In addition, our previous study showed that the similar exergaming training significantly improved balance in individuals with Parkinson’s disease12. Consistently, people with SCA3 improved more of their ataxia concerning gait and posture, as indicated by their significant improvements in gait-posture SARA subscores. The mild effect of exergaming training on the limb-kinetic subscore, however, may be due to the small sample size or short training duration. A larger sample size and longer training may support additional benefits for exergaming training.In the current study, we recorded not only the total SARA score but also the limb-kinetic and gait-posture SARA subscores. Our results showed that only the exergaming training significantly reduced the gait-posture subscore. Limb ataxia also showed a marginal effect after the exergaming training. In the past, the beneficial effects of motor training had been challenged by the fact that the cerebellum is crucially involved in motor adaptation and motor learning5. In this study, we used the directional control during the limit of stability test to indicate trunk ataxia. It is not surprising that neither the exergaming or conventional training had a beneficial effect here as intensive rehabilitation programs with balance and coordination exercises are necessary in individuals with cerebellar ataxia10. Our previous study suggested that balance-based exergaming training using the Kinect sensor for 8 weeks resulted in positive effects on postural stability, with particularly strong effects on the directional control in people with Parkinson’s disease12. It has also been noted that intensive coordinative training that focuses on static and dynamic balance not only decreases the severity of ataxia but also improves gait speed and step length in people with cerebellar ataxia11. Gait performance, however, did not improve significantly after the exergaming or conventional training in the present study, although the severity of gait-posture ataxia decreased, especially after the exergaming training. We therefore suggest that the duration of balance training should focus on improving trunk and gait performance, especially for people with SCA3.Although SARA is the most frequently used tool to document the severity of ataxia, it does not focus on posture disordersOf note, all participants in the exergaming group were highly motivated throughout the whole training period. None of the participants reported adverse events. Thus, incorporating technology in rehabilitation  should be encouraged to reduce the severity of symptoms and/or improve functions. Training programs could be complemented with newly developed exergames or commercially available video games, an idea that offers a novel, advantageous training strategy for people with cerebellar ataxia.7. Larger randomized controlled clinical trials should be conducted in the future to measure functional performance after exergaming training and over the long term.There are several limitations in this study. First, the sample size was small, limiting the strength to interpret our results. Second, the disease was more severe among controls as indicated by nine-hole peg test and walking speed. Third, the study lacks an evaluation of a long-term period of follow-up to determine the impact on function over time, although the retention effects were proved in a previous studyThis study showed that a 4-week period of exergaming training decreased ataxia in people with SCA3. The results support the potential therapeutic use of exergaming for people with SCA3. Further studies on the use of exergaming are needed to verify the clinical implications of these results.The Institutional Review Board of Taipei Veterans General Hospital, Taiwan, granted ethical approval for this study. The study was registered at ClinicalTrials.gov Identifier NCT02900508 on September 8, 2016.This study was an assessor-blinded, randomized controlled trial. The study protocol was explained to all subjects before their participation (supplementary information). Those finally participating in the study gave their consent. The study was performed in accordance with the Declaration of Helsinki. All participants were randomly assigned by block randomization to the exergaming or control training group. They learned of their assignment via a sealed envelope. Participants in the exergaming and control training groups underwent either a 4-week exergaming intervention or 4 weeks of conventional training, respectively. Measures of ataxia, upper-limb function, and gait performance were recorded before and after the training period.Participants were recruited from a medical center in Taipei. Information on age, sex, the more affected side, and disease duration were obtained through patient interviews and from medical charts. All participants met the following inclusion criteria: (1) genetically confirmed diagnosis of SCA3; (2) ability to walk independently with or without walking aids; (3) age ≥20 years; and (4) a score of ≥24 on the mini-mental state examination. The exclusion criteria were as follows: (1) uncontrolled medical conditions  and (2) a history of other neurological, cardiovascular, or orthopedic diseases that affect motor performance and balance. In total, 16 individuals were identified as potential participants for the study. Among them, 9 gave informed consent and participated in the study.Participants in both groups underwent training for 40 min per session at three sessions per week for 4 weeks. Each training session began with a 5-min warm-up and ended with a 5-min cool-down. The warm-up period focused on stretching exercises of the trunk and extremities. The cool-down period focused on walking on a treadmill.Reaching task, for balance training: Participants were asked to reach the arm toward a stationary target at a given location.Pointing task, for lower-limb coordination training: Participants were asked to point the foot toward a stationary target at a given location.Following task, for upper-limb coordination training: Participants were asked to track an airplane with a hand as the airplane flew in 3D space.Avoiding task, for trunk coordination training: Participants were instructed to avoid upcoming obstacles that approached from varying directions at random by moving the body right/left or up/down.Participants in the exergaming group underwent a 30-min exergaming intervention using the Kinect sensor . The Kinect sensor incorporates infrared light and a video camera, which creates a three-dimensional (3D) map of the area in front of it. This device captures full-body 3D motion. Four exergaming training programs were designed to incorporate an appropriate level of challenge to match the ability and fitness of people with SCA Fig. .ReachingDuring the training duration, the challenge level was increased progressively by adjusting the posture , changing the base of support, amplitude, frequency, speed, complexity, and