Catch-up growth, an important risk factor for later obesity and type 2 diabetes, is often characterized by a high rate of fat deposition associated with hyperinsulinemia and glucose intolerance. We tested here the hypothesis that refeeding on a high-fat diet rich in essential polyunsaturated fatty acids (ePUFA) improves glucose homeostasis primarily by enhancing insulin sensitivity in skeletal muscles and adipose tissues.Rats were caloric restricted for 2 weeks followed by 1–2 weeks of isocaloric refeeding on either a low-fat (LF) diet, a high-fat (HF) diet based on animal fat and high in saturated and monounsaturated fatty acids (HF SMFA diet), or a HF diet based on vegetable oils (1:1 mixture of safflower and linseed oils) and rich in the essential fatty acids linoleic and α-linolenic acids (HF ePUFA diet). In addition to measuring body composition and a test of glucose tolerance, insulin sensitivity was assessed during hyperinsulinemic-euglycemic clamps at the whole-body level and in individual skeletal muscles and adipose tissue depots.Compared to animals refed the LF diet, those refed the HF-SMFA diet showed a higher rate of fat deposition, higher plasma insulin and glucose responses during the test of glucose tolerance, and markedly lower insulin-stimulated glucose utilization at the whole body level  and in adipose tissue depots (by 2–5 folds) during insulin clamps. While refeeding on the ePUFA diet prevented the increases in fat mass and in plasma insulin and glucose, the results of insulin clamps revealed that insulin-stimulated glucose utilization was not increased in skeletal muscles and only marginally higher in adipose tissues and at the whole-body level.These results suggest only a minor role for enhanced insulin sensitivity in the mechanisms by which diets high in ePUFA improves glucose homeostasis during catch-up growth. While the mechanisms linking catch-up growth to the pathogenesis of these chronic diseases remain elusive, the dynamic state of catch-up growth is often characterized by a disproportionately greater rate of body fat recovery relative to that of lean tissue, with hyperinsulinemia as an early feature of this ‘preferential catch-up fat’ phenomenon6.Catch-up growth has long been considered as an essential feature of recovery from the deleterious effects of perturbed growth on development and health. Since the turn of this millennium, the analysis of large epidemiological databases and clinical studies have suggested that catch-up growth is also a risk factor for the development of type 2 diabetes and cardiovascular diseases later in life8. This is achieved by an increase in insulin-stimulated glucose uptake in adipose tissues which, together with an enhancement in de novo lipogenesis, provide a quantitative important glucose sink8. Refeeding on a typical ‘western’ diet high in saturated fat, however, blunts the increased sensitivity of adipose tissues to insulin-stimulated glucose uptake, as well as its capacity for de novo lipogenesis9, so that dietary fat offsets the ability of adipose tissue to buffer against glucose spared as a result of skeletal muscle insulin resistance, leading to exacerbated hyperinsulinemia and glucose intolerance.Using a rat model of semistarvation-refeeding which exhibits preferential catch-up fat associated with hyperinsulinaemia even in the absence of hyperphagia, we previously showed that despite insulin resistance in skeletal muscle, the animals refed on a low-fat diet nonetheless achieved blood glucose homeostasis in that they show normal plasma glucose similar to those of the controls whether in the post absorptive state or in response to a glucose load11 involving refeeding isocaloric amounts of high-fat diets varying in fatty acid composition, have revealed major differences among the effects of fats derived from various sources of animals’ or plants’ origin on the recovery of fat and lean mass. Of particular interest was the demonstration that refeeding with diet enriched with oils rich in essential polyunsaturated fatty acids (ePUFA)—linoleic acid and/or α-linolenic acid—prevented the excessive fat deposition and exacerbation of hyperinsulinemia and impaired glucose homeostasis observed during catch-up fat on high-fat diets high in saturated and monounsaturated fatty acids12.In the search for dietary fat types that may counter such impaired glucose homeostasis and excessive fat deposition during catch-up fat, previous work in our laboratoryIn order to gain insights into mechanisms by which the high ePUFA diet improves glucose homeostasis during catch-up fat, we investigated here the impact of the high ePUFA diet specifically on glucose metabolism in skeletal muscle and adipose tissue. In particular, we tested the hypothesis here that refeeding on the high ePUFA diet would increase insulin sensitivity in both skeletal muscle and adipose tissues by measuring the in vivo glucose utilization in these tissues during hyperinsulinemic-euglycemic clamps, associated with the labeled 2-deoxy-glucose technique.Sprague-Dawley rats , 6 weeks old males, were adapted to room and cage environments for at least 5 days prior to the start of each experiment. They were caged singly in a controlled room (22 ± 1 °C) with a 12-h light–dark cycle, and maintained on a commercial pelleted chow diet  consisting, by energy, of 24% protein, 66% carbohydrate, and 10% fat, and had free access to tap water. Animals were maintained in accordance with the regulations and guidelines of the Department of Medicine, University of Fribourg, for the care and use of laboratory animals; all experimental procedures were performed under conditions approved by the Ethical Committee of the State of Fribourg Veterinary Office. Hyperinsulinemic-euglycemic clamps were conducted at the University of Geneva according to the procedures approved by the animal care and experimentation authorities of the Canton of Geneva, Switzerland.11. During the food restriction (semistarvation) period, the animals were fed a fixed ration diet of 14 g chow daily represented ~50% of their spontaneous ad libitum daily food intake. After 2 weeks of semistarvation that induced growth arrest, the animals with body weight in the range of 230–250 g were distributed into three groups (n = 8–9) such that each group had similar mean body weight and standard deviations, and was provided (non-blinded) with an isocaloric amount of one of three different diets: a low-fat (LF) diet or two high fat (HF) diets made either of lard, i.e. low in polyunsaturated fatty acids and high in saturated and monosaturated fatty acids (HF-SMFA diet), or a HF diet rich in essential polyunsaturated fatty acids (HF-ePUFA diet), the fat source consisting of a 1:1 mixture of safflower oil and linseed oil; the nutrient composition of these diets is provided as supplementary information (SI 12. All groups were thus provided with the same amount of ME intake (355 kJ/day/rat), which corresponds to that consumed during spontaneous food intake on pelleted chow; each refed rat consumed all the food provided on a daily basis throughout the entire refeeding period. The level of fat in the HF diets (58% of energy intake) corresponds to dietary fat levels often utilized in rehabilitation (energy-dense) diets of malnourished infants and children in order to meet their high-energy requirements for catch-up growth14. Four experiments semistarvation–refeeding of similar design were conducted with these three diets refed isocalorically. Experiment I was conducted to assess energy balance and body composition over 2 weeks of refeeding, with a test of glucose tolerance conducted on days 7–8. Experiments II and III were two insulin clamp studies (with high and low dose of insulin), each clamp study was performed after 7–8 days of refeeding. The last experiment (IV) was conducted to harvest adipose tissues for assessing key de-novo lipogenic enzymes after one week of refeeding on these three diets.The design was similar as previously reported using our rat model of semistarvation, with the rats studied being in an age range characterized by a high rate of weight gain during spontaneous growth15. Body water was calculated as the difference between body weight before and after drying, and the dry lean mass was determined by subtracting total body fat and body water content from body weight.After the animals were sacrificed, the whole carcasses were dried to a constant weight in an oven maintained at 70 °C and were subsequently homogenized for analysis of total fat content by the Soxhlet extraction method9. Food was removed early in the morning (07:00 h). At 6–7 h later, i.e. in the post-absorptive phase, blood was drained from the tail vein and immediately followed by an intraperitoneal injection of glucose (2 g/kg body weight). At intervals of 30 min for the next 2 h period, blood samples were taken from the tail vein in heparinized tubes and transferred on ice. The blood samples were then centrifuged, and the plasma was frozen and stored at −20 °C for later assays of plasma glucose and insulin. Plasma glucose was determined using a Beckman Glucose Analyzer , while plasma insulin was assessed using a rat insulin ELISA kit .Glucose tolerance tests were performed on days 7 and 8 of refeeding, according to the protocol described previously® . Surgeries were performed as previously described in detail17 and body temperature of rats was maintained at 37 °C throughout the study by means of a heating blanket. Glucose infusion rate (GIR) to maintain euglycemia was measured under basal and insulin-stimulated  conditions, as previously described17. Insulin was infused at a dose (18 mUI/kg/min) known to ensure complete suppression of hepatic glucose production16. In another separate insulin clamp experiment conducted at the same time-point of refeeding (day 7–8), the dose of insulin infused was halved (9 mUI/kg/min) such that plasma insulin could be clamped at values corresponding to peak plasma insulin values (12–15 ng/mL) previously found after a glucose load in this rat model showing catch-up fat9.On the day of insulin clamp experiments (day 7 or 8 of refeeding), rats were anesthetized (09:00 h) with Nembutald-[1-3H] glucose . A bolus of 2-deoxy-glucose was injected through the jugular vein and aliquots of arterial samples were regularly collected. After 30 min, rats were killed by decapitation and tissues rapidly removed and stored at –80 °C. The 2-deoxy-d-[1-3H] glucose-specific activity was determined in ZnSO4 and Ba(OH)2 deproteinized blood samples17. Measurement of tissue concentration of 2-deoxy-d-[1-3H]glucose-6-phosphate allowed calculation of the in vivo glucose utilization index of individual tissues, and was expressed in ng/min/mg of tissue. Plasma glucose and insulin levels were determined under basal and clamps conditions by the glucose oxidase method  and enzyme immunoassay , respectively.At the end of the insulin clamps, the in vivo insulin-stimulated glucose utilization index of individual tissues was determined using 2-deoxy-9; the details are provided as supplementary information (SI Fatty acid synthase (FAS) and glucose-6-phosphate dehydrogenase (G6PDH) activities were measured as previously reported9. All data are presented as means with their standard errors. The data was analyzed by one-way analysis of variance (ANOVA), followed by post-hoc pairwise comparisons using Scheffe’s test across the three diet groups; and differences were considered to be statistically significant at p < 0.05. The statistical treatment of data was performed using the computer software STATISTIX, version 8.0 . Statistical tests and group sizes are specified in the table legends.All rats were included in the analysis; none