We replaced the presynaptic neuronal firing rate by repeated electrical stimulation (frequency between 3.125 and 100 Hz) applied to the lateral olfactory tract. Our results revealed a considerable enhancement of postsynaptic response amplitude for stimulation frequencies in the beta and gamma range. A phenomenological model of STP fitted to the data suggests that the experimental results can be explained by the interplay between three mechanisms: a short-term facilitation mechanism (time constant ≈160 msec), and two short-term depression mechanisms (recovery time constants <20 msec and ≈140 msec). Increasing calcium concentration (2.2 mM) resulted in an increase in the time constant of facilitation and in a strengthening of the slowest depression mechanism. As a result, response enhancement was reduced and its peak shifted toward the low beta and alpha ranges while depression became predominant in the gamma band. Using environmental conditions corresponding to those that prevail in vivo, our study shows that STP in the lateral olfactory tract to layer Ia synapse allows amplification of olfactory bulb inputs at beta and gamma frequencies.Neuronal activity is characterized by a diversity of oscillatory phenomena that are associated with multiple behavioral and cognitive processes, yet the functional consequences of these oscillations are not fully understood. Our aim was to determine whether and how these different oscillatory activities affect short-term synaptic plasticity (STP), using the olfactory system as a model. In response to odorant stimuli, the olfactory bulb displays a slow breathing rhythm as well as beta and gamma oscillations. Since the firing of olfactory bulb projecting neurons is phase-locked with beta and gamma oscillations, structures downstream from the olfactory bulb should be driven preferentially at these frequencies. We examined STP exhibited by olfactory bulb inputs in slices of adult mouse piriform cortex maintained  EEG and intracerebral LFP recordings have disclosed a variety of oscillatory phenomena in the brain. Although the precise ranges differ depending on the species and structure examined, several types of oscillations with characteristic frequencies and dynamic features have been associated with different sleep/waking states and with various perceptual and cognitive processes. For example, non-REM sleep is characterized by the presence of a slow sleep rhythm < 1Hz), of delta oscillations 1–4 Hz) and short epochs of sleep spindles 11–15 Hz) Hz, of de. Theta w Hz and s Hz Hz, oAlthough they are clearly associated with a variety of perceptual and cognitive processes, the functional significance of oscillations, in particular beta and gamma oscillations, has remained elusive. At one extreme, it has been proposed that oscillations are merely an epiphenomenon of the functioning of cerebral networks e. g., –25) whil whil25])consequences of oscillatory activity on synaptic transmission. Indeed, synaptic transmission is not steady: on a short time range  postsynaptic responses usually display reversible dynamic changes, referred to as short-term plasticity (STP). Synaptic efficacy can thus be modulated by two broad types of STP: short-term facilitation (STF) and short-term depression (STD) . Severain vitro, while applying electrical stimulation in the lateral olfactory tract (LOT), which contains olfactory bulb efferent axons. The stimulation frequency range (0.1 to 100 Hz) encompasses that observed in natural brain rhythms.We examined STP in the piriform cortex, one of the main relays of olfactory information after integration in the olfactory bulb e. g., –32). Thr. Thr32])in vitro: 1) use of several stimuli in a train in order to be able to observe the dynamics of STP, rather than the classical stimulus pair that limits the analysis to paired-pulse ratios; 2) model based, quantitative analysis of the data to obtain relevant parameter values for mechanisms of STD and STF, to be able to go beyond the mere phenomenological description of changes in response amplitude; 3) use of adult animals and of an extracellular medium that most closely mimics the one encountered in the adult brain in vivo; in particular, most in vitro experiments use an extracellular concentration of calcium of 2–2.5 mM while that measured in vivo is of the order of 1 mM; given the importance of calcium in synaptic transmission, this factor-two difference is likely to strongly affect STP and its dependence on stimulation frequency. Although 1 or 2 of these conditions have been met in the numerous studies devoted to STP in piriform cortex, our study would be the first where all 3 requirements were fulfilled: our experiments were performed using brain slices issued from adult mice and were maintained in an in vivo-like ACSF; we used stimulation trains of 5 stimuli and extracted the parameters of STP by fitting the data with a model derived from that initially developed by Tsodyks and Markram (1997)  on average in comparison to the initial model. The second variant was one in which parameter E was allowed to vary in the different calcium concentration conditions. For the 8 experiments in which the 2 calcium concentrations were tested, the mean RMSE was significantly (P = 0.008) lower (mean difference: -15% [-24%—-6%]), but this had the inconvenience of assuming that E, the total synaptic resource, varies with calcium concentration. The third variant was a model in which, in addition to E, the time constants of recovery from depression and facilitation were shared between the two calcium concentrations. This third variant performed less well on average: the RMSE was significantly (P = 0.03) increased by +32% [+11%—+53%] on average in comparison to the original model.The overall quality of the model fit was estimated by the RMSE. At the population level the mean RMSE was 0.047 [0.036–0.058] (n = 17). We tested other variants of the model. The first variant was a model with only one depression mechanism as in: , 82). Th. Th82]).++.Distribution of model parameter values are presented in E corresponds to the maximum potential response, that is, a ceiling level for the response amplitude given the experimental conditions of the study. The mean value returned by the model was 2.825 [2.422–3.228]  in 2.2 mM Ca++, with a mean at 0.548 [0.456–0.639]. U was observed in all cases and was highly significant .The parameter 9–0.424] . U was l++  increase of τF in 2.2 mM Ca++  where both 1.1 and 2.2 mM calcium were tested .The depression mechanism with slow recovery displayed a recovery time constant,  mM Ca++ . DespiteP = 0.2, . The sloThe parameters returned by the model allowed reconstructing STP over a continuum of stimulation frequency. In comparison to that observed in 1.1 mM, the dynamic range was reduced in 2.2 mM calcium, with a maximal enhancement at +22% with the second pulse and at +14% only with the fifth pulse. The frequency at which the maximum RA was reached was lower with the second pulse (17 Hz) and furthermore, it progressively drifted toward lower frequency with the following stimulation pulses (8 Hz with the fifth pulse). The half maximal amplitude frequency range drifted in parallel . Finally, except for second pulse response, RA decreased below first pulse amplitude for frequencies within the gamma range (third pulse) and within the beta band (fifth pulse) whereas stimulation frequencies >100 Hz would be required to induce such reduction in 1.1 mM calcium.in vivo-like ACSF revealed a larger amplification of synaptic responses especially when stimulation frequency straddled the range reported to be associated with odor-induced oscillations in the olfactory bulb.Altogether these data show that the features of STP at the LOT-layer Ia synapse endows piriform cortex with the ability to amplify its afferent olfactory inputs. Yet using a “classical” extracellular calcium concentration (2.2 mM) resulted in underestimating both the amplification and the frequencies over which amplification took place. Using an in vivo, response dynamics at the LOT-layer Ia synapse is characterized by a considerable response enhancement. This enhancement was maximal in the beta frequency range, although it also impinged on the gamma frequency range and on the range of frequencies associated with active sniffing. The STP model fitted to the data indicated that this enhancement was essentially achieved through the interaction of 3 mechanisms: a facilitation mechanism with a recovery time constant of 157 msec on average, that resulted in a progressive increase in response amplitude when stimulation frequency was > about 2 Hz, a fast depression mechanism with a recovery time constant of 19 msec that curtailed facilitation when stimulation frequency was > 23 Hz, and, in some cases, a depression mechanism with slower recovery. Increasing calcium concentration to 2.2 mM unsurprisingly increased the amplitude of responses evoked at low frequency but it also reduced the dynamic range of response enhancement. Furthermore, depending on pulse ordinal number, the frequency at which enhancement was maximal drifted from the low beta band to the alpha band. Finally, at stimulation frequency >30–50 Hz, depression took over to the extent that response