number of hints. The details of the exergaming programs are shown in Table Participants in the control training group underwent a 30-min conventional balance and coordination training session. The training program included reaching activities as well as upper-limb, lower-limb, and trunk coordination activities. The general training protocols used for the control training group were the same as those used for the exergaming group except for capturing the 3D motion. The challenge level was increased progressively by adjusting the posture and changing the base of support, speed, and complexity Table .Table 4C18. Possible total scores using the SARA range from 0 to 40 points, with a higher score indicating more severe ataxia. Four of the eight items in the SARA involve limb-kinetic function  and three involve gait and posture 20. We calculated the limb-kinetic subscore from the sum of finger chase, nose–finger test, fast alternating hand movements, and heel-shin slide and the gait-posture subscore from the sum of gait, stance, and sitting.The primary outcome measure for ataxia was the Scale for the Assessment and Rating of Ataxia. This scale, which consists of eight items, is a valid, highly reliable tool for evaluating SCAThe directional control of the limit of stability test by the Smart Balance Master  was first used to indicate possible trunk ataxia. The directional control is defined as the amount of movement in the intended direction minus the amount of extraneous movement. A directional control score of 100% indicates that the participant does not deviate from a straight path during the test. Therefore, the directional control was used to indicate possible trunk ataxia quantitatively during balance control in the standing position in this study. To assess the limit of stability, participants stood on a force plate and shifted their center of gravity to reach a maximum distance in the target direction as quickly and accurately as possible without moving their feet. The directions assessed included forward, less affected side, more affected side, and backward, in random order.The nine-hole peg test was used to assess upper-limb function. During the test, participants picked up the pegs one at a time and put them into the holes, in any order, until the holes were filled. They then removed the pegs one at a time and returned them to the container. The time needed to complete the test was recorded. Both upper limbs were tested twice, and the mean of the two tests for each limb was calculated. The more affected and less affected sides were identified based on the time needed to complete the test.The GAITRite system  was used to evaluate gait performance. It comprises a portable carpet walkway  with 16,128 embedded sensors along its length. The sampling rate of the system is 80 Hz. When the participant walks on the carpet, the sensors under the carpet collect data on spatial and temporal gait parameters. Participants were instructed to walk at a comfortable pace for five trials. The collected data were then averaged. Gait parameters of interest were velocity (cm/sec) and step width (cm).χ2 and Mann-Whitney U test for categorical and continuous variables, respectively. The Wilcoxon signed-rank test was performed for within-group comparisons. To adjust for between-group baseline differences, percentage changes of the variables were calculated and analyzed by the Mann-Whitney U test for between-group comparisons. Statistical significance was set at P ≤ 0.05.All analyses were performed using the SPSS 20.0 statistical package . The distributions of the variables were expressed as the medians and interval. The general characteristics of two groups were compared using the CONSORTClinical trial protocol


A fast and user-friendly computational for predicting the regioselectivity of electrophilic aromatic substitution reactions of heteroaromatic systems is presented. via a web server at ; http://www.regiosqm.org. RegioSQM should therefore be of practical use in the planning of organic synthesis.While computational prediction of chemical reactivity is possible it usually requires expert knowledge and there are relatively few computational tools that can be used by a bench chemist to help guide synthesis. The RegioSQM method for predicting the regioselectivity of electrophilic aromatic substitution reactions of heteroaromatic systems is presented in this paper. RegioSQM protonates all aromatic C–H carbon atoms and identifies those with the lowest free energies in chloroform using the PM3 semiempirical method as the most nucleophilic center. These positions are found to correlate qualitatively with the regiochemical outcome in a retrospective analysis of 96% of more than 525 literature examples of electrophilic aromatic halogenation reactions. The method is automated and requires only a SMILES string of the molecule of interest, which can easily be generated using chemical drawing programs such as ChemDraw. The computational cost is 1–10 minutes per molecule depending on size, using relatively modest computational resources and the method is freely available  Consequently, organic chemists tend to install the halogens early in the synthesis because they are not comfortable with late-stage functionalization.Heteroaromatics and benzene derivatives constitute important structural classes in drug discovery, agrochemistry, and material science. Their halogenated derivatives are often applied as substrates in carbon–carbon and carbon–heteroatom cross-coupling reactions such as Suzuki–Miyaura, Heck, and Buchwald–Hartwig couplings.1H and 13C chemical shifts can be applied to retrospectively account for the regiochemical outcome of ca. 80% of 130 EAS reactions from the literature. For many of the remaining cases, the regioselectivity was rationalized by visual inspection of the HOMO orbital computed using DFT, bringing the success rate up to ca. 95%.It was recently reported that empirically calculated et al.N-bromosuccinimide (NBS) to avoid any substrate protonation that might occur when using for example bromine in acetic acid. This precaution may not have been necessary as the vast majority of cases where both conditions have been applied led to the same regiochemical outcome of the halogenation reaction. The computational work is based on the observation by Streitwieser and others that the rates of many EAS reactions correlate well with equilibrium values for protonation in solution.i.e. the protonated regioisomer with the lowest free energy) corresponds to the most probable site for EAS, as was demonstrated by Wang and Streitwieser for several polycyclic aromatic hydrocarbons.Ka values of ionizable groups.In this paper the Kruszyk N-methylimidazole. For pyrazole the predictions point exclusively to the 4-position; indeed literature examples of the other regioisomer being formed were not identified. The situation is more complex for N-methylimidazole with both 4 and 5 positions as potential