satisfied the pre-determined exclusion criteria of abnormal behavior such as hyperactivity or lack of food intake. A sample size of a minimum of seven animals per diet group was based on previous experiments that detected significant differences in key parameters of body composition and glucose homeostasis between low-fat and high-fat dietsp < 0.001), but similar gain in dry lean mass than animals refed the LF diet. The HF-ePUFA refed group, by contrast, showed significantly less gain in body fat but more gain in lean mass than those refed on the HF-SMFA diet. Thus, the HF-ePUFA diet abolished the differences in body fat between the HF-SMFA and LF groups, and resulting in a higher lean mass relative to the two other groups.The body weight and body composition profiles of the groups are shown in Fig. The results of the glucose tolerance test, conducted on days 7–8 of refeeding, are shown in Fig. p < 0.01), with the value for the HF-ePUFA group being lower than in the HF-SMFA by ~24% and 18% in the high-dose and low-dose clamp experiments, respectively. In both clamp experiments, there was also a marked difference across diets for the glucose infusion rate (GIR) with the two groups refed high-fat diets showing 25–50% lower GIR than in the LF group (p < 0.001). Relative to animals refed the LF diet, those refed the HF-SMFA and HF-ePUFA diets showed a 48% and 47% reduction in the GIR, respectively, in the high-dose clamp study, and a 32% and 23% reduction in the GIR, respectively, in the low-dose clamp study. Direct comparison between the two high-fat diets indicate a tendency for a higher GIR in the HF-ePUFA group than in the HF-SMFA group, particularly in the low-dose clamp study; however, no significant differences could be detected by a post-hoc pair-wise comparison test after ANOVA.Table The data on tissue-specific insulin-stimulated glucose utilization index (GUI) are presented in Fig. t-test shows borderline statistical significance.The data on GUI in adipose tissues indicate highly significant differences across diets for all adipose tissue depots studied , but on the HF-ePUFA, this reduction relative to the LF diet is less pronounced . Concerning G6PDH activity in EWAT, we observe the same tendency, namely a decrease of 41% on HF-SMFA diet and 19% for HF-ePUFA diet, with ANOVA analysis indicating significant difference across diets (p < 0.05). In IWAT, comparisons across all three diets indicate that FAS activity is significantly decreased on HF-SMFA and HF-ePUFA diets relative to LF rats , and there is a tendency for FAS activity to be higher on the HF-ePUFA diet than on the HF-SMFA diet. For G6PDH activity in IWAT, a significant decrease is observed in HF-SMFA rats relative to LF rats , but not in HF-ePUFA rats. Overall, the reduction in activity of these two key de novo lipogenic enzymes in the adipose tissues of rats refed the high fat diets (relative to the LF diet) is less pronounced during refeeding on the high ePUFA diet.The results of FAS and G6PDH activities in adipose tissues  are shown in Fig. 8. Isocaloric refeeding on a typical ‘western’ high fat (HF-SMFA) diet results in an even higher efficiency of fat deposition, exacerbated hyperinsulinemia9, persistent skeletal muscle resistance but blunting of adipose tissue insulin hyper-responsiveness pertaining to glucose utilization9. However, this exacerbation of the efficiency of fat deposition and excessive hyperinsulinemia observed with the HF-SMFA diet could be prevented by isocaloric refeeding on diets rich in ePUFA12. In our study here, we confirm the latter demonstrations and furthermore show that, contrary to our hypothesis, the efficacy of the e-PUFA diet in countering the impaired glucose homeostasis resulting from the HF-SMFA diet, does not reside in increased insulin sensitivity in skeletal muscle and is only marginally related to improved insulin sensitivity in adipose tissue.Previous studies in this rat model of semistarvation-refeeding have demonstrated that compared to spontaneous growing control animals, the efficiency of fat deposition during refeeding is increased on a low fat diet and is accompanied by an early development of hyperinsulinemia, skeletal muscle insulin resistance but increased adipose tissue insulin sensitivity, as assessed by the insulin clamp techniqueExamination of the data on plasma glucose and insulin in the basal post-absorptive state (i.e. prior to the test of glucose tolerance or prior to the insulin clamp studies) indicate that although basal plasma glucose was similar across the three diet groups, basal plasma insulin, however, tended to be lower in the ePUFA group. While a lower basal insulin requirement for maintaining basal glucose levels may be interpreted as being consistent with a state of higher whole-body insulin sensitivity, this remains merely an association and does not provide direct proof for a higher whole-body insulin sensitivity. Using the hyperinsulinemic-euglycemic clamp, which is widely considered the gold standard method for assessing insulin action in vivo, we found the glucose infusion rate (GIR) to be markedly lower in both HF groups than in the LF group. Although GIR tended to be higher in the HF-ePUFA group than in the HF-SMFA group, this difference was small and not statistically significant. Thus, the ability of the HF-ePUFA diet to lower basal insulin and to counter the higher insulin and glucose responses observed with the HF-SMFA diet during the test of glucose tolerance is not reflected in a significant improvement in whole-body insulin sensitivity assessed during the insulin clamps.12. However, the improvement in GUI in all WAT depots studied here is very modest as the HF-ePUFA diet only minimally prevented the marked drop in adipose tissue GUI observed in the HF-SMFA group relative to the LF group. Furthermore, the higher GUI in adipose tissues from animals refed the HF-ePUFA than in those refed the HF-SMFA diet is found to be either of borderline statistical significance or failed to achieve statistical significance in the WAT depots studied.The examination of data on tissue-specific insulin-stimulated glucose utilization index (GUI), assessed by the additional use of labeled 2-deoxyglucose at the end of the clamps, revealed that there is no increase in insulin sensitivity in skeletal muscle by the high ePUFA diet. Indeed, animals refed the HF-ePUFA diet did not show an increase in GUI in any of the six different skeletal muscles studied, thereby underscoring the fact that this lack of increase in muscle insulin sensitivity by HF-ePUFA refeeding is independent of skeletal muscle type and fiber composition. By contrast, our results in WAT may be interpreted as providing partial support to our hypothesis that the HF-ePUFA diet increases insulin sensitivity in adipose tissue, given the tendency for GUI to be higher than in WAT from animals refed the HF-ePUFA diet than on the HF-SMFA diet. These latter findings would be consistent with the tendency for a higher activity of key de novo lipogenic enzymes (FAS and G6PDH) in tissues from the HF-ePUFA than in the HF-SMFA group as demonstrated here in EWAT and IWAT, and also as previously reported by Crescenzo et al. in EWATTaken together, the results presented here suggest that while refeeding on the high ePUFA diet reduces basal plasma insulin and prevents the higher insulin and glucose response curve to a glucose load found during refeeding on the HF-SMFA diet, these improvements in glucose homeostasis are unlikely to be primarily attributed to an increase in insulin action on glucose uptake and utilization, as evidenced in the skeletal muscles, adipose tissues, and at the whole-body level. Consequently, the question arises as to what could be the other mechanisms in these or other tissues that may dispose of glucose, and account for the major impact of refeeding HF-ePUFA diet in reducing hyperinsulinemia and allowing the achievement of blood glucose homeostasis to that found in low-fat refed animals.18. The contraction of skeletal muscle in vitro increases glucose transport in the absence of insulin19, and insulin and muscle contraction stimulate the translocation of GLUT4 glucose transporters to the cell membrane (and consequently glucose uptake) through distinct pathways20. Among the signaling factors that may mediate pathways for glucose uptake in contracting skeletal muscle are the activation of AMP-activated protein kinase (AMPK)22, calcium concentrations23, nitric oxide25, and reactive oxygen species27, several of which have also been implicated in muscle glucose uptake independently of both insulin and muscle contraction18. They can be modulated by the neuroendocrine system , and could also be involved in the mechanisms by which ePUFA may influence the regulation of glucose and fatty acid metabolism in peripheral tissues. Such a contention would be consistent with the effects of diets enriched in n-3 PUFA in the prevention of weight gain and insulin resistance28, and which are thought to be mediated by adiponectin and leptin, two adipokines that regulate glucose and lipid metabolism, through AMPK activation31. Furthermore, the direct effects of leptin on skeletal muscle thermogenesis have been shown to require glucose entry through insulin-independent mechanisms that require intact phosphatidylinositol 3-kinase and AMPK signaling32.One explanation may reside in insulin-independent mechanisms by which high levels of ePUFA may stimulate glucose uptake and utilization in the skeletal muscle and other tissues. In fact, the existence of insulin-independent pathways for glucose uptake in skeletal muscle has long been known33. In particular, there is some evidence that PUFA may serve as a natural regulator of glucose uptake in vivo and these effects are mainly through PPARγ function34. Furthermore, isomers of conjugated linoleic acids have been shown to act on pathways that are both insulin-dependent and independent via PPARs, and that they can mimic insulin action by stimulating glucose uptake and GLUT4 trafficking through isomer-specific effects in stimulating PI3-kinase or AMPK signaling35.Whether one or more of these factors implicated in insulin-independent glucose uptake in response to muscle contraction or to leptin and adiponectin in muscle or other tissues may be stimulated by ePUFA is not known, but PUFAs are known to regulate the expression of genes encoding proteins involved in the regulation of energy metabolism by acting as agonist ligands of peroxisome proliferator-activated receptors (PPARs)Finally, it should be pointed out that the effects of ePUFA in diets based on safflower and linseed oils (as utilized here) might be mediated via their essential fatty acids (linoleic acid and α-linolenic acid) per se as well as via their elongated-desaturated products such as arachidonic acid, docosahexaenoic acid, and eicosapentaenoic acid. Whether the non-insulin-dependent pathways by which ePUFA (linoleic or linolenic acids) themselves or their metabolites are also involved in the efficacy of the HF-ePUFA diets to increase lean mass as shown here and previously is unknown, but a higher lean mass in its own right would increase the glucose buffering capacity and glucose clearance and hence contribute to improving glucose homeostasis.In conclusion, our study indicates that during refeeding on the high-fat rich in ePUFA, in vivo assessed insulin sensitivity pertaining to glucose utilization is increased only marginally in adipose tissues, and not at all in skeletal muscle, resulting in a marginal improvement in whole-body insulin sensitivity. It is argued on the basis of these findings that the efficacy of ePUFA in improving glucose homeostasis is only marginally contributed by increased insulin sensitivity, and that the major contribution to glucose disposal and blood glucose homeostasis during refeeding on a high ePUFA diet could reside in (i) insulin-independent mechanisms that stimulates glucose entry in skeletal muscle and other tissues and (ii) in increased protein retention in skeletal muscle and in other organs/tissues, thus providing an increased lean mass effect as glucose buffering capacity.Supplementary materials