amplitude could be similar to that obtained in 1.1 mM calcium. The STP model provided three explanations for the changes induced by increasing calcium concentration: the longer time constant of facilitation accounted for part of the shift of enhancement toward lower frequencies; the slow depression mechanism, more prominent than in 1.1 mM calcium, also contributed to the shift of response enhancement toward lower frequencies; finally larger depletion of synaptic resources with the first pulse resulted in reduced reserve availability, in turn leading to stronger depression at high frequency.Our experimental data showed that, provided extracellular calcium concentration corresponds to that measured That postsynaptic response amplitude depends on extracellular calcium concentration is a well established fact that has been reported in numerous studies. Quantitative studies exploring this relationship initially examined the initial portion of the calcium concentration vs. response amplitude curve that can be linearized in logarithmic representation, while later studies relied on fitting the whole relationship with, among others, Hill equation as in the present study . These sin vivo and in vitro. Without exception all the studies relying on paired-pulse protocols revealed paired-pulse facilitation at the LOT-layer Ia synapse )τ, can associate two mechanisms, summarized by the equation: τ = τ0 × (1+κE), where τ0 corresponds to the time constant of the calcium extrusion mechanism and κE to the calcium binding capacity of an endogenous calcium binding protein. An increase in τf would readily be explained if κE increases with extracellular calcium concentration.We found that the time constant of recovery from facilitation was longer with higher calcium concentration Figs  and 7. AOur results also suggest that two different depression mechanisms operate at the LOT-layer Ia synapse in our experimental conditions Figs  and 7: tAs for facilitation, several models have been proposed for explaining STD. Few of these mechanisms are postsynaptic and most are presynaptic. As these different mechanisms are associated with different time courses, several may operate at a given synapse.One postsynaptic mechanism of STD, namely AMPA receptor desensitization, has been proposed to operate at some central synapses , 120. InMore traditionally, STD has been attributed to presynaptic mechanisms . Fu. Fu131])In between fast and slow recovery mechanisms, depression mechanisms recovering with a time constant of the order of a few hundred msec, as our second depression mechanism, have been reported in multiple structures   revealedIt is also now well established that STP is strongly impinged by the developmental stage. For example, studies in neocortex demonstrated that STD, which prevails in immature animals, is reduced in adults while STF becomes more prominent , 138–143in vivo [in vitro. Part of the difference might be explained by the presence of spontaneous activity that results in a steady state depression in vivo [in vivo and classical in vitro studies is calcium concentration: 1–1.2 mM in vivo vs. 2–2.5 mM in most in vitro studies. One reason initially invoked for justifying the use of high calcium concentration in vitro was that it improved the stability of intracellular recordings [in vitro calcium concentration and in in vivo-like calcium concentration showed larger facilitation and/or reduced depression with the later [Yet, even with adult animals, studies examining STP in vivo , 147–151 in vivo , 152. Ancordings . Hence ahe later –156.Our study is a further demonstration of the importance of calcium concentration in STP, here in piriform cortex. Noticeably, had we stuck to the traditional approach of using young animals and high calcium concentration, we would have missed the large response enhancement in response to stimuli delivered in the beta frequency. Linking short-term plasticity in piriform cortex to rhythmic activity in the olfactory bulb would have then been more tentative.in vivo-like ACSF the maximal enhancement was observed in the beta frequency range but was still large in the gamma frequency range (Our results indicate that the interplay of three short-term plasticity mechanisms results in a frequency dependent enhancement of postsynaptic response amplitude at the LOT-layer Ia synapse in piriform cortex. In the cy range . Beta anS1 Dataset(XLS)Click here for additional data file.


ZNF804A, CDK1, and MANF) have not previously been associated with autoimmunity. Random forest classification also allowed prediction of patients at risk for lupus nephritis with an area under the curve of 0.94. By allele-specific gene expression analysis we detected cis-regulatory SNPs that affect the expression levels of six of the top 40 genes designed by the random forest analysis, indicating a regulatory role for the identified risk variants. The 40 top genes from the prediction were overrepresented for differential expression in B and T cells according to RNA-sequencing of samples from five healthy donors, with more frequent over-expression in B cells compared to T cells.Genome-wide association studies have identified risk loci for SLE, but a large proportion of the genetic contribution to SLE still remains unexplained. To detect novel risk genes, and to predict an individual’s SLE risk we designed a random forest classifier using SNP genotype data generated on the “Immunochip” from 1,160 patients with SLE and 2,711 controls. Using gene importance scores defined by the random forest classifier, we identified 15 potential novel risk genes for SLE. Of them 12 are associated with other autoimmune diseases than SLE, whereas three genes ( SLE is considered as a model for systemic autoimmune diseases, in which most organ systems of the human body can be affected. SLE is characterized by the presence of autoantibodies, immune complex formation, and organ inflammation. The disease phenotype varies between individual patients from relatively mild manifestations of skin and joints to organ-threatening renal involvement (lupus nephritis)2, but a large proportion of the genetic contribution to SLE susceptibility still remains unknown. Although the genetic background of specific manifestations of SLE is less well known than that of SLE in general, several single nucleotide polymorphisms (SNPs) have also been associated with the subgroup of patients with lupus nephritis4.Genome-wide association studies (GWAS) have identified over 60 genetic loci that confer risk for SLE5. Using the collective information from many single nucleotide polymorphisms (SNPs) it should be possible to identify novel disease-associated genes. There are multiple machine learning methods that could be applied to SNP genotype data, such as logistic regression, artificial neural networks6, support vector machines7, and random forests8. Information from the GWAS catalogue has been used for predicting risk of 18 common diseases using a logistic regression model evaluated on SNP allele frequency and odds ratios9, but SLE was not included in this study.SNPs that reach genome-wide, or close to genome-wide, significance in genetic association studies have been compiled in the GWAS catalog8, as this method has been shown empirically to perform well in many scenarios where predictions are needed10 and is applicable to genetic association data12. We used genotype data from the Immunochip14 (Illumina), which targets about 200,000 SNPs in genes that are relevant for diseases of the immune system, to predict disease status in a large set of Swedish patients with SLE and controls and to identify risk genes for SLE. A high disease probability from the classifier indicates higher genetic risk for SLE for an individual compared to the general population, and a high gene importance score in the model indicates a gene region that contains SNP alleles that confer risk of SLE.In the current study our aim was to use machine learning based on random forests to design a SNP genotype classifier to predict risk of SLE and to predict previously unknown genes and genetic variants that confer risk of SLE. Out of the multiple prediction algorithms available, we chose to use the random forests machine learning method15 and RNA sequencing to assess functions of the candidate genes for SLE predicted by the random forest algorithm, in B and T cells from peripheral blood of healthy donors. The genotype data from the Immunochip combined with cell type-specific gene expression and ASE in B and T cells yield information on the regulation of gene expression of the putative risk genes for SLE defined by the random forest algorithm.SLE is a systemic autoimmune disease characterized by activated T cells and autoantibody production by B cells. We therefore applied analysis of allele-specific gene expression (ASE)16, offers the advantage of combining the specificity and sensitivity measures into one accuracy without setting a fixed threshold for evaluation of the accuracy. The AUC can range from 0 to 1, where an AUC-value of 0.5 equals a random prediction and an AUC of 1 represents a perfect prediction. The AUC for the random forest prediction of SLE was 0.78  located in or close to 12,500 genes related to