sites for the reaction. This correlates well with the moderate yield of 42%. Indeed the 4,5-dihalogenated adduct can be obtained in synthetically useful yields.N-methylimidazole can be halogenated which is not surprising given the fact the calculations suggest that the three possible arenium ions have similar standard free energies.N-benzylimidazole, which is in line with the predictions.et al.We used 118 reactions reported by Kruszyk  is computed using either PM6-DH+, is computed with the COSMOnrot, 20) where nrot is the number of rotatable bonds. Increasing 20 to 50 has no discernible effect on the accuracy as shown in ESI.N,N-dimethylformamide . Structures where protons have transferred and/or other bonds were broken are removed from the analysis. Gas phase, chloroform, and DMF were evaluated using PM3 and the COSMO solvation model in MOPAC. PM3/COSMO was chosen because it gave the best results in a previous study of amine pKa values.versus two cases for chloroform and DMF, respectively. Predictions with chloroform as a solvent resulted in six to eight incorrect predictions using PM6 and PM7 or AM1 and PM6-DH+ -dione and 64 bicyclic systems. Important aromatic systems like benzene and pyridine as well as indazole and 7-azaindole are well-represented with 16–214 examples, but the analysis also includes a number of less common heteroaromatics like pyridazin-3(2H)-one and imidazopyrimidine with 1 and 2 examples, respectively. This diversity of (hetero)aromatic cores serves to illustrate the applicability of RegioSQM as a predictive tool to guide organic synthesis. The full dataset is provided in the ESIThe analysis is based on more than 525 EAS reactions compiled from the literature. RegioSQM can rationalize the regiochemical outcome of 90% and 96% of these when using a 1.0 and 3.0 kcal moltert-butyl oxo-carbonyl (Boc), benzyl oxo-carbonyl (CBz) and tosyl/nosyl sulfonamides as well as synthetically useful functional groups like silyl ethers, amides, esters and lactones, succinate esters (HOSU-esters), alkynes and olefins, aldehydes, ketones, and ketals. The set also covers conjugated epoxides and acrylonitriles, Weinreb amides, enol ethers, primary alkyl and benzyl halides, phosphonates, and unprotected catechols. Indeed, the dataset was assembled to cover not only a diverse set of ring systems but also a wide range of typical as well as less common functional groups to demonstrate the usefulness of RegioSQM as a synthesis planing tool.In terms of functional groups attached directly to reacting aromatic rings the analysis covers more than 50 substituents that range from the four halogens to more exotic functionalities such as propellanes, imines, azides, and trifluoroborates. A subset of these groups is provided in 3, 4, 6, 11, and 12 are examples where only a single position is proposed at 1 kcal mol–1 leading to correct predictions (this applies to ca. 400 compounds in the dataset). Similarly there are ca. 40 cases including 5 with two proposed sites at 1 kcal mol–1 and where the corresponding bis-halogenated products were obtained. The incorrect prediction for 7 may reflect sterical hindrance due to tert-butyl-diphenylsilyl group or be a consequence of deprotonation of indazole under the somewhat unusual basic reaction conditions (KOtBu/N-chlorosuccinimide (NCS)). The corresponding tert-butyl-dimethylsilyl analog 8 illustrates that the 3-position of this ring system is also a reactive site. Furthermore, chlorination and nitration of 6-hydroxy-indazole occur at the 7-position in agreement with the predictions.9 and 10. Conversely, it is difficult to rationalize the regioselectivity for 13 although a directing effect of the primary alcohol cannot be ruled out. Imidazole 12 is reported to react with NCS at the 4-position in agreement with the predictions. However, bromination of the same substrate with Br2 has been reported to occur at the 2-positions of the imidazole ring.14.Broad functional group compatibility  is imporRegioSQM consists of several python scripts executed using two Linux Bash scripts, one that automatically generates all MOPAC input files given a list of SMILES strings and one that extracts energies from the MOPAC output files, identifies the most reactive arene atom(s), and generates a 2D representation of the molecule and highlights the likely site of reaction (see ESIH-imidazol-5-yl)pyridine (11) requires 2–3 minutes on a single CPU. A relatively large and flexible molecule like 6 requires 45–60 minutes on a single CPU. Each energy minimization is run on a single CPU, so the computation time is reduced almost linearly with the number of CPUs. For example by using 24 CPUs the time requirement for 6 is reduced to 2–3 minutes. The computational cost could be further reduced by using Merck molecular force field (MMFF)–1 energy cutoff.The computational cost per molecule depends on the size of the molecule and the number of energy minimizations, where the latter is a function of the number of rotatable bonds and the number of protonated isomers for a given molecule. The prediction of the most reactive site of a relatively small molecule like 3-.RegioSQM is a new computational method that identifies the aromatic carbon with highest proton affinity  using thfinities , RegioSQhttp://www.regiosqm.org.The method is based on the MOPAC software package, which is free for academic research, and the RDKit and OpenBabel toolkits, both of which are open source. The scripts that automate the calculation and the associated python codes are available on GitHub under the MIT open source license (see ESIUnlike our previous NMR-based method,http://www.regiosqm.org.The underlying dataset used to develop and test RegioSQM contains more than 80 different mono- and bicyclic aromatic cores bearing more than 50 functional groups directly on the rings that undergo the reaction as well as and more than 30 commonly used protective groups and important reactive functionalities in organic synthesis. This structural array not only illustrates the high chemoselectivity of EAS chemistry, but also that RegioSQM is applicable to essentially any molecule of interest. Consequently, chemists can apply the method in the planning of synthesis routes and confidently begin to contemplate novel approaches to target molecules where the strategic halogen atom be introduced at any stage of the synthesis pathway either to introduce a halogen in the final molecule, as protective groups to block more reactive sites to ensure the desired regiochemical control, or to introduce a halogen handle for a subsequent step. In other words, chemists would be in the position to predict the site selectivity of EAS reactions and focus on the synthesis of key scaffolds suitable for late-stage functionalization, for example to support efficient SAR explorations. Readers are encouraged to test RegioSQM on the webserver There are no conflicts to declare.Supplementary informationClick here for additional data file.