High-throughput phenotyping (HTP) technologies can produce data on thousands of phenotypes per unit being monitored. These data can be used to breed for economically and environmentally relevant traits ; however, incorporating high-dimensional phenotypes in genetic analyses and in breeding schemes poses important statistical and computational challenges. To address this problem, we developed regularized selection indices; the methodology integrates techniques commonly used in high-dimensional phenotypic regressions  into the selection index (SI) framework. Using extensive data from CIMMYT’s  wheat breeding program we show that regularized SIs derived from hyper-spectral data offer consistently higher accuracy for grain yield than those achieved by standard SIs, and by vegetation indices commonly used to predict agronomic traits. Regularized SIs offer an effective approach to leverage HTP data that is routinely generated in agriculture; the methodology can also be used to conduct genetic studies using high-dimensional phenotypes that are often collected in humans and model organisms including body images and whole-genome gene expression profiles. Modern agricultural production systems use HTP data to optimize management practices4, forecast agricultural outputs5 and to assess the quality  of agricultural commodities6. HTP data can also be a valuable input for breeding programs. For instance, extensive HTP may enable an expansion of genetic testing that can lead to higher intensity of selection and faster genetic progress. Moreover, HTP data may offer opportunities to improve traits such as drought tolerance that are otherwise difficult to measure and breed for.High-throughput phenotyping (HTP) technologies have been adopted at a fast pace in agriculture; applications range from the use of HTP in highly controlled environments  in plant and animal breeding, respectively.Sensors can generate data on hundreds or thousands of phenotypes per unit being monitored. For example, hyper-spectral cameras can generate reflectance of electromagnetic power at hundreds of wavelengths in the visible and infrared spectrum. These measurements can be considered as indicator phenotypes that can be used to predict other traits. An extensive body of research deals with the use HTP data to predict phenotypes such as grain yieldyi  using information from another set of measured traits . A linear SI is a weighted sum of the measured phenotypes with weights derived to maximize the correlation between the genetic merit for the selection target and the SI. Thus, the SI methodology offers a natural framework for integrating HTP data into breeding decisions. However, when the measured phenotype is high-dimensional, the naïve application of the SI can lead to overfitting and sub-optimal accuracy of indirect selection.A SI seeks to improve a target trait 14. Using extensive multi-environment crop imaging data from CIMMYT’s wheat breeding program we show that regularized SIs offer improved accuracy of indirect selection in both optimal and stress environments.To address this problem, we developed regularized selection indices  that are tailored to achieve accurate prediction of genetic values using high-dimensional phenotypes. The proposed methodology integrates into the SI framework methods often used to prevent overfitting in high-dimensional phenotypic regressionsxi = ′, of the form β = ′ is a vector of regression coefficients whose entries define the weights of each of the measured phenotypes in the SI. In a standard SI the weights are derived by minimizing the expected squared deviation between the genetic merit for the selection goal (A selection index is a linear combination of Methods section):16.The solution to this optimization problem is (see The SI is by construction the best linear predictor (BLP) of the genetic merit for the selection goal; this property holds when 14 are two approaches commonly used to confront overfitting in high-dimensional regression problems. These methodologies were developed for regression problems involving an observable phenotype  and penalized regressionMethods). Thus, a reduced-rank SI (referred to as PC-SI) can be derived following these steps: (i) extract, using the singular value decomposition, ii) estimate the genetic covariances between the first iii) use these estimated (co)variances to derive coefficients associated with the top iv) transform these coefficients into coefficients for the measured phenotypes. This process can be done using optimal rank for the PC-SI can be chosen to maximize the accuracy of indirect selection.In principal components (PC) regression, the response is regressed on a reduced number :Using 2 in Eq.  renders 19), or in combination with the L2-norm, 18). Unlike the L2-PSI, the LASSO and elastic-net SIs  do not have a closed-form solution14. However, solutions for PSIs involving an L1-penalty can be obtained using iterative procedures such as the coordinate descent20 and the least angle regression21 (LARS) algorithms (see Methods). As with the PC-SI, an optimal PSI can be obtained by choosing the values of the regularizing parameters In many applications, variable selection  may be desirable. This property can be obtained using penalties involving the L1-norm, either alone, 16. To avoid estimation bias i) partitioned the data into training and testing sets, (ii) derived the coefficients of the SI in the training set, (iii) applied these coefficients to image data of the testing set (iv) estimated 16.Indirect selection accuracy is defined as the correlation between the index used to rank genotypes and the genetic merit of the selection objective, that is, mentclass2pt{minimn = 3,276) from the CIMMYT’s Global Wheat Program consisting of grain yield (ton ha−1) and hyper-spectral image data. The data were collected at CIMMYT’s experimental station in Ciudad Obregon, Sonora, Mexico  from 39 yield trials in which a total of 1,092 genotypes were tested. Rainfall in Obregon is very limited; therefore, four different environments were generated representing a combination of planting methods (Flat or Bed), controlled irrigation , and planting dates (optimum or early-heat). As expected, average yield decreased as drought stress intensity increased (see Table We applied the methodology described in the previous section to data (d ton ha− and hypeMethods section).Image data was collected using an infrared and an hyper-spectral camera and consisted of reflectance of electromagnetic power at 250 wavebands within the visible and near-infrared spectrums (392–850 nm). Separate images were collected at 9 time-points covering vegetative (VEG), grain filling (GF), and maturity (MAT) stages of the crop  are presented in Fig. Bed-2IR). Results from Tukey’s Honest Significance Difference confirmed that the accuracy of the regularized SIs is statistically different (higher) than the standard SI at a 5% of significance (Supplementary Table Bed-2IR) time-point/environment. Regularization increased the selection accuracy across time-points and environments. Regularized SIs (either PC-SI or L1-PSI) had an accuracy of indirect selection that was in average 10–40% higher than the accuracy achieved by a standard SI. These gains in accuracy were stronger in the optimal environment (Bed-5IR with a median of 36%) and smaller in the stressed environments (Flat-Drought and Bed-EHeat with a median of 16%). Interestingly, there were no sizable differences between the accuracy of indirect selection achieved with the optimal L1-PSI and that of the optimal PC-SI. Compared with a standard SI, regularized SIs had higher heritability  had considerably sparser indices with only a reduced number of wavebands in the solutions; these were mostly located in the violet, blue and red regions of the spectrum. In stressed environments (Flat-Drought and Bed-2IR) there were also a few wavebands in the green and infrared regions that were active. In all the indices, there were wavebands from several time-points that were active in the optimal solution, suggesting that data from both early and late phenological stages are informative about the genetic merit for grain yield.Figure 22, biomass, and chlorophyll content24. We considered two vegetation indices: the Red and Green Normalized Difference Vegetation Indices (RNDVI25 and GNDVI26 respectively). For each of these indices we estimated the genetic correlation with grain yield, as well as their heritability and accuracy of indirect selection  and compared the accuracy of indirect selection of these phenotypic prediction methods with that of penalized SIs. Overall, the L1-Phen achieved an accuracy of indirect selection very close to that of the L1-PSI  using HTP data as inputs. This approach is well-suited for phenotypic prediction; however, the same approach can be sub-optimal for selection because the best predictor of a phenotype is not always the best predictor of the genetic merit of the same trait.The best (linear) phenotypic predictor is the sum of the best linear predictor of the genetic merit ; it was exactly in those time-points and environments that the L1-PSI achieved higher accuracy of indirect selection than the L1-Phen method . Thus, the two methods yield different results whenever the patterns of phenotypic correlations are sufficiently different from the patterns of genetic correlations. In our data set, environmental conditions were highly controlled, with relatively low un-controlled within-trial variability in environmental conditions. Consequently, the patterns of phenotypic and genetic correlations were very similar  is, by To prevent overfitting, we considered integrating ideas commonly used in high-dimensional regression into the SI methodology. Our empirical results show that regularization consistently improves the accuracy of indirect selection relative to standard SIs. We verified this for various environmental conditions and for crop imaging data collected at 9 different time-points. The optimal PSI and the optimal PC-SI achieved almost the same accuracy of indirect selection for all the environments and time-points, suggesting that either type of regularization can be effective.27 or genomic-enabled28) using standard multi-trait models.Reduced-rank selection indices are appealing because after dimension reduction the problem of deriving a SI is trivial and can be dealt with methods commonly used to derive standard SIs. Moreover, after HTP has been reduced to a few derived-traits (say the top 10 PCs), these traits can be integrated into genetic evaluations . Building upon this idea, Dagnachew, Meuwissen & Ådnøy30 suggested predicting the genetic merit for milk fatty acids using FTIR-derived PCs as ‘traits’ in a genetic evaluation. However, when mapping from genetic predictions of PC-lodgings onto genetic predictions for the selection objective the authors used coefficients derived from a phenotypic  regression. This does not guarantee that the resulting index is maximally correlated with the genetic merit of the selection target. The penalized and PC-SI presented in this study address that problem by using coefficients that are derived using genetic (and not phenotypic) covariances.Principal components-based methods have