the immune system from 1,160 patients with SLE and 2,711 healthy controls. The random forest classifier yields a probability that a sample originates from a patient with SLE for each individual. This probability value was used to calculate the area under curve (AUC) as a measure of the prediction accuracy. The AUC, based on a receiver operating characteristic (ROC) curveThe explained heritability for different models was calculated using the AUC value for each model in conjunction with the disease prevalence and the sibling recurrence risk for SLE. The SNPs reported in the GWAS catalog account for 5% of the heritability for SLE, compared to 11% using the genetic risk score obtained by logistic regression of the case-control association data from our study, and 16% obtained using the random forest model for our data.18 for which there are associated SNPs that reach genome wide significance in the GWAS catalog, we constructed a random forest classifier also for this subgroup of patients. Analysis of the 274 samples from SLE patients with lupus nephritis in our cohort yielded a high success in the random forest prediction, with an AUC of 0.91 compared to 0.78 for prediction of SLE in the whole cohort. Predictions using the risk score calculated from the regular SNP association analysis (logistic regression) for the same patients with lupus nephritis yielded a significantly lower AUC of 0.70  criteria0.0003*X corresponding to a 0.07% increase in score for each rank from the lowest rank to approximately gene rank 500, after which the slope is 3.3 times steeper (Y = 100.001*X), and at around gene rank 40 the slope is additionally 8.1 times steeper (Y = 100.008*X). Based on this observation, together with the fact that there are as many as 25 known SLE associated genes among the top 40 predicted risk genes, we chose the 40 top genes from the random forest prediction for further investigation.We used the random forest algorithm to calculate “importance scores” based on the genotype data from the Immunochip. This score describes to what extent a gene region confers risk of SLE based on the classification performance of the SNPs in the gene region . The gene importance scores for all genes from the random forest prediction are listed in Supplementary Table PSMG1, PTGER4, CPEB4, EGR2, RFX3, IL1R1, LRRK2, GPR183, ZMIZ1, ELMO1, TNFSF11, SATB2 are associated with other autoimmune diseases than SLE according to the GWAS catalog  and kidney biopsy specimens from SLE patients and is correlated with the expression of three representative IFN-inducible genes . Additionally, a CDK1 inhibitor was shown to reduce the expression of interferon-stimulated genes in PBMCs from SLE patients and in renal cells from mice with SLE19.MANF is located 1 kb downstream of DOCK3. Dysfunctional response to unfolded proteins in the endoplasmic reticulum (ER) was found in SLE patients with upregulated levels of MANF. Stress of the ER is closely correlated with inflammation and/or immune diseases. However, it is still unknown whether aberrant ER stress is involved in SLE pathogenesis20.Based on the known involvement of ZNF804A, CDK1 and MANF in important pathways that are affected in SLE, these three genes are strong novel candidate risk genes for SLE. The 12 putative novel SLE genes with associations reported to other autoimmune diseases than SLE in the GWAS catalog are also of great interest due to their potential involvement in the pathogenesis of SLE Table . Of the SLC2A13, ZMIZ1, TRIB1, RASGRP3, RMI2, and IPMK. RASGRP3 has so far been associated with SLE only in Asian populations27, where the incidence of lupus nephritis is higher than in Caucasians. The five other genes are associated with multiple autoimmune diseases, but not with SLE.Creating a classifier for patients with lupus nephritis and treating the other SLE patients as a control group, allows identification of genes that distinguish patients with lupus nephritis from other SLE patients. Somewhat surprisingly, this “case-case” prediction reached a similar accuracy (AUC of 0.94) as when using the healthy blood donors as a control group (AUC of 0.91). Many of the top genes in the “case-case” classification overlap with the genes from the “case-control” classifications , included 24 known SLE genes, 11 known autoimmune genes and 5 genes without any known connection with autoimmunity .We used expression patterns of the genes high-lighted by random forest classification as a functional validation in B cells and T cells from healthy donors. Allele-specific expression of a gene in a relevant cell type or tissue gives information on cis-rSNPs using ASE calculated from the Immunochip data of the top 40 genes from the random forest prediction. We found that 30 out of the 40 top genes were expressed in B cells or T cells   Table . Six SLE−8 were 2-fold over-represented  among the top 40 genes with the highest importance scores from the random forest prediction of SLE. The over-representation was 3-fold  when only genes expressed at a higher level in B cells than in T cells were considered  after multiple testing correction (Bonferroni p < 0.05). The genes with the highest differential expression using a p-value threshold of 10red Fig. . The oveOf the 30 genes that were expressed in either cell-type, 15 were expressed at a higher level in one of the cell-types. However, when only genes with significant differential expression between B cells and T cells were considered, B cell-specific genes were more common among the top 40 genes  between the genetic variants. However, as the over-all gene importance score from the random forest prediction is a sum of many individual importance scores, and each individual importance score is based on an average over many trees and cross validations, the gene importance score should remain unaffected by LD.29. The high level of accuracy to discriminate between SLE patients with and without lupus nephritis could be useful to identify patients at high risk of lupus nephritis before the manifestation is apparent. Risk patients could thereby be monitored more closely and possibly receive treatment at an earlier stage. At a sensitivity of 70% the specificity is 95%, which implies that with the prevalence of 24% for lupus nephritis defined in this study, a genetic test would identify 70% of the lupus nephritis patients, while 17% of the patients without lupus nephritis would be false positives. As lupus nephritis is acquired over time it is uncertain if these patients would develop or already have developed lupus nephritis after the time of phenotype data collection or if they are true false positives. Approximately 1,000 SNPs were selected in each fold of the random forest classification, making this a technically feasible test to use in a clinical setting. The relatively high prediction success in lupus nephritis probably originated from the comparative homogeneity of this subgroup of SLE patients compared to the entire group of SLE patients.The accuracy of the prediction of genetic risk for complex diseases varies greatly between diseases, depending on the heritability of the disease, on the uniformity of the disease phenotype, and the power and the number of investigated variants. The reported genetic predictability of SLE is low, compared to rheumatoid arthritis and several other diseases of the immune systemAlthough the number of samples is relatively high in our study, it is too small for detecting significant association signals to common SNPs with low risk and to rare SNPs with moderate risk. Also, the Immunochip is not a classical GWAS SNP-chip as it only targets autoimmunity loci. Thus the classifier could miss relevant genes for SLE that are not included on the chip. Novel genes that we identified should be subjected to independent replication as confirmation. Including other immune cell-types than B and T cells would allow more comprehensive detection of functional risk-SNPs for SLE.ZNF804A gene was expressed at a several-fold higher level in B cells than in T cells, which combined with functional evidence from the literature24 renders ZNF804A a strong novel candidate gene for SLE. For lower ranked genes the relative expression levels in B cells and T cells were equally distributed. In our study we observed an over-representation of SLE and autoimmunity genes for each of the three measures related to regulation of gene expression. One fourth of the top 40 predicted risk genes for SLE were differentially expressed in B cells versus T cells, for 15% of the risk genes we detected an associated cis-rSNP in at least one of the cell types, 30% of the genes had measurable ASE. In total 53% of the risk genes for SLE displayed one of these gene regulatory features, which is an enrichment compared to the expected frequency of 33% for randomly chosen genes . This observation confirms from a new perspective that genes with cell type-specific regulation are more prone to be involved in SLE and other autoimmune diseases, where the risk of a gene being involved in disease is not only dependent on its function, but also on its regulatory control.Our data confirms a strong involvement of B cells in the pathogenesis of SLE. We observed an enrichment of genes among