The study of genomic regions that contain gene copies and structural variation is a major challenge in modern genomics. Unlike variation involving single nucleotide changes, data on the variation of copy number is difficult to collect and few tools exist for analyzing the variation between individuals. The immunoglobulin heavy variable (IGHV) locus, which plays an integral role in the adaptive immune response, is an example of a complex genomic region that varies in gene copy number. Lack of standard methods to genotype this region prevents it from being included in association studies and is holding back the growing field of antibody repertoire analysis. Here we develop a method that takes short reads from high-throughput sequencing and outputs a genetic profile of the IGHV locus with the read coverage depth and a putative nucleotide sequence for each operationally defined gene cluster. Our operationally defined gene clusters aim to address a major challenge in studying the IGHV locus: the high sequence similarity between gene segments in different genomic locations. Tests on simulated data demonstrate that our approach can accurately determine the presence or absence of a gene cluster from reads as short as 70 bp. More detailed resolution on the copy number of gene clusters can be obtained from read coverage depth using longer reads . Detail at the nucleotide resolution of single copy genes (genes present in one copy per haplotype) can be determined with 250 bp reads. For IGHV genes with more than one copy, accurate nucleotide-resolution reconstruction is currently beyond the means of our approach. When applied to a family of European ancestry, our pipeline outputs genotypes that are consistent with the family pedigree, confirms existing multigene variants and suggests new copy number variants. This study paves the way for analyzing population-level patterns of variation in IGHV gene clusters in larger diverse datasets and for quantitatively handling regions of copy number variation in other structurally varying and complex loci. Regions of the human genome that vary in gene copy number are challenging to identify and analyze. This is particularly true for the immunoglobulin heavy variable locus (IGHV), which codes for a component of the antibody molecule. Previous approaches to interrogate the IGHV locus using locus-specific assays have provided detailed information about genetic variation, but tend to be low-throughput. Here, we introduce a method that leverages the increasing availability of large whole-genome sequencing datasets to genetically profile all functional IGHV genes in terms of a reference set of operationally defined gene clusters. We demonstrate this approach both on simulated data and on reads from a sixteen-member family of European descent. In the European family, not only did we find instances of known copy number variants, but also evidence of new variants. As larger, more diverse, datasets become available, our approach will allow the investigation of inter-individual copy number variation in larger samples for this and similarly hypervariable regions. The variation between human genomes in gene copy number is understudied and poorly characterized. One such region where this variation is known to exist is the immunoglobulin heavy variable (IGHV) locus. It is a vital component of the adaptive immune system, containing the V genes that code for a component of the heavy chain of antibody molecules. Like other multigene receptor families, the gene segments in this region have been accumulated over time through a process of gene duplication and diversification –3. As suThe human IGHV locus lies at the telomeric end of chromosome 14 and is approximately 1 Mb in length. In this 1 Mb region, there are about 40 functional genes, each approximately 300 bp in length. There are also approximately 80 non-functional pseudogenes in the region, so-called because they are either truncated or contain premature stop codons. Known allelic variants of individual IGHV genes are currently curated in the International Immunogenetics Information System (IMGT) Repertoire database [Given the role of the IGHV locus in the adaptive immune response, IGHV genotypes are obvious candidates as genetic determinants for susceptibility to infectious disease. Several early targeted studies of the IGHV locus have implicated allelic variation and copy number in determining expressed antibodies repertoires and understanding disease susceptibility , 15–18. Lack of tools for genotyping the IGHV locus also hampers the burgeoning field of antibody repertoire sequencing –24, whicThe increasing availability of whole-genome sequencing (WGS) provides a new opportunity to investigate genetic variation in the IGHV locus. Specifically, the large sample sizes of these WGS datasets and the high-throughput manner in which the data can be analyzed could provide valuable information. This approach can complement and guide genotyping efforts based on locus-specific assays –12. Yet Here, we address this pressing need for methods that quantitatively characterize the IGHV locus from WGS short-read data. By leveraging the IMGT database of known alleles, we construct a pipeline that gives a systematic description of the IGHV locus from short-read data. This description is in terms of a set of operationally defined gene clusters, so called because each cluster comprises IGHV alleles that are operationally indistinguishable. Our method does not attempt to reconstruct the organization of the locus or sequence the intergenic regions, both of which are important and challenging tasks. It does, however, allow the quantification of coarser measures of genetic variation. With reads as short as 70 bp and with coverage of 30×, our pipeline accurately detects the presence of gene clusters from simulated reads of the two known IGHV reference sequences (GRCh37 and GRCh38). With sufficiently long read lengths (250 bp), the pipeline also outputs accurate nucleotide sequences of gene segments present in single copy. We then run the pipeline on an empirical dataset of whole-genome sequencing reads from a sixteen member family, obtaining for the first time distributions of copy number in this family. Our copy number calls are consistent with the family pedigree and confirm known multigene variants of the IGHV locus. Our results also suggest evidence of copy number variants that are mosaics of the existing reference haplotypes and variants that might be transitional between them.The main difficulty in accurately genotyping the IGHV locus is the high level of similarity between alleles of different gene segments. This problem also occurs with other gene segments: across all full-length functional IMGT alleles, there is a 10.6% overlap in the distribution of nucleotide differences between alleles with the same segment name and alleles with distinct segment names . Reads fTo determine these gene clusters in a systematic manner, we perform hierarchical clustering within each family of