been considered before in the analysis of Fourier-transformed infrared (FTIR) spectra derived from milk samples. For instance, Soyeurt, Misztal & GenglerA disadvantage of the PC-SI is that the methodology does not naturally provide variable selection, a feature that may be desirable when the measured phenotype is high-dimensional.Penalized selection indices can perform variable selection based on genetic covariances. While the derivation of a PSI is a bit more challenging than that of the PC-SI, the computational burden involved in the derivation of a PSI is not extremely high.The SIs considered here predict genetic merit for a selection target from a set of traits measured on an individual  may enable the expansion of the number of genotypes tested/measured as well as the number of locations where those genotypes are tested. This could lead to an increase in selection intensity which will in turn increase selection gains. For instance, if the use of HTP enables doubling the number of genotypes tested, the increase in selection gains that could be achieved with HTP may range from 20% (in the case where the PSI has RE of 60%) to 80% (for the traits/environments with RE of 90%).According to breeders’ equationThe discussion in the preceding paragraph is entirely based on breeders’ equation, which does not contemplate the long-term impacts of selection in genetic diversity. A more accurate and more intensive selection may affect diversity and long-term response to selection. To address this problem, attention to diversity will be needed with regularized SIs as with any other selection criteria.High-dimensional phenotypes are also becoming increasingly available in genetic studies involving human subjects and model organisms. Performing genetic studies  on high-dimensional phenotypes is challenging and the burden of multiple testing across hundreds or thousands of phenotypes  may critically compromise power. The PSI and PC-SI presented in this study could be used to extract genetic patterns from high-dimensional phenotype data such as brain imaging or whole-genome gene expression profiles and these patterns can then be used as traits in genetic studies.We proposed two novel methods for predicting the genetic merit for selection objectives from high-dimensional phenotypes. These phenotypes are becoming increasingly available as the adoption of HTP in crop and animal production increases. The proposed methods integrate regularization procedures commonly used in high-dimensional regressions into the SI methodology. Regularization prevents overfitting and increases the accuracy of the index. The methods proposed here can be used to extract genetic patterns from almost any kind of high-dimensional phenotype, including not only HTP data emerging in agriculture but also high-dimensional phenotypes that emerge in genetic studies involving human subjects and model organisms.The weights on a SI are derived as the solution to the optimization problem of Eq. :\documenThe right-hand side can be expressed as mentclass2pt{minimDifferentiating the right-hand side with respect to vector Recall that the singular value decomposition of a real-valued matrix, Using The objective function of the penalized SI is given by Eq. . Here weUsing an L2-norm as penalty, , in Eq.  leads toTherefore:The second and third right-hand side terms can be combined to obtain:The coefficients for the elastic-net family are obtained by considering an objective function as in Eq. , with \d14). These algorithms can be implemented either by ‘partial residuals’ or using ‘covariance updates’32. In our case, the objective function is entirely based on (co)variance terms. The objects et al.14) entirely based on covariance updates.The L1-PSI and L2-PSI are particular cases corresponding to Flat-Drought (sowing in flat with irrigation of 180 mm through drip system), Bed-2IR (sowing in bed with 2 irrigations approximately 250 mm), Bed-EHeat , and Bed-5IR . In 2013, all the trials were planted by mid-November , on the Bed-2IR and Bed-5IR) and on the Flat-Drought. Trials for Bed-EHeat were planted on October −1, total plot yield after maturity) was recorded.The data set consists of 1,092 inbred wheat lines grouped into 39 trials and grown during the 2013–2014 season at the Norman Borlaug experimental research station in Ciudad Obregon, Sonora, Mexico. Each trial consisted of 28 breeding lines that were arranged in an alpha-lattice design with three replicates and six sub-blocks. The trials were grown in four different environments: Reflectance data were collected from the fields using both infrared  and hyper-spectral  cameras mounted on a Piper PA-16 Clipper aircraft on 9 different dates (time-points) between January iid) normal with null mean and effect-specific variances. Likewise, the error terms were assumed to be iid with null mean and common error variance.Within each environment, grain yield phenotypic records were pre-adjusted by fitting the following mixed model,Grain yield data were pre-adjusted by subtracting from the phenotypic record ; for ease of exposition, hereinafter we refer to the adjusted-scaled phenotypes (including grain yield and the image data) simply as phenotypes.After pre-adjusting standardization, we analyzed phenotypes using a mixed model of the formm in Eq. ) of the 33. In each training-testing partition, out of the 29 trials available, 26 trials  in validation problems in previous studies involving milk spectra dataThe training-testing procedure described above was repeated 100 times by randomly assigning trials to training and testing sets. From these analyses, we reported the mean of heritability, genetic correlation, and selection accuracy; and their standard deviation across training-testing partitions.The population phenotypic (co)variance matrix The genetic covariance s in Eq. . We fittTo assess the accuracy of indirect selection we applied the regression coefficients derived in the training set to image data from the testing set to derive 34, version 3.5.1. Linear mixed models were implemented using the ‘lmer‘ function from the LME435 R-package. The software that implements the LARS and coordinate descent algorithms are available through the SFSI R-package (https://github.com/MarcooLopez/SFSI).All the aforementioned analyses were implemented in the R software environmentSupplementary information.


Enterovirus-induced infection of the central nervous system (CNS) results in acute inflammation of the brain  and constitutes a significant global burden to human health. These viruses are thought to be highly cytolytic, therefore normal brain function could be greatly compromised following enteroviral infection of the CNS. A further layer of complexity is added by evidence showing that some enteroviruses may establish a persistent infection within the CNS and eventually lead to pathogenesis of certain neurodegenerative disorders. Interestingly, enterovirus encephalitis is particularly common among young children, suggesting a potential causal link between the development of the neuroimmune system and enteroviral neuroinvasion. Although the CNS involvement in enterovirus infections is a relatively rare complication, it represents a serious underlying cause of mortality. Here we review a selection of enteroviruses that infect the CNS and discuss recent advances in the characterization of these enteroviruses with regard to their routes of CNS infection, tropism, virulence, and immune responses. Enteroviruses belong to the family Picornaviridae, a highly diverse group of small, non-enveloped, icosahedral-shaped viruses with single positive-strand RNA genomes. Based on the sequence diversity, they have been divided into 15 species: enterovirus A to L and rhinovirus A to C. Human enteroviruses containing four enterovirus species (A to D) and three rhinovirus species (A to C) infect millions of people worldwide every year. Although infections are frequently asymptomatic, human enteroviruses can cause a variety of symptoms comprising fever, headache, respiratory illness, sore throat, and, occasionally, vomiting and diarrhea. Importantly, several members of human enteroviruses are neurotropic pathogens with a wide range of clinical disorders ranging from aseptic meningitis to more severe encephalitis. In the United States, enterovirus has been shown to be the most common etiology of meningitis/encephalitis . About 5cis-acting RNA element that forms secondary and tertiary structures to allow cap-independent initiation of translation (pol) as well as some stable precursors, which function in virus replication and disrupting the host cellular immune system. Viral RNA replication is catalyzed by the virally encoded RNA-dependent RNA polymerases, 3Dpol, which utilizes Vpg as a protein primer to initiate the replication process. The synthesis of viral RNA takes place on the virus-induced remodeling of intracellular membranes that form replication organelles and leads to double-stranded RNA formation, which in turn will be transcribed into positive single-stranded RNA (The enterovirus genome contains approximately 7.5 kb with a single open reading frame flanked by 5′- and 3′-untranslated regions (UTR) and is enclosed in a capsid. While the 5′ end is covalently attached to a viral protein genome-linked (VPg) required for replication, the polyadenylated 3′ terminus is important not only for the negative-stranded RNA synthesis but also for translation and RNA stability . The lifnslation . Translanded RNA . Newly strans-synaptic propagation  infected by EV-A71 does not display cytopathic effect and the viral particles adopt a non-lytic exit pathway through autophagy  about 50 years after the primary infection. Some studies have shown that post-polio syndrome is correlated with the presence of viral RNA in the CNS, suggesting that PV can persist and cause long-term damage in the CNS . In cons animals . Interesutophagy . Furtherutophagy . Taken tSome enteroviruses can cause diseases within the nervous system. The most thoroughly studied neurovirulent enterovirus is PV. To eradicate global poliomyelitis, much effort has been made to develop polio vaccines including an inactivated PV given by injection and an attenuated PV given by mouth. Although both types of polio vaccines are effective, oral polio vaccines are superior not only in administration but also in providing longer-lasting immunity. The attenuated PV was developed based on the observation that the virus no longer caused disease after many passages in different animals and cell cultures, but replicated sufficiently to induce protective immunity. Genetic analysis of the attenuated PV has shown that a point mutation within the IRES of the vaccine strains is a critical determinant of the attenuation phenotype . For insEV-A71 has emerged as a serious threat to public health across the Asia-Pacific region. EV-A71 causes hand, foot and mouth disease (HFMD) and herpangina, and occasionally severe neurological disorders. Unlike PV in which a point mutation within the IRES can attenuate neurovirulence, the association between EV-A71 neurovirulence and viral genome sequences remains largely unknown. So far, there are only a few reports showing that the neurovirulence can be weakened by mutations in the viral genome of EV-A71. For example, studies have shown that defined genetic manipulation of the EV-A71 genome based on the temperature-sensitive determinants of a poliovirus vaccine strain results in attenuated neurovirulence in monkeys . The genEV-D68 is another non-polio enterovirus that has an association with a polio-like neurological disorder known as acute flaccid myelitis (AFM) with symptoms such as dysneuria and muscle weakness although