the top 40 predicted risk genes for SLE that were expressed at significantly higher level in B cells than in T cells, compared to all genes on the Immunochip . For example, the in vivo experiments on humans. The patients (included in the study) were 87% female, of Caucasian origin, and on average 36 years old at SLE onset. The patients fulfilled at least four American College of Rheumatology (ACR) 1982 criteria for SLE18, with the exception of eight patients who fulfilled the Fries criteria for SLE30. A total of 274 patients fulfilled the ACR-82 criterion for lupus nephritis. Healthy blood donors were 70% female and had an average age of 43 years at the time of blood donation.DNA was extracted from peripheral whole blood of 1,411 SLE patients visiting the rheumatology clinics in Uppsala, Karolinska (Stockholm), Lund, Linköping and the four northern-most counties in Sweden. All patients were examined by a rheumatologist and medical records were reviewed. Control DNA was extracted from whole blood of 3,361 healthy volunteer blood donors visiting the university hospital in Uppsala , Lund and Stockholm (Karolinska). SLE patients and blood donors provided informed consent to participate in the study, and the study was approved by the Regional Ethics Committees of the involved institutions. The study did not include any CD19+ B cells from 53 samples and CD3+ T cells from 54 samples were fractionated from buffy coats of 60 healthy voluntary blood donors from Uppsala by Ficoll-Hypaque  density-gradient centrifugation for isolation of PBMCs, followed by positive selection with a cell type-specific antibody (Miltenyi Biotec). Purity of the isolated cell population (>95%) was confirmed by control sampling by flow cytometry .14. Genotyping was performed by the SNP&SEQ Technology Platform at Uppsala University, Sweden.DNA samples from SLE patients and controls were genotyped using the Illumina Infinium assay on the Immunochip, which detects about 200,000 SNPs selected based on GWAS of diseases of the immune systemThe genotype data was first subjected to quality control (QC) on the sample level, whereby samples from second-degree or closer relatives, samples that did not cluster with Europeans in principal component analysis (PCA), samples with heterozygosity rates exceeding five standard deviation from the average and samples with genotype call-rates below 95% were removed. After these QC parameters, 1,160 SLE patients and 2,711 controls remained for further analysis. Genotype data from the sex-chromosomes and from insertion-deletions were excluded. The QC on the SNP level removed SNPs with an average sample call-rate below 98%, SNPs with a Hardy-Weinberg equilibrium (HWE) p-value below 10E-4, and SNPs with a minor allele frequency below 1%. After QC, genotype data for 134,523 SNPs remained for further analysis. Genes in the HLA region are not reported in the association analysis and the random forest prediction due to high gene density and strong LD making it very hard to determine the causative gene.31. SNPs were annotated to overlapping genes or to the closest downstream gene within 100 kb for intergenic SNPs. When SNPs in high linkage disequilibrium (LD) were associated with several genes, only the SNP-gene combination with the lowest association p-value was kept for further analysis. For associated SNPs that showed a significant difference in number of missing genotypes between controls and patients  or a HWE p-value < 0.05, the genotype cluster plots were inspected manually and SNPs were filtered out if the called genotypes appeared to be based on low quality data.The genotype data for individual SNPs from the Immunochip was analyzed for association with SLE using logistic regression in PLINK version 1.07The accuracy of the logistic regression to predict disease status was assessed by splitting the association data into training data sets (80%) and test data sets (20%) in five different folds. Only SNPs with an association p-value < 0.01 were used in the prediction. Based on the training data, a risk score was calculated for the test data defined by the difference between the total number of risk and protective alleles in each individual. Using the risk score, the predictive performance was evaluated in the merged test data from the five folds using the AUC measure.8. The computations were run using the R package Emil , version 1.1-6.), which in turn uses the RandomForest R-package32.Prediction of SLE status and calculation of a gene importance score based on the genotype data from the Immunochip was performed using a random forest machine learning methodSLE status was predicted based on genotype data in three iterations with five cross-validation folds per iteration, where each of the 15 cross-validation runs used 80% of the data for training of the classifier and 20% for testing, using on average the 3,000 most informative SNPs per classification fold. The SNPs were selected based on training data only within each fold. Fisher’s exact test was calculated for each site and only sites with a p-value < 0.01 were included.11.The number of variables selected per tree (mtry) and number of trees (tree) for the random forest algorithm were set to 300 and 1,000, respectively. A larger number of trees did not improve the prediction. mtry was set to approximately 0.1 times the number of selected variables, which is recommended for sparse data33. We also searched for autoimmune disease annotated genes close to high scoring genes without an autoimmune disease annotation in the GWAS catalog. When relevant genes were found, the gene region was expanded into a gene region including all relevant genes as candidate genes.The Gini importance measure was used to determine the importance of each SNP. This measure is calculated by the random forest algorithm and describes the classification performance of a variable averaged over all trees and nodes where the variable was used. An importance score for each gene was defined by summing the Gini importance measure of individual SNPs within a gene and its 10 kb flanking regions. Finally, the summed importance score for each region was averaged over the 15 cross validation folds. Gene regions were obtained from the Reference sequence (RefSeq) database at NCBI29. The SLE prevalence in the Swedish population was set to 68 in 100,00034 and the sibling recurrence risk for SLE that is 20 times higher than for non-siblings. For lupus nephritis the prevalence in the Swedish population was set to 23 in 100,00034.To determine the heritability explained by the different models, we used the AUC for the respective statistical model in conjunction with disease prevalence and sibling recurrence risk of SLE15. DNA and RNA were prepared from B and T cells using the AllPrep DNA/RNA Mini Kit (Qiagen). cDNA was synthesized from 1–5 µg of RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). Double-stranded cDNA was purified using the MinElute PCR purification kit (Qiagen).Fractionated B cells from peripheral blood of 53 healthy donors and T cells from 54 donors were subjected to ASE analysis by SNP genotyping as described previously for human monocytes37 or as in this study by quantitative genotyping of heterozygous SNPs on the RNA level38. Genomic DNA (gDNA) and complementary DNA (cDNA) from B cells and T cells were genotyped in parallel using the Immunochip. Genotypes were called in gDNA using the Genome Studio version 2009.2 (Illumina) with a call rate of 99% as the threshold for SNP genotype calls and 98% sample success rate. SNPs were further filtered on deviations from HWE with a p-value cutoff of 10−6 (Chi-squared test). ASE-levels were determined using the genotype data calculated for each gene region as described in Almlöf et al.15. In short, the ASE-levels were calculated for each heterozygous SNP as the difference in normalized allele fractions between cDNA and gDNA: [Allele1cDNA/(Allele1cDNA + Allele2cDNA)] − [Allele1gDNA/(Allele1gDNA + Allele2gDNA)]. Cis regulatory SNPs (cis-rSNPs) were called in each gene region and 100 kb flanking regions, having at least five heterozygous SNPs with a fluorescence intensity over 5,000, by logistic regression analysis against the ASE-levels essentially as described by Ge et al.39. The analysis was performed on 2,604 gene regions in B cells and 2,582 gene regions in T cells. Additionally, an ASE-value for entire gene regions was determined using the median of the absolute ASE-levels for SNPs within each gene region for all individuals. To minimize the number of false positive signals ASE was only called in gene regions with more than 20 observations of SNP-individual combinations in expressed gene regions  was depleted from 1 µg of total RNA using the Ribo-Zero Magnetic Gold Kit (Epicentre). Strand-specific RNA-sequencing libraries were constructed from rRNA-depleted RNA with the ScriptSeq V2 Kit (Epicentre). The libraries were sequenced using an Illumina Hiseq. 2000 instrument using paired-end 50 bp reads, which yielded 14M-89M read pairs per sample (median 58 M). The reads were aligned with TopHat and transcript assembly was performed by Cufflinkshttp://www.ncbi.nlm.nih.gov/gds.Genotyping summary data are available from the corresponding author on reasonable request. ASE summary data are available from the corresponding author on reasonable request. Raw and normalized FPKM values generated from RNA-sequencing data are available in the GEO repository with IDs: GSM1978773–GSM1978782 at Supplementary Figures S1-S5 and Tables S2,S3,S5Supplementary Table S1Supplementary Table S4