full-length, functional IMGT alleles . By grouSome families have clearly defined gene clusters. In family 1, the gene clusters correspond to segment name, as long as duplicate segments 1-69D and 1-69 are merged . In famiSurprisingly, the same clustering algorithm that leads to clean gene clusters in the other families fails to identify clear-cut gene clusters in family 4 . Not onlgene cluster to refer to our operationally defined clusters and IMGT V gene segment to refer to the standard IMGT nomenclature. When an operationally defined gene cluster is the same as an IMGT V gene segment—e.g., in the case of segments in gene families 5, 6, and 7—we use the terms interchangeably. It may help the reader to keep in mind that with the exception of family 4, the majority of gene clusters coincide either with the IMGT V gene segment names, or with IMGT V gene segments merged with their duplicates .With the caveat that family 4 gene clusters are more speculative, The operationally defined gene clusters  address At the coarsest scale, we ask whether the pipeline correctly identifies the presence or absence of each gene cluster. We find the pipeline to be highly accurate, with precision of 100% for all coverage depth  and read length  combinations. This means that all the gene clusters identified by our pipeline are present in the reference. The recall, the fraction of gene clusters in the reference that are identified by our pipeline, is 100% for all but two of the coverage depth/read length combinations .At the next level of resolution, we ask whether the pipeline can correctly determine the copy number of each gene cluster. We use the read coverage depth of the assembled contig as our point estimate for copy number. At the highest level of resolution, we compare the assembled contig obtained from the pipeline to the known nucleotide sequence for each gene cluster. When a gene cluster is only present in single copy in the locus, and if the read lengths are 250 bp, the recall of the nucleotide sequence is 100% in all but one of the simulated datasets . With shWe next apply the pipeline to the publicly available Platinum Genomes dataset , a set oA summary of copy number and allelic variation in IGHV gene clusters in this dataset is shown in Thirteen out of the forty two gene clusters (about 30%) found in the family are each represented by the same single allele in all sixteen members of the family. The unique alleles corresponding to these clusters are denoted in We next looked for the presence in family members of two copy number variants that differ between the GRCh37 and GRCh38 reference haplotypes and that involve more than one gene segment or cluster . Using kIn the case of alternative haplotypes 1-8/3-9  on one chromosome but not the other . The insInterestingly, although all the children are homozygous for the GRCh37 1-69/2-70) haplotype without the insertion, three of them have the GRCh38 3-64D/5-10-1) haplotype on at least one chromosome. This implies that there are IGHV haplotypes different from both reference genomes and that these haplotypes are possibly mosaics of the reference genomes. Analysis of these two variants therefore not only confirms their presence in the Platinum Genomes sample, but also demonstrates that different configurations are present in the same ethnic population and suggests that many more configurations may exist. This is in line with resequencing efforts that have discovered novel sequences not found in GRCh37 –44, and D/5-10-1 /2-70 hapAmong all the gene clusters, 3-30 exhibited the most variation in coverage depth . This coUsing pedigree information as a constraint, we reconstructed the putative copy number of gene cluster 3-30 on each chromosome in each member of the family . Its abuWith the exception of 7-4-1, we found exact matches to IMGT alleles among all the segments in the Platinum Genomes dataset. In the case of 7-4-1, none of the assembled alleles exactly matched an existing IMGT 7-4-1 allele. To eliminate the possibility that the allele calls were confounded by reads from known pseudogenes, these alleles were determined after applying an extra filtering step to reads that mapped to the 7-4-1 segment . One of With the approach introduced here, we can begin to obtain population-level statistics on the IGHV locus from WGS data and systematically quantify variation with respect to operationally defined gene clusters. Given the small sample size of the Platinum Genomes data, we have focused here on quantifying variation in genes known to vary in copy number. As larger whole-genome sequencing datasets become available, it will be possible to compare IGHV copy number profiles at the population scale. These profiles can then be studied to find correlations between multiple gene segments and clusters and to discover new copy number variants. Even with the coarse measure of presence/absence of gene clusters, we can begin to address basic open questions such as whether there is a minimal number of IGHV gene clusters required for a healthy immune system and whether there is a common core set of IGHV gene clusters that are shared by all individuals.Our study makes clear that read depth information can be used to accurately determine the presence and absence of gene segments and clusters. However, complications remain for ascertaining copy number and allelic content to high accuracy. The first complication arises from the cell type on which whole-genome sequencing is commonly performed. The Platinum Genomes data were generated from immortalized B lymphocytes. The IGHV locus in these cell types have undergone VDJ recombination. This rearrangement, which truncates the IGHV locus, confounds the correlation between read coverage depth and copy number of a gene cluster. We can see this from the pipeline output, where coverage depth tends to decrease towards the centromeric end of the locus. The extent of this decrease can be quite marked, for example in the case of NA12877, or not noticeable at all, for example in NA12891 . If one The second complication is that the majority of whole-genome sequence reads are generated from diploid cells. Because the majority of segments on both chromosomes are of different alleles, the single allele call generated by our pipeline may be composed of sequence from all the alleles present or represent just one of the alleles. Allele calls can thus hide the heterozygous state of an individual. We note that there are many existing methods for estimating copy number based on coverage depth using whole-genome sequencing –52. ThesTrue determination of IGHV haplotypes must ultimately come from sequencing the 1 Mb region in its entirety and in multiple individuals. Indeed, because the GRCh37 reference is a chimera of three diploid haplotypes , there iThe fundamental strategy applied here is not specific to the IGHV locus. Reads from whole-genome sequencing datasets can similarly be used to characterize other gene families and in other species, where the genes are of comparable length and similar level