the most common clinical symptom of EV-D68 infection is respiratory illness . EV-D68 About 3% of enterovirus infected people will develop encephalitis while most people never show evidence of CNS infection . Since eEnterovirus encephalitis is reported to occur more frequently in younger children. Although the exact cause remains unknown, it has been suggested that a compromised or immature immune system may be involved in the observed effect. Studies have shown that expression of CD40-ligand on activated T cells and interleukin 4 (IL-4) production are significantly lower in EV-A71 infected children with meningoencephalitis than those without it . CD40-liAlthough genetic susceptibility may provide clues to why enterovirus encephalitis only occurs in some children but not in others, it cannot explain why younger children infected with enteroviruses are at higher risk for developing encephalitis. It has been suggested that the immaturity of the BBB in early development may account, at least in part, for the age-related difference in viral neuroinvasion . The devThe CNS immune system may also be involved in the age-related difference in developing enterovirus encephalitis. Microglia, the primary resident immune cells of the brain, play a key role in regulating signaling pathways during CNS inflammation . Althougin vivo is largely unknown and how enteroviruses gain access to and spread in the well-protected CNS remains to be explored. For example, a common feature of enteroviral encephalitis is the involvement of brainstem (Enterovirus infection is a major public health concern considering the increase in outbreaks of serious neurological complications. Although there has been considerable progress in studying the complex interplay between enteroviruses and the infected cell in a culture dish, more complicated interaction between virus and host rainstem , but somrainstem , suggestAll authors contributed to the writing and discussion of this review article and approved the final version of the manuscript. H-CL made the figure and table.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Juvenile idiopathic arthritis (JIA) affecting temporomandibular joints (TMJ) in growing patients results in maxillofacial deformities, especially if only one condyle has been affected by the rheumatic disease. Mandibular hypoplasia is the most common issue and it may be associated with maxillary hypoplasia. The aim of this retrospective case-control study is to evaluate the effects of rapid maxillary expansion (RME) in these patients. Methods: 25 growing patients affected by maxillary hypoplasia, currently in a quiescent phase of JIA for at least one year and monolateral involvement of the TMJs, were treated with RME. Data gathered from posteroanterior and lateral cephalograms before and after 1 year from RME were compared to those of 25 non-JIA controls. Results: Nasal cavity width, maxillary width and upper and lower intermolar width statistically increased. Maxillary and mandibular symmetry indexes presented a statistically significant increase, so did the skeletal class. No signs or symptoms of TMJ activity of JIA occurred according to Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) criteria. No difference was found when comparing JIA and non-JIA patients apart from the better improvement of several mandibular symmetry indexes in the affected TMJ side of JIA patients. This event is allegedly due to a worse baseline asymmetry in JIA patients that underwent a bigger relative improvement after treatment. Conclusions: Results suggest that solving maxillary hypoplasia and, therefore, premature contacts are likely to have allowed mandibular repositioning and condylar growth. RME is a safe and effective solution that can substantially improve maxillary and mandibular symmetry in growing patients affected by JIA with TMJ involvement. Juvenile idiopathic arthritis (JIA) include a heterogeneous group of conditions of uncertain aetiology defined as the chronic inflammation of articular connective tissue for a minimum of 6 weeks, before the of age 16 ,3. JIA iThe temporomandibular joint (TMJ) is involved in 38–72% of cases, depending on the JIA subtype examined and on the diagnostic method used ,5,6. TMJMedical literature on orthodontic treatment in patients suffering from this rheumatic condition is scarce . Recent As far as the authors are aware, there have been no previous studies which have evaluated rapid maxillary expansion in JIA patients. Effects of interceptive orthodontic treatment on the maxilla of JIA patients are not reported in literature. In 2017, Menezes et al. described a case of a patient treated with a quad-helix appliance in the maxilla and a Nance lingual arch in the mandible. However, they did not evaluate the effect on the maxilla apart from cross-bite correction . JIA is RME have already been demonstrated to cause an augmentation of SNB angle, which measures the antero-posterior positioning of the mandible in relation to the cranial base (partly due to condylar repositioning inside the glenoid fossa and partly to condylar growth) , to reduA retrospective case-control study was performed analyzing postero-anterior and lateral cephalograms before and after maxillary expansion of patients affected by JIA and comparing them with non-JIA controls.The study protocol was approved by the Ethical Committee of the Fondazione IRCCS Ca’Granda, Ospedale Maggiore, Milan-Italy (protocol n.573/15). All patients’ parents provided written informed consent allowing the anonymous use of the patients’ medical records for research purposes regarding all the procedures performed. This study was performed according to the Declaration of Helsinki for human studies.The records of 25 Caucasian patients with a diagnosis of JIA with TMJ involvement  were selected from the archives of the Department of Biomedical Surgical and Dental Sciences, University of Milan, Italy. Inclusion criteria for the selection of patients affected by JIA were: diagnosis of JIA with unilateral TMJ involvement; transverse maxillary hypoplasia  treated with a rapid maxillary expansion protocol with a hyrax type expander; treatment beginning during a JIA quiescent phase that lasted for at least 12 months; TMJ involvement classified as Grade 3 according to Cahill classification (juxta-articular erosions without any sign of an active inflammatory process in MRI scans) ; presencInclusion criteria for the control group in order to obtain a clinically meaningful comparison were: transverse maxillary hypoplasia  treated with a rapid maxillary expansion protocol with a hyrax type expander; skeletal class II, no malformation of the craniofacial area; no previous orthodontic treatment; presence of lateral and postero-anterior radiographs before and after expansion. The records of 25 non-JIA Caucasian patients matched with sex and age  treated with RME with the same diagnosis served as controls.Exclusion criteria in both groups were congenital anomalies, dental anomalies , and previous orthodontic treatment.All patients from both groups were treated with Hyrax palatal expander to correct maxillary transverse hypoplasia, to treat posterior cross bite and to increase the space within the arch, between 2013 and 2016 in the Orthodontic Department of Fondazione IRCCS Ca‘Granda Ospedale Maggiore Policlinico of Milan (University of Milan).A Hyrax-type rapid maxillary expander was bonded to the maxillary second primary molars using a glass-ionomer orthodontic luting cement . Two activations of the hyrax screw were performed by the orthodontist, and two by the patient’s parents as training immediately after appliance luting (0.25 mm per activation). On the following day, activations were prescribed twice a day (0.50 mm) until the next follow-up appointment after 7 days when patients were re-evaluated, and the clinician would decide whether to end or continue the activations. In the latter case a maximum of 14 additional activations would have been prescribed and patients would have been re-evaluated each for 7 days until the clinician decided to end activations. The therapeutic goal was achieved when the patients presented a dental overcorrection, that is when the contact between the palatal cusps of maxillary first permanent molars and the lower first permanent molars occurred at the edge of the lingual side of buccal cusps . The actTeleradiographs in lateral and postero-anterior projection of all patients’ skulls were obtained from the same machine Orthophos XG with fixed focus sensor distance (150 cm), at the Dental and Maxillofacial Department of the Fondazione IRCCS Ca ´ Granda, Ospedale Maggiore Policlinico, Milan, Italy. Digital cephalometric tracings were performed by an operator using Dolphin Imaging software , which computed all reported measurements. Three weeks later, the tracings were made by a different operator and retraced with the first one in order to evaluate intra- and inter-operator variability on 15 randomly selected lateral and postero-anterior radiographs. Intraclass correlation coefficients (ICC) were calculated to compare intra- and inter-operator variability. Correlation coefficients for the skeletal measurements were greater than 0.92. Linear measurement errors averaged 0.4 mm (standard deviation (SD) 0.4 mm) and angular measurements averaged 0.6° (SD 0.5°). Method error was considered negligible.Fifteen points from postero-anterior cephalograms  were conFourteen points from lateral cephalograms  were con® 23 for Windows  was used for the statistical analysis.Sample size was calculated a priori using G*Power (version 3.1.9) based on a primary outcome (SNB angle) to obtain a statistical power of the study greater than 0.80 at an alpha of 0.05, using the mean values and standard deviations of SNB with a lateral cephalometric study by Baratieri et al.  The valut-test was applied to assess the difference before and after maxillary expansion on lateral and postero-anterior cephalograms in JIA patients and in the non-JIA control group.The Shapiro–Wilk test showed that data were distributed normally. Measurements have been reported as mean and standard deviation. A paired t-test was used to evaluate whether the difference before and after treatment of each parameter between the two groups was significantly different, and whether the difference between the Non-affected and Affected side in JIA group and between the right and left side in the control group were significantly different. A p value <0.05 was considered as statistically significant.An independent samples 2-tailed Descriptive statistics and statistical comparisons of postero-anterior and lateral measurements before (T0) and after treatment (T1) with RME in patients affected by JIA are shown in p < 0.05) when comparing measurements on postero-anterior cephalograms both before and after RME for the following parameters: maxillary transverse dimension (Mx r-l); upper intermolar width (Cvm+ r-l); nasal cavity width (NL r-l); linear distance between the affected condyle and the axis of symmetry (Cd A-MID); linear difference between affected/non-affected condyle and the axis of symmetry (ΔCd NA/A-MID); linear distance between the non-affected gonion and the axis of symmetry (Go NA-MID); linear distance between the affected gonion and the axis of symmetry (Go A-MID); linear difference between affected/non-affected gonion and the axis of symmetry (ΔGo NA/A -MID); linear distance between menton and the axis of symmetry (Me-MID); linear distance between incision inferior frontale and the axis of symmetry (Iif-MID); linear distance between incision superior frontale and the axis of symmetry (Isf-MID) (p >0.05) were noticed for the linear distance between