It is commonly thought that phonological learning is different in young children compared to adults, possibly due to the speech processing system not yet having reached full native-language specialization. However, the neurocognitive mechanisms of phonological learning in children are poorly understood. We employed magnetoencephalography (MEG) to track cortical correlates of incidental learning of meaningless word forms over two days as 6–8-year-olds overtly repeated them. Native (Finnish) pseudowords were compared with words of foreign sound structure (Korean) to investigate whether the cortical learning effects would be more dependent on previous proficiency in the language rather than maturational factors. Half of the items were encountered four times on the first day and once more on the following day. Incidental learning of these recurring word forms manifested as improved repetition accuracy and a correlated reduction of activation in the right superior temporal cortex, similarly for both languages and on both experimental days, and in contrast to a salient left-hemisphere emphasis previously reported in adults. We propose that children, when learning new word forms in either native or foreign language, are not yet constrained by left-hemispheric segmental processing and established sublexical native-language representations. Instead, they may rely more on supra-segmental contours and prosody. It is often stated that children learn a new language differently from adults, just by exposure and imitation. Indeed, the ability to form memory representations for meaningless word forms and reproduce them has been shown to be important especially for the early stages of vocabulary learning , 2. The It is well documented that the ability to learn to perceive phoneme contrasts outside one’s native language decreases during development, reflecting the influence of linguistic environment on the speech processing system . Native Neuroimaging studies have revealed striking differences in the overall cortical sequence of auditory processing in children compared to adults. Auditory evoked responses undergo many changes from childhood to adulthood, with mature features appearing somewhat earlier in the right than left hemisphere –28. ThesThe reorganization of the speech processing system due to native language exposure may also be reflected in cortical learning effects. Cortical familiarity effects at 200–400 ms after stimulus onset have been observed in infants for known or newly-learned words paired with referents in comparison with unknown words, initially bilaterally but becoming more left-lateralized with increasing proficiency in the language , 9, 35. The present study addresses learning of new meaningless foreign and native-like word forms in a narrow age group of 6–8-year-old Finnish-speaking children. We used an incidental learning task with overt repetition and repeated exposure to meaningless word forms, developed in our earlier studies on adults , 36 as wBehavioral experiment. In the MEG experiment, data were collected from another group of 13 children  with mean age of 7 years 2 months (ranging from 6 years 3 months to 7 years 10 months). Participants in both experiments were Finnish-speaking monolinguals. Three children participating in the Behavioral experiment had some experience with foreign languages (not Korean or related languages) in school or daycare. Four additional children participated in MEG measurements, but were excluded from analysis because of large motor artifacts or failure to complete all brain imaging sessions. All participants were dominant native speakers of Finnish. Participants were right-handed as established with an adapted version of the Edinburgh Handedness Inventory  (th block of Day 1 and on Day 2 . Children correctly recognized 73% of the Recurring Korean word forms in the recognition test at the end of Day 2 of the Behavioral experiment. However, the recognition performance only approached significantly better than chance .The = 0.014] . In pairMEG experiment learning effects did not reach significance in the available subsample of children when tested separately for each language, but in visual inspection seemed similar to the behavioral experiment in both languages, although overall the performance was higher for the Native language  = 4.9, p = 0.04]. In paired comparisons, the difference in repetition accuracy between Recurring and New words was statistically significant in the 4th block of Day 1 as well as on Day 2 .In the language . Poolingst block of Day 1 / 4th block of Day 1 / Day 2): F = 10.6, p = 0.011]. In paired comparisons, the difference in repetition accuracy between Recurring and New words was significant in the 4th block of Day 1 .To obtain a reference point of Foreign word repetition accuracy in adults, data obtained in a previous experiment  using a The spatiotemporal pattern was comparable for all stimulus categories and across languages, in both the MEG sensor signals and the distributed source-level cortical estimation . Transient activation was seen bilaterally in the superior temporal cortex, peaking at ~140 ms and returning to baseline at ~300 ms, followed by a strong sustained response from ~300 ms onwards . For mosThe maximum amplitude of the transient response at ~140 ms in the temporal sources was determined individually for each child in a time window from 100 to 170 ms. The declining slope of the response was described with mean source strength in a time window from 170 to 300 ms and individual zero-crossing latency. The earliest statistically significant effect was that of Experimental day in the right hemisphere : The peak amplitude of the transient activation at ~140 ms was lower on Day 2  than on Day 1 [25.3 ± 2.9 nAm] , Table 1For the ECD components modeling the sustained temporal and frontal responses, mean activation values were considered in three 300-ms time windows , which captured the onset, plateau and decline of the sustained responses. Activation in the sustained left temporal responses was weaker for Foreign than Native language  , Table 1In the right temporal sustained response, activation was reduced for the Recurring stimuli compared to the New stimuli, similarly for both languages . In the left temporal sustained responses there was a trend for increased responses for Recurring compared to New stimuli . The effects of Stimulus type were more consistent across subjects in the right than left temporal responses .In the left frontal sustained response, a significant Stimulus type x Language interaction indicated an increase of activation for the Recurring stimuli compared to the New stimuli for Foreign but not for Native language . In paired comparisons of Recurring and New stimuli, the effect approached significance only for the Foreign language .st to the 4th presentation of the Recurring items correlated significantly with the behavioral improvement in repetition accuracy from the 1st to the 4th repetition during Day 1 : The more the signal strength decreased during learning, the better the participant had become at repeating the Recurring items  in the right superior temporal cortex was weaker on day two, akin a similar effect found in adults . BilaterWe observed an effect of word-form-level familiarity as a reduction of right superior temporal activation for the recurring word forms. This effect mirrored the decreased left superior temporal activation found in adults, in both spatial properties and timing –6. FurthSeveral studies on normally developing school-aged children have demonstrated bilateral activation or learning effects during language processing and learning in younger children, with a shift to left hemispheric dominance as age increases , 32, 55.Tentatively, the observed hemispheric differences in the familiarity effects between children and adults could be indicative of a general qualitative shift in the way phonological stimuli are processed . StudiesSpeech comprehension and production can both be thought to be mediated by shared phonological representations that emerge from the mapping of acoustic input to motor output . Less deThe current results reveal a salient right-hemispheric contribution to learning new word forms in children, similarly for native and foreign language. We propose that this might be due to a qualitatively different processing in children compared to adults: in processing new word forms, children rely on suprasegmental prosodic information supported by right-hemispheric processing, and have not yet grown fully constrained to segmenting the input into established native-language phoneme representations in the specialized left temporal areas. It remains for future studies to investigate, possibly over longer periods of exposure, whether the differential cortical learning effects in comparison to adults might reflect differences in language learning potential.