of diversity. Some examples include T cell receptor genes and olfactory receptor genes. The use of whole-genome sequencing data therefore need not be restricted to single nucleotide variants, but can also be applied to study regions exhibiting copy number variation.IGHV genes are named according to their “family” and genomic location. The families, numbered 1 to 7, comprise genetically similar genes. The segment 6-1, for example, is in IGHV family 6 and is the first gene in the locus, counting from the centromeric end. Gene names with a suffix “D” denote a duplicate gene, for example 1-69D, while an appended number, for example 1-69-2, indicates that the gene was discovered subsequent to the original labeling and is located between 1-69 and 2-70. An allelic variant of an IGHV gene is denoted by a *01, *02, etc., as in 1-69*01, 1-69*02.hclust function in R [Nucleotide sequences for IGHV gene alleles were downloaded from the IMGT database . Only fuion in R  (method ion in R ) and indhttps://github.com/jyu429/IGHV-genotyping. We assume the WGS data is in BAM or SAM format [de novo assembler [Our scripts and example datasets are available at: M format , with reM format  is used ssembler  is run ossembler  to deterTo test the capabilities and quality of our methods, ART  was usedFor the Platinum Genomes data, which comprises paired-end reads, we apply an additional filtering step to remove reads from pseudogenes that share a common subsequence with a functional gene. One way to identify reads of a pseudogene is to compare its mapped position with the position of its mate. If the mate read maps to a region that is substantially farther from the region the first read maps to (we use a threshold of 1000 bp to be conservative) then there is a chance it comes from a pseudogene and the original read is discarded. de novo assembler.Alleles of 7-4-1 have high nucleotide similarity to subsequences of pseudogenes 7-81, 7-40, and 7-34-1. The mate-pair filtering step above does not apply to 7-4-1 because the Platinum Genomes reads are aligned to GRCh37, which does not contain 7-4-1. To filter out reads from these pseudogenes for 7-4-1, we ran stand-alone IgBLAST on reads mapped to segment 7-4-1. The reads that had the highest match to a pseudogene were removed. The remaining reads were then used as input for SPAdes S1 FigHeatmap color scale is same as in the main text, with red = 0% nucleotide differences, white = 10% or more.(PDF)Click here for additional data file.S2 FigHeatmap color scale is same as in the main text, with red = 0% nucleotide differences, white = 10% or more.(PDF)Click here for additional data file.S3 FigAllele labels are in cladogram above matrix. Heatmap color scale is same as in the main text, with red = 0% nucleotide differences, white = 10% or more.(PDF)Click here for additional data file.S4 Fig(A) Simple average of ‘TN93’ evolutionary distance and indel distance. (B) Hamming distance. Allele labels are in cladogram below matrix. Because TN93 and indel distances cannot be interpreted in terms of nucleotide similarity, the distances in each matrix have been normalized by the maximum value in the matrix for comparison. Heatmap color scale ranges from cyan = 0 to blue = 1. The clustering that uses ‘TN93’ and ‘indel’ distances has clearer block diagonal structure and fewer conflicts with IMGT nomenclature. It was therefore used to define the gene clusters for family 4 in (PDF)Click here for additional data file.S5 FigThe data points are the same as in (PDF)Click here for additional data file.S6 FigThe absence of 1-8/3-9 and 5-10-1/3-64D variants does not appear to be due to VDJ recombination because positive copy number calls are made for gene clusters left of 1-8 and 3-9 (and toward the recombination site). Y axis is normalized read coverage depth.(PDF)Click here for additional data file.S7 FigThe allele 7-4-1*04_5 was found in individuals NA12877, NA12878, NA12879, NA12883, NA12884, NA12886, NA12888, NA12891, and NA12893. Pairwise alignment was performed using the online IgBLAST tool .(PDF)Click here for additional data file.S8 FigAllele calls are arranged according to family pedigree. Only gene clusters for which there were two alleles in the family are shown (colored grey and white). Individuals for whom the gene cluster is not present are denoted by boxes with dashed outlines.(PDF)Click here for additional data file.S9 FigL), where L is the read length, reads originally from pseudogenes or similar functional segments are incorrectly mapped to 3-48, as seen by multiple vertical strips of dots. (B) With the threshold increased to 20 + 70 ln(L), a single diagonal row of dots indicates that only reads from 3-48 are mapped to segment 3-48. (C) When the threshold is increased to 20 + 85 ln(L) however, this is too restrictive and too few reads are mapped. Assessing analogous plots for the rest of the segments led to a threshold of 20 + 70 ln(L) being chosen. The README of the package provides more detail on how to modify the threshold. (Coordinates are for chromosome 14 on GRCh37).Axis values are centered at position chr14:1,062,766,005. (A) With default Bowtie2 local alignment threshold of 20 + 8.0 ln((PDF)Click here for additional data file.S10 FigPlots are shown for 30×, 40×, and 50× coverages and are only displayed for GRCh37 (plots for GRCh38 are similar). Note the high error rates for 100 bp and 70 bp reads. This difference is attributed to the fact that ART automatically selects one of several built-in read quality profiles according to the read length provided. Mutation rates are computed by first calculating, for each position in the read, the number of mismatches between the position of the simulated nucleotide and the original nucleotide. The number of mismatches was then divided by the total number of reads.(PDF)Click here for additional data file.S11 FigTo make the profiles for 70 bp and 100 bp comparable to that of 250 bp, the parameter for quality score shifting (-qs and -qs2) was used: 12.896 for 100bp and 7.99 for 70bp.(PDF)Click here for additional data file.S12 FigFor each individual, we calculated the fraction of reads that more closely match a pseudogene than an IMGT allele (blue). We then did the same calculation after filtering reads using the location of the paired read (green).(PDF)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S1 File(R)Click here for additional data file.S1 TableReads simulated from GRCh37 map to multiple IMGT alleles. Note that the in cases where reads map exactly to one allele, i.e. 3-72*01, 2-26*01, 1-24*01, and 3-20*01, these are the only full-length and functional alleles corresponding to a segment. Simulated reads are 100 bp long and have coverage depth of 30x.(PDF)Click here for additional data file.S2 TableWhen there is one allele listed for a gene cluster, that gene cluster is considered to be in single copy. If there are two alleles listed, the gene cluster has two copies.(PDF)Click here for additional data file.S3 TableData points in (TSV)Click here for additional data file.