the non-affected condyle and the axis of symmetry (Cd NA-MID), and the linear distance between incision superior frontale and the axis of symmetry (Isf-MID)  . No statIsf-MID) .p < 0.05)  .Descriptive statistics and statistical comparisons of postero-anterior and lateral measurements before (T0) and after treatment (T1) with RME in the non-JIA control group are shown in p < 0.05) has been noticed comparing postero-anterior cephalograms before and after RME in the control group for the following parameters: maxillary transverse dimension (Mx r-l); upper intermolar width (Cvm+ r-l); nasal cavity width (NL r-l); linear distance between the condyle closer to the axis of symmetry and the axis of symmetry (Cd A-MID); linear distance between incision inferior frontale and the axis of symmetry (Iif-MID). No statistically significant difference (p > 0.05) was found for all the other measurements on postero-anterio cephalograms  has been found for all the measurements on the lateral cephalogram in the control group  has been noticed comparing the effect of maxillary expansion between the two groups for: linear difference between affected and non-affected condyle and the axis of symmetry of JIA group and linear difference between the condyle more distant from and the one closer to the axis of symmetry in control patients (ΔCd NA/A-MID), linear distance between the affected gonion and the axis of symmetry in the JIA group and the homologous parameter in control group (Go A-MID), linear difference between the affected/non-affected gonion and the axis of symmetry in JIA group and the homologous parameter in control group (ΔGo NA/A-MID); linear distance between menton to the axis of symmetry (Me-MID) (p > 0.05) between the two groups for all the other measurements evaluated on postero-anterior cephalogram  . RME shohalogram .p > 0.05) has been found for all the measurements traced on lateral cephalogram comparing the effect of RME in the JIA and control groups . All patients responded negatively according to RDC-TMD IIIa criteria . No reocCurrent available evidence on interceptive orthodontic treatment in patients suffering from JIA is low ,17,41. MResnick et al. reported the results of an international consensus conference that took place in 2019 stating that in developing patients with no or minimal active TMJ disease, interceptive functional orthodontic treatment is recommended. This therapeutic choice may help in avoiding orthognathic surgical therapy at the end of growth or at least in reducing the complexity of surgical intervention .Maxillary transverse hypoplasia may be observed in patients affected by this rheumatic condition . The autIn the present study, a statistically significant augmentation in maxillary transverse dimensions  after RME was found both in JIA and in control subjects. The aforementioned values are comparable to those reported by previous studies that described the effect of rapid maxillary expansion appliances . No releSolving cross bite and premature contact connected to dental crowding promotes asymmetry reduction and avoidance of dental interferences during function that could cause a worsening of joint inflammation and therefore of mandibular asymmetry ,50,51,52Our study on postero-anterior cephalograms evaluated whether JIA patients presented any sign of mandibular asymmetry improvement after treatment with RME. The results showed how both condyle (Cd A-MID) and gonion (Go A-MID) of the side involved in the rheumatic degeneration underwent a statistically significant improvement in their distance from the axis of symmetry. Both Δ Cd NA/A-MID and Δ Go NA/A-MID, which measure the asymmetry as the difference in linear distances from the axis of symmetry between affected and non-affected condylar and gonion points respectively, showed a statistically significant improvement (reduction) after treatment with RME. . Taking The menton (Me) and the incision inferior frontale (IIF) points improved by a statistically significant amount  as they Non-JIA patients did not show any statistically significant improvement for all the asymmetry indexes assessed on postero-anterior cephalograms, because they did not present any significant asymmetry prior to the expansion . The onlRME could also have positive effect on TMJ health of JIA patients, as the increase in condylar space and the promotion of forward repositioning of mandibular posture could reduce condylar functional stress  and promThe comparison between the affected and unaffected side in the JIA group  showed aCertain limitations of the study should be noted, including: the analysis focused only on the bidimensional radiographs, not considering the growth of the mandible because of the obvious limitations of two-dimensional cephalometric analysis.The high significance achieved for all measurements allows us to clearly conclude that JIA patients undergoing RME have experienced benefits both in treating maxillary hypoplasia and in partially solving positional asymmetry caused by premature contacts and cross-bite. As already proven, besides promoting reduction of asymmetry indexes maxillary expansion improved mandibular growth thus reducing skeletal second class.The number of patients included in this study, although sufficient to identify statistically significant differences, is small in order to generalize its findings. Future studies should investigate a larger sample of subjects affected by JIA considering also the subtype of JIA present.Orthodontists in cooperation with pediatric rheumatologists should be trained to recognize dentofacial alterations in patients suffering from JIA. Regular clinical examination and early treatment, together with progressive monitoring of the craniofacial development, is advised. Treatment of JIA patients in the quiescent phase with maxillary constriction using RME is a suitable option for all patients. that show no signs of disease relapse at TMJ level for about 1 year. Clinical indications are the same as for non-JIA patients. In both, it was possible to observe an improvement in nasal and maxillary width. Maxillary hypoplasia and cross-bite were solved, and as additional positive effects both mandibular asymmetry and skeletal class II improved.The most important effect of RME was the improvement of mandibular asymmetry and the resolution of mandibular shift. Further studies on RME treatment of patients affected by JIA should be carried out to thoroughly understand its three-dimensional effects on maxillofacial conditions that affects these patients as the present results appear promising.


Genomic selection with genome-wide distributed molecular markers has evolved into a well-implemented tool in many breeding programs during the last decade. The resistance against Fusarium head blight (FHB) in wheat is probably one of the most thoroughly studied systems within this framework. Aside from the genome, other biological strata like the transcriptome have likewise shown some potential in predictive breeding strategies but have not yet been investigated for the FHB-wheat pathosystem. The aims of this study were thus to compare the potential of genomic with transcriptomic prediction, and to assess the merit of blending incomplete transcriptomic with complete genomic data by the single-step method. A substantial advantage of gene expression data over molecular markers has been observed for the prediction of FHB resistance in the studied diversity panel of breeding lines and released cultivars. An increase in prediction ability was likewise found for the single-step predictions, although this can mostly be attributed to an increased accuracy among the RNA-sequenced genotypes. The usage of transcriptomics can thus be seen as a complement to already established predictive breeding pipelines with pedigree and genomic data, particularly when more cost-efficient multiplexing techniques for RNA-sequencing will become more accessible in the future. Fusarium graminearum, Fusarium culmorum, and Fusarium sporotrichioides are the most prominent and agronomically important causal agents conferring large losses in grain yield and contamination of harvested grain with mycotoxins g~N or the mock-treatment and one Fusarium-treatment at a time (r = 0.79). This observation was also made when varying the number of the employed predictors that were used to generate these relationship matrices, where the transcriptomic data showed a general advantage over molecular markers at same number of predictors if at least two feature matrices were combined  in comparison to the pairwise combination of feature matrices based either on the two combined . AverageFusarium isolates  between two replicates of the Fusarium-treatment, it suggested a low-to-moderate repeatability of RNA-sequencing data without biological replicates. A combination of feature matrices from both biological replicates of the Fusarium-treatment into a common matrix was promising since the average reliability of the predictors increased from the estimate of h²rep = 0.36 to h²rep = 0.52. This gain was furthermore higher than the added noise even when averaging across the mock- and Fusarium-treatments resulting in a repeatability of h²rep = 0.54. Increasing the number of biological replicates has also been reported to have a larger impact than library size on the power of differential gene expression and ontology enrichment analysis [The average correlation among the magnitude of the expression from the individual genes between the mock-treatment and analysis . A transHowever, as costs are likewise higher for RNA-sequencing than DNA-fingerprinting, and this cost difference increases with each additional library, a more cost-efficient option might be given by obtaining gene expression data only from a subset of genotypes in combination with complete SNP marker data in a given set of genotypes. Exploiting the single-step methodology developed in animal breeding,  showed tImplementing transcriptomics into the predictive breeding framework has a potential to obtain more accurate predictions. The current cost-restrictions are making this endeavor mostly realizable in the single-step prediction framework or other methods for integrating multiple kernels. The use of transcriptomics can thus be seen as a complement to already established pipelines with pedigree and genomic data. This is particularly valid when more cost-efficient multiplexing techniques for RNA-sequencing ,57 will