Pakistan is one of only three countries where poliovirus circulation remains endemic. For the Pakistan Polio Eradication Program, identifying high risk districts is essential to target interventions and allocate limited resources.Using a hierarchical Bayesian framework we developed a spatial Poisson hurdle model to jointly model the probability of one or more paralytic polio cases, and the number of cases that would be detected in the event of an outbreak. Rates of underimmunization, routine immunization, and population immunity, as well as seasonality and a history of cases were used to project future risk of cases.The expected number of cases in each district in a 6-month period was predicted using indicators from the previous 6-months and the estimated coefficients from the model. The model achieves an average of 90% predictive accuracy as measured by area under the receiver operating characteristic (ROC) curve, for the past 3 years of cases.The risk of poliovirus has decreased dramatically in many of the key reservoir areas in Pakistan. The results of this model have been used to prioritize sub-national areas in Pakistan to receive additional immunization activities, additional monitoring, or other special interventions.The online version of this article (doi:10.1186/s12916-017-0941-2) contains supplementary material, which is available to authorized users. The Global Polio Eradication Initiative (GPEI) has seen great success since its launch in 1988. At the time of this writing, only Pakistan, Afghanistan, and Nigeria remain endemic for polio with only 37 cases of wild poliovirus serotype 1 (WPV1) recorded in 2016. The 20 cases reported in Pakistan in 2016 represent a historically low case count for a calendar year and a 63% reduction in cases compared to 2015. However, transmission is still occurring on a considerable geographic scale, with four of eight provinces reporting WPV1 cases in 2016. Pakistan has approximately 25 million children under the age of 5 years [In the Eastern Mediterranean Region of the World Health Organization (WHO) key strategies of the of GPEI included (1) achieving high coverage of at least three doses of oral polio vaccine (OPV), 2) implementation of supplementary immunization activities (SIAs), and 3) the development of sensitive epidemiological and laboratory surveillance using standard WHO definitions [ implemen the deveSince 2011, the Pakistan program has been implementing and enhancing a National Emergency Action Plan (NEAP) for polio eradication to improve management and accountability strategies, highlight core reservoirs of transmission, and to ensure the program is creating and using high quality data , 6. To aOur modeling efforts rely on the Pakistan AFP surveillance data, which is managed by WHO and is the source for information on paralytic cases of polio. AFP cases are identified through the extensive surveillance network in Pakistan, which relies on both passive and active (sweeps and case search) surveillance with oversite at the district, provincial, and national level . HoweverUsing the NPAFP dose histories we constructed estimates of district-level vaccination rates, such as zero-dose RI (received zero OPV doses from RI) and underimmunization (defined as children who have received three or fewer doses of OPV from birth until AFP investigation). However, the number of NPAFP observations in each district within a 6-month period can be quite small, ranging from 0 to 74 in our data, resulting in differences during a 6-month period that are implausible when considering children under 5 years of age. To alleviate this problem, we used a hierarchical Bayesian space–time model with a temporally structured space–time interaction  to generA dynamic immunity model, originally developed for Northern Nigeria , was impPolio incidence has been rare and spatially heterogeneous in Pakistan since 2003, with 91% (3804 out of 4185) of 6-month district-level observations reporting zero WPV1 cases. To account for this large number of zeros we implemented a spatial Poisson hurdle model  using a Model selection was carried out in two stages. In the first stage, we fit models based on the 64 combinations of seasonality (low season is January through June), vaccine-derived immunity for type 1 poliovirus, underimmunized fraction, zero dose RI fraction, recent neighboring cases , and recent cases  for the Bernoulli portion, with only random effects in the truncated Poisson model, and selected the model that minimized the deviance information criteria (DIC) . In the The predictive accuracy of our model was assessed by comparing our predictions to held out data. The selected model was first fit using data from 2003 until the first 6 months of 2009 and then used to predict cases in the second half of 2009. This procedure was repeated, using all historical data, for each subsequent 6-month period with the final predictions for the second half of 2016 relying on data from 2003 through the first 6 months of 2016. For each set of predictions, the area under the curve (AUC) for the receiver operator characteristic (ROC) curve was calculated. The AUC for the ROC curve is a single value that summarizes the shape and position of the ROC curve and has the useful interpretation as the probability that a randomly selected district with a case will have a higher risk score than a randomly selected district without a case . AUCs beAll modeling was completed in R 3.2.1 . The SIAAFP surveillance in Pakistan collected data on 43,301 NPAFP cases between January 2003 and June 2016, with an average annual rate increasing from 4.3 to 11.4 NPAFP per 100,000 children under the age of 5 years from 2003 to 2016. Space–time smoothing models fit to the NPAFP vaccination dose history data indicated that zero dose RI and underimmunized rates (fewer than three doses) are highly heterogeneous across Pakistan , and local knowledge of access and security status of districts and Union Councils, as well as sociocultural links within Pakistan and across the border with Afghanistan, were used to classify the 155 districts into four tiers Fig. . Tier 1 Our results indicate that seasonality, immunity, underimmunization rate, recent cases, and recent cases in a neighboring district are most predictive of at least one WPV1 case. The number of cases given at least one case is similarly predicted by seasonality, underimmunization rate, zero RI dose rate, recent cases, and recent neighboring cases. The point estimates of all associations were in the expected direction of lower immunity and dose history as well as recent cases being associated with increased risk of cases and larger outbreaks.Our modeling efforts suggest that the large outbreaks in 2014 and the recent improvements over the past 2 years can be described by population immunity driven primarily by SIAs in FATA and KP provinces. For example, the dramatic decrease in serotypes 1 and 2 immunities in KP beginning in 2010 were partially the result of declining SIA coverage rates (despite the high frequency of campaigns). Furthermore, type 2 immunity declined due to infrequent trivalent OPV campaigns, whereas type 1 immunity declined because SIAs were primarily using bivalent and trivalent OPV, which has a lower efficacy for type 1 than monovalent OPV type 1 vaccine. Finally, improvements beginning in 2015 were due to improved vaccinator access driven by military intervention in FATA .Based on the recommendations in the 2016–2017 NEAP, finalized in May of 2016, Tier 1–3 districts participated in four bOPV SIAs in addition to the five national bOPV SIAs that covered all districts. Additionally, Tier 1 districts scaled up the community-based vaccination strategy, which employs local individuals, primarily women who are thought to have better access to children within homes, as permanent vaccinators within their communities. In the 6-month period between July and December, 2016, only two of the 44 Tier 1 and 2 districts experienced cases (one each), which reflects well on the efforts focused on those districts. Two of the three other districts, which reported cases during this time frame, were classified as Tier 4 districts (four of five cases in Tier 4), although they were ranked in the top 30 per the risk model; the remaining Tier 4 district, in northern KP province, would be considered relatively surprising from a modeling and programmatic perspective. We emphasize that even Tier 4 districts received considerable programmatic attention, with five planned SIAs across 2016.Our approach does have several limitations. We have developed a model on a 6-month time scale, which is programmatically relevant but does not align with the approximately 1 month infectious period