We present a proof-of-principle study describing an alternative animal model of malignant infarction of the middle cerebral artery (MCA) in the common pig and illustrate some of its potential applications. We report on metabolic patterns, ionic profile, brain partial pressure of oxygen  using a frontotemporal approach. The core and penumbra areas were intraoperatively monitored to determine the metabolic and ionic profiles. To determine the infarct volume, 2,3,5-triphenyltetrazolium chloride staining and immunohistochemistry analysis was performed to determine SUR1 and TRPM4 expression.2 monitoring showed an abrupt reduction in values close to 0 mmHg after MCA occlusion in the core area. Hourly cerebral microdialysis showed that the infarcted tissue was characterized by reduced concentrations of glucose (0.03 mM) and pyruvate (0.003 mM) and increases in lactate levels (8.87mM), lactate-pyruvate ratio (4202), glycerol levels (588 μM), and potassium concentration (27.9 mmol/L). Immunohistochemical analysis showed increased expression of SUR1-TRPM4 channels.PtiOThe aim of the present proof-of-principle study was to document the feasibility of a large animal model of malignant MCA infarction by performing transcranial occlusion of the MCA in the common pig, as an alternative to lisencephalic animals. This model may be useful for detailed studies of cerebral ischemia mechanisms and the development of neuroprotective strategies. Stroke is the second most common cause of death and the third most common cause of disability-adjusted life years worldwide. One-thiThe term ‘malignant’ middle-cerebral artery (MCA) infarction was coined by Hacke et al. in 1996 to describe a form of IS that involved at least 50% of the MCA territory, followed an uniform clinical course, and resulted in transtentorial herniation and death in most patients despite optimal medical treatment. HoweverSus scrofa domestica) and to elucidate some of its potential advantages as an alternative to the few gyrencephalic models already available. Furthermore, this model will allow the use of all the modern neuromonitoring clinical tools that cannot be used easily in rodents.Pigs have been described as excellent experimental animals for medical research because of the similarities between human and porcine biology. The pig brain is gyrencephalic and has a white-gray matter ratio similar to that of the human brain–12. In a2), and the ionic profile of the extracellular space. We also explored at a molecular level the expression of sulfonylurea receptor 1 (SUR1) and transient receptor potential melastatin 4 (TRPM4), previously described in the brain of patients with multiple sclerosis brain), pyruvate ([Pyr]brain), glucose ([Glu]brain), and glycerol ([Gly]brain) were monitored using the point-of-care IscusFlex analyzer . After hourly measurements were completed, microvials were placed on a rack designed to seal them and prevent evaporation . All racks were stored at -20°C until ion analysis was carried out.Polarographic Clark-type electrodes  were connected to a tissue oxygen pressure monitor  to determine the infarct volume. Each brain slice was placed in a 60-mm dish, covered with 1% TTC solution , and incubated at 37°C under dark conditions for 30 minutes. Using TTC staining, viable gray matter was stained red or pink and infarcted tissue remained a pale cream or white color  were obtained. All samples were dehydrated with different alcohol solutions . Brain tissue samples were cryoprotected using 30% sucrose and embedded in a Tissue-Tek optimal cutting temperature compound . From these blocks, 10-μm sections were obtained using a cryostat , mounted on glass slides, and stored at -20°C until analysis was carried out. For immunohistochemistry analysis, sections were incubated in a blocking solution containing 2% donkey serum  and 0.2% Triton-X  in 0.1 M phosphate-buffered saline for 1 h. Next, cryosections were incubated for 1 hour at room temperature and then for 48 hours at 4°C with the primary antibodies goat anti-SUR1 1:100  and chicken anti-TRPM4 1:500  from the Image J 1.47v program . Using the total number of neurons and vessels , the percentage of SUR1/TRPM4-positive cells was calculated.To calculate SUR1/TRPM4-positive neurons and endothelial cells, between 4 and 8 randomly captured 440×330-μm2 images from the cortex (NEURONS) and the white and grey matter (VESSELS) were taken with an epifluorescence BX61 Olympus microscope. The primary antibodies used were mouse anti-NeuN 1:100  for neurons and CD31: mouse anti-CD31 1:100  for vessels. Next, all images were quantified using the plugin Cell Counter . Next, the whole section was quantified by a single observer using a semi-quantitative scale to count the following: 1) GFAP-positive cells  and 2) SUR1-positive cells and TRPM4-positive cells of each type .After hourly microdialysis measurements were completed, the microvials were placed in a rack designed to seal them and prevent evaporation . All racks were stored at -20°C until analysis could be performed. Prior to analysis, the microvials were defrosted on ice and the ionic profile was determined using an ICP-MS analyzer  with collision cell technology using He as inert gas at 5 mL/min. All concentrations obtained were corrected using a previously-defined linear model described previouslybrain. In all animals, the drop in [Glu]brain was followed by a significant drop in [Pyr]brain to undetectable levels and a significant increase in [Lac]brain, with a consequent increase in the lactate/pyruvate ratio (LPR)  . A typicbrain (~50%) with respect to the baseline values was observed in most animals. In 2 animals (#3 and #4) the decrease was more marked at the end of the experiment, indicating recruitment of the penumbra into the core. [Lac]brain increased in the penumbra, but it was less marked than in the core , while penumbra presented increased Na+ levels . After MCA clipping, ionic data were obtained hourly for the entire period in both brain regions. Due to the minimum required dialysate for the ionic analysis, it was not possible to obtain the medians, minimums, and maximums of the 3 ions during the baseline period. During the ischemic period the ionic profile in the core was compiled from 24 valid determinations, and in the penumbra area the ionic profile was made from 19 valid determinations. The ionic profile was significantly different for each monitored brain area. The ischemic core was characterized by an increase in median Kp = 0.01 in both cases). A summary of the semi-quantitative findings for SUR1-positivity and TRMP4-positivity are shown in p = 0.02) (All sections were examined by a single observer (LC), who carried out a quantitative or semi-quantitative analysis, depending on the cell type, for SUR1 and TRPM4 expression . In neur = 0.02) .Most treatments used to manage patients with IS  seek to obtain rapid recanalization of the occluded artery and reperfuse the ischemic brain to reduce the final amount of necrotic brain, 21. HowThere is a wide consensus that developing new therapeutic strategies in animal models is crucial despite the ethical concerns that animal experimentation raises. As some authors emphasize, ethical issues and animal welfare constitute an important limitation that should be clearly weighed against their scientific potential, medical benefit, and the availability of appropriate alternative approaches, 28. ForSus scrofa) but there are a large number of breeds with significant anatomical differences., 30.13, The second important difference between pigs and humans is that the posterior communicating artery in pigs is comparable in size to the ICA. As a result, the connection between the anterior and posterior circulation systems