The data describe supplementary materials supporting the research article entitled “Worldwide geographical mapping and optimization of stand-alone and grid-connected hybrid renewable system techno-economic performance across Köppen-Geiger climates” . Hybrid renewable energy systems are increasingly adopted worldwide as technically and economically effective solutions to achieve energy decarbonization and greenhouse gas reduction targets. This data article includes the results of worldwide techno-economic optimization of stand-alone and grid-connected photovoltaic-wind hybrid renewable energy systems designed to meet the electrical energy needs of an office district. The technical simulations have been performed in TRNSYS 17 (Transient Energy System) environment. A total of 48 different locations around the world have been chosen across Köppen-Geiger climates with different latitudes and homogeneously distributed over the whole globe, considering very different climates. The analyses have been conducted for 343 different system power configurations, considering both stand-alone and grid-connected systems. A total of 16464 dynamic simulations were performed, summarized in yearly energy output from each component and in energy and economic indicators. In addition, they provide the best localities in the world to host SA and GC HRES. Finally, data constitute a concrete device and database to detect the techno-economic performance of the optimal hybrid system around the world.•Maps are useful for rapid evaluation and comparison of the distribution of the techno-economic performance of the optimal HRES from a geographical point of view.•Data can be used by other researchers as a reference to compare investigations in any location worldwide, by stakeholders, entrepreneurs and policymakers.•The database can be extended to further localities worldwide, can be used to design installations of a hybrid system and to draw up economic incentive plans.•Dataset represents a device to inform people, researchers, stakeholder, entrepreneurs and policymaker where it is reliable and profitable to install an HRES.•In remote or isolated locations or underdeveloped countries, without access to the electricity, data can help to spread reliable and profitable SA installations. In urban areas with a grid connection, data are a valid guideline to reduce energy interaction between the user and grid, and consequently to avoid a grid overload, and to create new profitable investments for a private owner or public users.1-eight locations belong to climate group A ;-six to group B (dry);-eighteen to group C (temperate);-sixteen to group D .The typical meteorological year file .tm2, available in the TRNSYS library, is used for all simulations.01 Supplementary material” contain techno-economic parameters and indicators related to:-worldwide energetically optimal SA PV-wind HRES;-worldwide economically optimal SA PV-wind HRES;-worldwide energetically optimal GC PV-wind HRES;-worldwide economically optimal GC PV-wind HRES.The datasheets reported in the file “-Nominal PV, wind and battery power;-PV, wind, battery, load and overall power fraction;-Yearly energy produced by the PV and wind generator;-PV and wind energy fraction;-Yearly energy: sent directly to the load, drawn from the battery, missing, drawn from the grid, dissipated, produced sent to the grid;-PV, wind, inverter and battery capital costs;-Overall system cost;-Cost saving;-PV and wind revenue;-Yearly benefit;-Satisfied load fraction;-Utilization factor;-Grid Energy Interaction Factor;-Benefit cost ratio.For all locations, the technical, energy and economic data are reported in terms of:All parameters or indicators are explained in detail in the nomenclature sheet.02 Supplementary material – Histograms” represents a graphical elaboration of some parameters or indicators reported in the 01 Supplementary material”. It contains the following figures:•Fig. S.1. PV and wind power fractions for the energetically optimal stand-alone and grid-connected systems.•Fig. S.2. PV and wind power fractions for the economically optimal stand-alone and grid-connected systems.•Fig. S.3. Battery power fractions and load power fractions for the energetically optimal stand-alone and grid-connected systems.•Fig. S.4. Battery power fractions and load power fractions for the economically optimal stand-alone and grid-connected systems.•Fig. S.5. Wind and PV energy generated by the energetically optimal stand-alone and grid-connected systems.•Fig. S.6. Wind and PV energy generated by the economically optimal stand-alone and grid-connected systems.•Fig. S.7. PV and wind fractions of the overall energy generated in the energetically optimal stand-alone and grid-connected systems.•Fig. S.8. PV and wind fractions of the overall energy generated in the economically optimal stand-alone and grid-connected systems.•Fig. S.9. PV and wind fractions of the overall energy generated in order of yearly average wind speed strength (decreasing from left to right) for the energetically optimal stand-alone and grid-connected systems.•Fig. S1.10. PV and wind fractions of the overall energy generated in order of yearly average horizontal solar radiation strength (decreasing from left to right) for the energetically optimal stand-alone and grid-connected systems.•Fig. S1.11. PV and wind fractions of the overall energy generated in order of yearly average wind speed strength (decreasing from left to right) for the economically optimal stand-alone and grid-connected systems.•Fig. S1.12. PV and wind fractions of the overall energy generated in order of yearly average horizontal solar radiation strength (decreasing from left to right) for the economically optimal stand-alone and grid-connected systems.The “•PV/wind energy generated by the energetically/economically optimal SA and GC systems;•overall energy generated by the energetically/economically optimal SA and GC systems;•energy generated by the energetically/economically optimal SA and GC systems sent directly to the load.•energy drawn from the battery in the energetically/economically optimal SA and GC systems;•energy missing in the energetically/economically optimal SA systems and drawn from the grid in the energetically/economically optimal GC systems.•energy in excess dissipated by the energetically/economically optimal SA systems and sent to the grid by the energetically/economically optimal GC systems.•cost of the energetically/economically optimal SA and GC systems.•benefit from the energetically/economically optimal SA and GC systems.The following The worldwide geographical maps of other energy and economic indicators are contained in the main paper 2This data article shows the results of worldwide techno-economic analyses of SA and GC PV-wind HRES designed to meet the electrical energy needs of a district composed of five identical office buildings. The load requires energy for electric lighting, office equipment and vehicle charging stations.•fg) and the yearly energy produced sent to the grid (Etg);in blue: the yearly energy drawn from the grid (E•m) and the yearly energy dissipated (Ed);in yellow: the yearly missing energy (E•fb) and the yearly energy sent to the battery (Etb);in green: the yearly energy drawn from the battery (E•tl);in pink: the yearly energy produced sent to the load (E•g);in purple: the yearly overall energy produced by the generators (E•pv);in red: the yearly energy produced by the PV generator (E•w).in light blue: the yearly energy produced by the wind generator (EOnce the energy produced is more than the energy needed by the load, the energy is sent to the load and the remaining part is taken to charge the battery. The energy in excess is dissipated for the stand-alone system and sent to the grid for the grid-connected system. On the other hand, when the energy generated is not able to meet the load, it is taken from the battery. The missing energy is supplied by alternative systems, using a diesel generator for the stand-alone system, or by withdrawing energy from the grid for the grid-connected system ,5.TRNSYS software The electrical-thermal performance of the PV plant is implemented with the Type 94a, the wind plant with the Type 90 and the lithium-ion battery with the Type 47. Type 48 models the regulator and inverter, Type 15 imports the .tm2 climatic data from TRNSYS weather library and Type 25 plots the results.A total of 343 HRES power configurations, in terms of different PV, wind and battery powers, were dynamically simulated for 48 localities worldwide. For the 16464 simulations, hourly powers from each component were summarized in yearly energies, benefits and costs, and energy and economic indicators. An energy optimization problem was formulated to find, in each locality, the most reliable system power configuration for the SA HRES. Another energy optimization problem was formulated to find, in each locality, the system power configuration with the minimum level of energy exchange with the grid for the GC HRES. Similarly, two economic optimization problems were formulated, respectively, for the SA and GC HRES, to select the system power configuration with the highest benefit-cost ratio.The authors gave their consent to apply the instrument and provide the necessary data.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Based on 74 ore-processing plants and their energy meter's coordinates in Zaruma-Portovelo, we determined the energy consumption profiles for each ore-processing plant following the methodologies of Gonçalves (2016) and Cabezas (2016). In doing so, the total energy consumption in comminution processes was calculated. Data show a rising trend in energy consumption in ore processing in Zaruma-Portovelo. The data can be of immense use in designing sectoral reforms for transparency in the mining sector and developing cross-sectoral coordination between ARCOM and ARCONEL. The data can also help the state agencies in assessing the quantity of ore processed and preventing the leakage of revenue from the extractive sectors . Finally, the scientific community may benefit from this data, particularly those working on energy consumption, efficiency, and its forecasts in extractive sectors . Specifications Table•The data show a rising trend of energy consumption in ore processing industries in Zaruma-Portovelo.•The data can be used to understand the issues related to ore-processing industries and design new sectoral reforms for promoting transparency, and cross-sectorial coordination between ARCOM and ARCONEL.•The data show significant positive correlation between ore processing and electricity consumption (billing). The electricity consumption data can be used by the state departments for estimating the actual quantity of ore processed by each industry and as such capturing the non-realised share of revenue.•The scientific community may benefit from this data, particularly those working on energy consumption, energy use efficiency, and planning and development of extractive sector.•The data can serve as baseline or point of reference to be used in future for evaluations of mining sector, performance of processing plants, official and estimated data (based on electricity consumption) on ore-processed.Value of the DataTable 1: Energy consumption in comminution processes by plants performing under "industrial artisanal" and "industrial hourly demand" energy tariffs (2010–2018). Data columns describe the ore-processing plants number ID and their coordinates in [WGS 84 S17]. Each ore-processing plant is described in terms of its yearly energy consumption. Some ore-processing plants have no data, indicating that they were inactive or non-present during the 2010–2018 period. Total processing plants (per year), total energy consumption (kWh) per year and energy consumption in comminution (kWh) per year are presented in the last row.To compute the energy consumption of ore-processing plants, we first identified the total number of potential ore-processing plants and obtained their energy meter code and thus their energy consumption. Using the technical report published in 2014 by the Ecuadorian National Institute of Geological Mining and Metallurgical Research (INIGEMM), we identified an initial sample of 65 potential ore-processing plants in Zaruma-Portovelo. The initial sample was verified at the National Electricity Corporation's geoportal in which we associated ore-processing location (coordinates) with those energy meters under the layer and attributes table “CNELEP - Conexión Consumido” .Once verified, we expanded the sample of potential ore-processing plants by including additional ore-processing plants within the exclusion zone of Zaruma. Based on the resulting 185 potential ore-processing plants, we requested the monthly energy consumption during 2008–2019 from ARCONEL, as well as their electricity tariffs. However, we received the data for the period 2010–2018. Next, we limited the original sample of potential ore-processing plants to 74 (from 185) by filtering in those with an industrial electricity tariff only. Based on the resulting 74 ore-processing plants and their energy meter's coordinates, we requested the following data from ARCOM: a) ore-processing tonnage declaration, b) type of ore benefit process and c) monthly refining and marketing of final products between 2008 and 2018. Having obtained the above-mentioned data, we proceeded to determine the energy consumption profiles for each ore-processing plant following the methodologies of Gonçalves The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