estimated for poliovirus . AdditioIn 2009, the Pakistan program initiated environmental surveillance (ES) to compliment AFP surveillance. Since 2009, the program has grown from 47 samples across 6 sites to 648 samples across 62 sites covering 33 districts in 2016 , 39. DueDespite the limitations, our modeling approach provides a principled framework for ranking districts for risk classification that performs well as measured by AUC and sensitivity. In practice, the risk analysis generally identifies the same Tier 1 districts as the aggregation of programmatic knowledge and scientific intuition of the N-EOC members and the greatest impact of the modeling approach is the promotion of districts that appear susceptible based on immunity profile, but have not yet had WPV1 cases, to a lower (higher risk) tier. These promotions impacted the allocation of resources by deploying the community-based vaccination strategy in additional districts and broadening the geographic scope of the four sub-national SIAs. Additionally, the method provides a metric for quantifying the absolute risk and changes in risk over time, which is not always captured well by cases or intuition exclusively.This study serves as the first use of a spatial model for risk prediction and sub-national prioritization to aid in polio eradication in Pakistan. This risk modeling approach has been applied to the WPV1 case history and NPAFP dose histories to generate risk predictions and a ranked list of districts within Pakistan, which were subsequently used by the N-EOC to help assign districts to risk tiers as part of their NEAP for polio eradication. This approach will help maximize the impact of the resources available for polio eradication efforts in Pakistan.


Use of cone beam computed tomography (CBCT) in orthodontics is increasing; however, some patients started treatment with conventional images. The objective of this study is to manipulate CBCT panoramic reconstruction to make it comparable to conventional panoramic image and to compare mesiodistal root angulations on both images.Concurrent conventional panoramics and CBCT volumes were obtained from 40 subjects. CBCT volumes were manipulated to generate pan-like images that mimic the occlusal plane angle of the corresponding panoramic, allowing comparison of mesiodistal root angulations and determination of the head-tilt required to produce the reconstruction.p < .05) in the mesiodistal root angulations between standard panoramics and CBCT reconstructions emerged for 13 out of 24 teeth (54%). Greatest variations were seen in the maxillary and mandibular sextants and in first molar regions. Ideal axial head-tilt for image acquisition was determined to be with Frankfort horizontal plane 3.3o nose down.Clinically meaningful differences (CBCT pan images must be used with caution due to variation between methods in specific areas of arches. The images can be useful for the assessment of mesiodistal root angulations if the volume is properly manipulated to create a pan-like image. During dental rehabilitation, whether it is by orthodontic or prosthodontic correction of malocclusion, the clinician is concerned with the teeth being oriented ideally within the bone in all three planes , 2. For Recently, cone-beam computed tomography (CBCT) has been introduced as a great advancement in dental radiography. This technique provides a three-dimensional (3D) volume of data which can be rendered at any desired angle from a single imaging session. Several types of images, such as two-dimensional (2D) lateral cephalograms and panoramic views can be generated by manipulating the 3D volume data. The exactness of CBCT 3D images has been confirmed in a number of studies -19. ThisThis study was based on the hypothesis that it is possible to derive 2D pan-like images from CBCT scanned data in such a way that direct side by side mesiodistal root angulation comparisons with conventional panoramics can be made. Furthermore, it was hypothesized that significant differences would be detected in the mesiodistal root angulation measurements between conventional pan and CBCT derived pan-like images. It was expected that the results of this study would determine a useful reference plane for positioning patients in the CBCT cephalostat, as well as provide an increased understanding of how mesiodistal root inclination assessment with the newer method varies compared to conventional pan radiography.The specific aims of this study are:To assist clinicians to generate 2-D pan-like reconstructions from CBCT volumes that arecomparable to traditional pan images.To determine if there is a clinically significant difference between mesiodistal root angulations of teeth measured on conventional panoramics and CBCT derived pan-like reconstructions.This retrospective, observational study was designed to allow the comparison of the mesiodistal root angulations on x-rays taken from subjects who had received both conventional panoramics and CBCT scans within the same time frame.priori statistical power analysis was performed assuming that a difference between imaging methods of 2.5˚ or greater is clinically meaningful , indicating that the measured root angles obtained under the two methods, pan versus pan-like images were different when averaging over all 24 teeth (mean for CBCT = 97.21˚ versus 95.06˚ for pan). The main effect of tooth [ F = 23.64. p < .001] was also significant indicating that root angle depends on which tooth in the arch. Furthermore, the interaction effect between imaging method and tooth was significant [ F = 5,84, p < .001), demonstrating that the difference between methods depends on which tooth is being considered. Therefore, the difference between methods was further studied by comparing CBCT pan-like images versus panoramics for each tooth. Table (2) displays the mean differences, standard deviations, and 95% confidence intervals for the differences between root angles derived from pan and pan-like images for each tooth.In the omnibus analysis of the measured tooth pairs, all main and interaction effects were statistically significant at the p< .05 level for maxillary teeth 11-15 and 24-26. In the mandible, only three teeth 35, 45, and 31 were not significantly different. All other mandibular teeth, particularly first premolar to first premolar (except 31) showed significant differences. Additionally, pan-like images tended to depict the maxillary roots from teeth 15-25 more distally and the mandibular roots from 34-45 more distally. The remaining posterior teeth, except for tooth 45, were depicted with mesial inclination relative to standard pan angulations.Compared to standard panoramics, measurements from pan-like images were not significantly different at the uncorrected 3 provides a graphic for angle measurement comparisons, where larger variation exists, and whether there is a mesial or distal tendency to the angle projection by CBCT relative to standard panoramics. Employing the Bonferroni correction to the p-value of each tooth, we multiplied each p-value by 24 (the number of teeth). Even by this highly conservative standard, statistically significant differences persisted for several teeth . The Bonferroni p-values are presented in (Table 2).The more notable differences between methods were found for the upper left anterior region with teeth 22 (4.1˚) and 23 (5.1˚). Stark differences were found especially in the mandible where, except for tooth 31, teeth 34-44 differed from 4.1˚ to 8.9˚. Not only were the differences highly significant, but the corresponding standard deviations were larger for these teeth as well. Table The results relate only to the panoramic unit used in this study (Planmeca Proline) and the use of the "On demand 3D software" for manipulation of the DICOM files. However, it has been shown that several panoramic units are similar in the projection of mesiodistal root angulations .The consThe purpose of this study was to determine a useful reference plane for positioning patients in the CBCT cephalostat, to be able to generate images that can be used to compare to panoramic images taken previously as well as provide an increased understanding of how mesiodistal root inclination assessment with the newer method varies compared to conventional pan radiography.In order to create pan-like images that are comparable for this study, CBCT DICOM files must be manipulated via computer software in such a way as to produce a 2D view of a 3D object. During the course of this study, it was observed that the ability to produce a high-resolution 2D image free from obvious root distortions varied depending on the axial orientation of the volume used and on the method of plotting points along the arches. Image