is very well developed when compared with humans. An additional important difference is that in each hemisphere 2 MCAs originate from the ICA, 1 coursing laterally and another rostrally over the olfactory tract.The same limitations raised by our group concerning the use of models in rodents led a team at the Aarhus University of Denmark to propose for the first time animal models of infarction using common pigs with transorbital occlusion of the MCA. That gr2 and cerebral MD probes to study the metabolic disorders induced by ischemia, as well as the ionic disturbances of the ischemic brain and its potential reversibility through use of various therapeutic strategies. The main disadvantages of using large animals are handling difficulties within experimental facilities and the increased workload for the research group resulting from multi-hour experiments. When considering which swine species to use when establishing an animal model, domestic pigs have several advantages over minipigs, including lower cost and greater availability.The main anatomical consideration in the model of permanent arterial occlusion we present is that MCA occlusion involves an open surgical frontotemporal approach and the clipping of both MCAs. After a learning curve, the total surgical procedure took approximately 60 minutes. We believe this model has many advantages over the classic rodent model. We found that our model gives a satisfactory representation of malignant IS with a dense, reproducible infarction that causes early death if untreated. Moreover, the percentage of infarcted volumes we obtained from animal modeling appears to be consistent and is comparable to the volumes obtained in the classical models of malignant infarction in rats–40. In a2 monitoring is a reliable surrogate measure of rCBF and can detect ischemic and non-ischemic causes of brain hypoxia, such as low-extractivity hypoxia, shunt hypoxia, or dysperfusion hypoxia[2 levels dropped immediately and were thus a reliable indicator of the complete occlusion of both MCAs. After a careful anatomical examination of the brain in the only animal in which PtiO2 values did not change, the probe’s tip was found to be located outside the ischemic lesion. To our knowledge, this is the first report of the ionic profile monitored hourly together with energy metabolism in a brain IS, with results that are consistent with the patterns found in the human brain[+ because it exclusively and non-selectively transports monovalent cations. In situations of ischemia there is an increase in the transcription of SUR1 that is accompanied by overexpression of TRPM4. TRPM4 channels are activated either by an increase in cytosolic Ca2+ or by a decrease in the ATP/ADP ratio in the cytosolic space. SUR1-TRPM4 is not constitutively present in cells of the central nervous system, but its transcription increases in neurons, the capillary endothelium, and astrocytes several hours after the onset of cerebral ischemia. In the absence of ATP this channel is activated, favoring edema and oncotic cell death due to the massive entry of ions[Continuous PtiOn hypoxia–43. In aman brain. Malignaman brain. These iman brain, 45. TRPy of ions, 18, 46.The use of brain MD in large animal models offers a unique opportunity to observe the dynamic ionic changes in the brain over time and opens up a new path to explore the ionic profile during edema formation and its changes after different therapies.brain increased with time and can be used in experimental models as a biomarker for the progression of the damaged brain. Glycerol is considered a biomarker of brain tissue damage and its concentrations rise during cellular energy failure and cell damage[brain is associated with the destruction of the cell membrane and cell death, or whether it is a marker of cell "suffering" with the possibility of reversal. To clarify this distinction, further studies using the same experimental model are needed.From a metabolic perspective, our model allowed us to reproduce the changes expected in permanent focal ischemia and detect the metabolic deterioration of the penumbra . Furtherll damage, 48. HowThe main limitation of our study was the small sample size inherent to most animal-based experimental studies. Both cost and ethical issues  constitute a limitation and the number of animals must be reduced to the minimum required to obtain answers to predefined scientific questions. The aim of this article is to present a proof-of-principle study, using a small sample of subjects as per Directive 2010/63/EU of the European Commission for the protection of animals used for scientific purposes. Due to its small sample size, our study did not try to look for statistically significant results, but rather described the feasibility of a large hemispheric stroke in the common pig and elucidated some of its potential advantages as an alternative to rodents. Therefore, we cannot rule out that increasing the sample size could introduce more variability in the final infarction size.A second limitation is that our study was not designed to reproduce a reperfused focal infarction, and therefore we cannot present data on the lesions found after reperfusion or report on the time needed to obtain a complete infarction versus a reversible focal lesion. Our main goal was to determine the feasibility of obtaining a reproducible malignant infarction, and transient ischemia was not an endpoint in our model. Another drawback is that our model was designed to study only the early phenomena occurring at the very first stages of complete arterial occlusion, and therefore we cannot present data on the survival of animals, the degree of brain swelling—which in humans with malignant stroke develops many hours after the ischemic insult—or the long-term neurological sequelae that have been extensively studied in rodents that survive an IS. Finally, we must also highlight as a possible limitation of our study the need for anesthetic agents with possible neuroprotective effects, including propofol, zolazepam, and mannitol. However, we emphasize the difficulty in limiting their use in big-animal models since they are needed to facilitate microsurgical access of the cisterns for the clipping of both MCA branches, thus minimizing injuries secondary to the cerebral retraction.We report on the development of a porcine model of malignant IS involving craniotomy and the clipping of both MCAs as a feasible model that allows for the study of both ischemia-induced metabolic disorders and disturbances in the ionic profile. We believe that this model provides an excellent opportunity to better understand the mechanisms of cerebral ischemia in a human-like gyrencephalic brain and the neglected importance of white matter in post-ischemic brain edema, aiding in the development of novel therapeutics that can potentially be translated to patients. In addition, this model may help in elucidating the mechanisms that lead to the recruitment of the penumbra to the infarction core and the pathophysiology of ischemic brain edema. Furthermore, it will allow researchers to test new therapeutic strategies that, alone or in combination, may target some of the many molecular cascades  that may be useful in reducing edema-induced brain swelling and therefore improving the functional outcome of patients with malignant IS. Our animal model allows for the study of the temporal profile of TRPM4 overexpression in the ischemic brain, as well as the other ionic and water channels involved in focal brain ischemia. More importantly, it may open up a clear line of research on the effects of SUR1-antagonists as neuroprotective drugs in gyrencephalic animals, the brains of which are very similar to those of humans.