The defined formation and expansion of droplets are essential operations for droplet-based screening assays. The volumetric expansion of droplets causes a dilution of the ingredients. Dilution is required for the generation of concentration graduation which is mandatory for many different assay protocols. Here, we describe the design of a microfluidic operation unit based on a bypassed chamber and its operation modes. The different operation modes enable the defined formation of sub-µL droplets on the one hand and the expansion of low nL to sub-µL droplets by controlled coalescence on the other. In this way the chamber acts as fluidic interface between two fluidic network parts dimensioned for different droplet volumes. Hence, channel confined droplets of about 30–40 nL from the first network part were expanded to cannel confined droplets of about 500 to about 2500 nL in the second network part. Four different operation modes were realized: (a) flow rate independent droplet formation in a self-controlled way caused by the bypassed chamber design, (b) single droplet expansion mode, (c) multiple droplet expansion mode, and (d) multiple droplet coalescence mode. The last mode was used for the automated coalescence of 12 droplets of about 40 nL volume to produce a highly ordered output sequence with individual droplet volumes of about 500 nL volume. The experimental investigation confirmed a high tolerance of the developed chamber against the variation of key parameters of the dispersed-phase like salt content, pH value and fluid viscosity. The presented fluidic chamber provides a solution for the problem of bridging different droplet volumes in a fluidic network.  During the last decade, droplet-based microfluidics  has beenDepending on the assay applications, the droplet volumes vary considerably. Picoliter droplets or droplets of one or a few nanoliters are mainly used for emulsion-based applications , but havThe plurality of biological assays are dose/response type experiments, in which an effect is determined as a function of an effector concentration. Therefore, a defined concentration graduation of the effector in the different samples is required. Hence, the motivation for the development of droplet dilution strategies is the generation of droplet series with concentration graduation. Various droplet dilution strategies have already been described to solve this problem, e.g., the adjustment of the effector concentration during the initial droplet formation ,35,36, oThe use of bypassed channels is a proven strategy for controlling droplet motion in fluidic networks without active elements. A bypass channel equilibrates the pressure between two points of a main channel which are connected by the bypass. Self-controlled operations, triggered by the moving droplets themselves were realized in this way. Only the pressure-driven flow drives the droplet operations in these cases ,39. DiffStable emulsion processing without undesired random droplet coalescence of contacting droplets requires surfactants for its stabilization ,54. On tRecently, we have developed an integrated fluidic network for bacterial screening purposes see . The genThe bypassed chamber device was designed for liquid/liquid two-phase applications using aqueous and fluorinated phases stabilized by fluorinated surfactants . The geoThe combination of the restricting junctions and the bypass position causes the release of the chamber volume in a self-controlled way. The different cross-sectional areas of the nozzles lead to the self-controlled order of droplet breakthrough. According to the young-la-places law, a droplet will preferably break through a nozzle with the larger cross-sectional area, effected by its characteristic radius. See description in The developed device offers four different operation modes see :(1) Self-controlled droplet formation A: The dr(2) Single droplet dilution mode B: Single(3) Multiple droplet dilution mode C: Severa(4) Multiple droplet coalescence mode D: Here, In the case of mode A–C the final droplet volumes and the respective dilution factors can be adjusted. As long as the releasing droplet passes the chamber outlet junction d the add2 LED-plate  below the device. For fluid actuation, the fluid inlet ports of the device were connected to a precision syringe pump system using a chuck with clamping connectors and 0.5 mm PTFE-tubing [The complete microfluidic Disc-device was fixed in a frame . The oriented surfactant molecules cause a monomolecular film at the interfaces and stabilize both liquid phases. However, under an electric field the stabilizing effect of the surfactants gets lost and contacting droplets merge.For the self-controlled droplet formation mode, the separation phase was feed to the chamber consciously through the inlet channel  droplets were generated by a T-junction and spaced by a second T-junction feeding the continuous phase  were varied. It was found that the coalescence rate is nearly independent on the media composition. Only the applied power has a significant influence. The application of minimum 450 V for the electrocoalescence pulses led to nearly quantitative coalescence rates. The presented chamber layout can be constructively transferred to new fluidic networks which require a volumetric transfer of droplets. We believe even a gentle dimension rescale will not change the fluidic functionality, if the electric field is considered. Especially applications using emulsion-based droplets in the low nL range may need for downstream processing a volumetric expansion. The combination of several drops into one compartment using the multiple-droplet coalescence mode offers new application possibilities by combinatorial mixing strategies. Both examples may give an outlook on future applications of the presented bypassed chamber.


Although cognitive training in healthy older adults (OA) has been controversial, specific and isolated cognitive skills such as semantic memory can be improved with appropriate designs. Semantic memory has been considered as a clinical marker for cognitive decline in dementia. The current study, as part of a larger touch-screen dual-language intervention program with cognitive training tools, aims to slow down the rate of cognitive decline in OA with dementia (OwD). A set of neuropsychological tests was conducted before and after the training program. After 24 training sessions over 8-12 weeks, OwD  improved significantly in their verbal working memory  while performance of the cognitive-healthy OA  remained the same post-intervention. Our findings suggest that touch-screen technology can help OwD improve their semantic memory. The strengths and limitations of our game design and intervention will be discussed. Part of a symposium sponsored by Technology and Aging Interest Group.


FMR1 gene contains a polymorphic CGG trinucleotide sequence within its 5′ untranslated region. More than 200 CGG repeats (termed a full mutation) underlie the severe neurodevelopmental condition fragile X syndrome, while repeat lengths that range between 55 and 200 (termed a premutation) result in the conditions fragile X-associated tremor/ataxia syndrome and fragile X-associated premature ovarian insufficiency (FXPOI). Premutations in FMR1 are the most common monogenic cause of premature ovarian insufficiency and are routinely tested for clinically; however, the mechanisms that contribute to the pathology are still largely unclear. As studies in this field move towards unravelling the molecular mechanisms involved in FXPOI aetiology, we review the evidence surrounding the two main theories which describe an RNA toxic gain-of-function mechanism, resulting in the loss of function of RNA-binding proteins, or a protein-based mechanism, where repeat-associated non-AUG translation leads to the formation of an abnormal polyglycine containing protein, called FMRpolyG.The  FMR1 gene on the X chromosome. This repeat sequence is polymorphic in length, with categorization into four different size ranges  translation, drawing on parallels in FXTAS and advances made in that disease.Fragile X-associated premature ovarian insufficiency (FXPOI) is among a family of disorders caused by the expansion of a CGG trinucleotide repeat sequence located in the 5′ untranslated region (UTR) of the e ranges . HealthyFOXL2  is defined as the loss of ovarian hormonal function with evidence of follicle depletion before the age of 40 years . It thusFOXL2 . AbnormaFor many women, the most immediate and significant consequences of POI are amenorrhoea and an inability to conceive, however, some women present with intermittent ovarian function and conceive naturally  inadequate formation of the ovarian reserve during foetal life and (ii) alterations in follicle recruitment and maturation that cause abnormally extensive or fast depletion of the ovarian reserve during post-natal  life. While information from women is scarce, the development and use of model systems has gone some way towards addressing this, with data available from three published knock-in mouse strains, harbouring 130   repeat lengths. While these data are only correlative, these findings suggest that FMR1 mRNA accumulation in granulosa cells may be an important cause of follicle dysfunction and loss. A preliminary report of in vitro-based studies further demonstrated that transfection of the human granulosa cell tumour line COV434 with plasmid DNA expressing 88 CGG repeats decreased cell viability after 48 h . However, although the proteins discussed here are predicted to be also relevant in the pathogenesis of FXPOI, there has been no validation as yet of these proteins in ovary-specific premutation models. Furthermore, while a degree of overlap is expected given the parallels with FXTAS, it is probable that some deregulated proteins will be specific to granulosa cells, thus identification of these ovarian targets will be essential as they are likely to be key in understanding the RNA gain-of-function mechanism that may potentially contribute to FXPOI.Identification of the proteins that bind and are sequestered by CGG RNA within intranuclear inclusions in the brain has been an important step towards understanding the contribution of RNA gain-of-function toxicity to the pathogenesis of FXTAS. Several proteins have been identified that bind to CGG repeats in FXTAS-affected cells, unlike other disorders associated with unstable repeat expansions, which usually have a principle protein deposit . RNA binFMR1 locus also supports RNA toxic gain-of-function as one of the possible pathophysiologic mechanisms underlying FXPOI. Using genomic approaches   RNAs originating from the Met takes place. The 40S ribosome is now committed to its selection of start codon and is joined by the 60S ribosomal subunit, allowing the first peptide bond to be formed, thus beginning translation elongation     and Wellbeing of Women (PRF005 to R.R.). R.R. declares no conflict of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and personal fees from Ferring Pharmaceuticals, IBSA, Merck Serono, outside the submitted work.