integrity was apparently lost particularly along the line of the tightest curve of the anterior mandible. This may account in part for why the greatest differences between imaging methods occurred in the anterior mandible. Refined operator technique to some extent, and further technological improvement in software rendering inevitably should improve image quality during software manipulation of the 3D volume into a 2D pan and reduce the resultant distortions.This study included 40 subjects, with possibly none exhibiting ideal occlusion. Although the inclusion criteria for the study did not include an assessment of each subject’s occlusion, many subjects were of orthodontic age and contemplating orthodontic treatment who presumably exhibited a degree of malocclusion. Although from an assessment of the 3D volume, it might have been possible to determine the degree to which the included subjects in the study presented with various features associated with malocclusions, it was only noted that features existed. The presence of such things as constricted arch forms, crowding in the maxilla and mandible, acute inter-incisal angles, and unusual occlusal plane angles seemed to influence the observer’s ability to obtain distortion-free pan-like images. Through the trial and error process of creating pan-like images, it was found that these various features, in addition to the previously mentioned axial head tilt and plotting of points for generating the 2D view, also seemed to influence the resolution of the images, the degree of distortions, and ultimately the mesiodistal angulations of the teeth in the anterior sextants.Notwithstanding the presence of features that influence the generation of a pan-like image, the best subjective depiction of the anterior teeth was obtained when the axial head tilt corresponded to Frankfort horizontal being about 3-4 degrees nose down from the true horizon. This is helpful to know when positioning the patient’s head in the cephalostat to preclude the need to reorient the 3D volume in the software prior to creating a 2D pan-like image, thereby decreasing the time it takes to create the image. It is suggested that clinicians or technicians performing the scans keep this head position in mind as a good position for minimizing post-scan rendering effort. However, if the scan happened to be taken with the head oriented too high or low, this recommended head orientation serves as a point to begin creation of the pan image within the software. Further research directed toward the determination of the ideal orientation of the patient’s occlusal plane from the cephalometric perspective relative to true horizontal would be most appropriate for reducing distortions in pan-like images. Although Frankfort horizontal (Po-Or) plane is convenient due to readily discernible external landmarks, it is the ideal orientation of the occlusal plane that would give rise to the greatest consistency in the creation of pan-like images for analysis of mesiodistal root angulations. A study determined the optimal orientation of the occlusal plane would also be informative.and clinically significant differences in measured root angulations in the mandibular anterior sextant [A pattern of statistically significant differences in the mesiodistal root angulations between standard panoramics and CBCT pan-like images emerged for 13 out of 24 teeth (54%). All first molars except for tooth 26, the upper left anterior (teeth 21-23), and the lower teeth from first premolar to first premolar, except for tooth 31 were significantly different. The magnitude of the differences in mesiodistal angles measured relative to the occlusal plane was larger in the mandible from first premolar to first premolar. If a tolerance limit of ±2.5˚ is applied, there remained statistically  sextant , 9, 16. et al explored an alternative method in the use of in vivo Dental 3D software to create custom sections from axial slices, with measurements of each tooth made from a facial view relative to the occlusal plane [Obviously, it is not a requirement to use CBCT pan-like image to assess mesiodistal root angulations. Other methods exist; Bouwens al plane . Ideal ual plane , 20, 21.The significance of the study is its ability to provide a method for acquiring CBCT volumes able to generate panoramic reconstructions that can be compared to pre- or post-orthodontic treatmentpanoramic images taken prior or after a CBCT volume.The following conclusions can be drawn from this study:CBCT-derived pan-like images can be made comparable to that of conventionally taken panoramic radiographs, allowing direct comparison of mesiodistal root angles from both types of images. To create pan-like images that resemble the conventional pan images, the patient’s head should be positioned with Frankfort horizontal plane oriented 3.3 degrees nose down to true horizontal. Alternatively, the 3D volume could be oriented within the software with the same orientation as a starting point for deriving the pan-like image.Greatest differences in the mesiodistal root angles between the pan and CBCT pan-like images were observed in the upper and lower anterior sextants, and in the first molar areas. CBCT pan-like images are less consistent than standard panoramics in their depiction of mesiodistal root angulation, especially in the maxillary and mandibular anterior regions, as well as in molar regions, and must be used with an understanding of the potential root distortion that occurs when generating these images. However, the images can be a useful adjunct for assessment of mesiodistal root angulations if the clinician is knowledgeable about its limitations and how the volume is manipulated in creating a pan-like image.A pattern existed with the CBCT scan depicting from maxillary first premolar to first premolar with a more distal angulation compared with standard panoramics, and second premolars and molars with a more mesial angulation. In the mandible, a similar pattern emerged, where the lower right second premolar to lower left first premolar was depicted more distally, and the rest of the posterior teeth were more mesial inclined.


ADAMTSL4 gene and results in a loss of expression in homozygous carriers. In addition, by taking this translocation as a model, we used a whole-genome next-generation mate-pair sequencing approach on pooled individuals to evaluate this strategy for high-throughput screening of structural rearrangements.Reciprocal translocations are the most frequently occurring constitutional structural rearrangements in mammalian genomes. In phenotypically normal pigs, an incidence of 1/200 is estimated for such rearrangements. Even if constitutional translocations do not necessarily induce defects and diseases, they are responsible for significant economic losses in domestic animals due to reproduction failures. Over the last 30 years, advances in molecular and cytogenetic technologies have led to major improvements in the resolution of the characterization of translocation events. Characterization of translocation breakpoints helps to decipher the mechanisms that lead to such rearrangements and the functions of the genes that are involved in the translocation. Here, we describe the fine characterization of a reciprocal translocation t  detected in a pig line. The breakpoint was identified at the base-pair level using a positional cloning and chromosome walking strategy in somatic cell hybrids that were generated from an animal that carries this translocation. We show that this translocation occurs within the  Reciprocal translocations are the most frequently occurring constitutional structural rearrangements both in humans, with an incidence ranging from 1/752 live-births . As in hThe first chromosome rearrangement involving large DNA fragments in pigs was identified and described by  using coADAMTSL4 gene and thus modifies its expression in translocation carriers. The flanking sequences were used to develop a PCR-based diagnostic test to genotype all the animals of the selected line.Here, we report the characterization of a reciprocal translocation t  that segregates in a Large-White pig line, which has been selected since six generations for residual feed intake (RFI) . The traOur experiments were conducted in accordance with the European Directive 2010/63/EU on the protection of animals used for scientific purposes, and approved by the Ethics Committee for Animal Experimentation of the Poitou Charentes region (France) (N°CE2012-2). The animals used in this study were raised in a conventional production system with little or no animal manipulation. The animals are checked for health and well-being every day, and they are prided with food and water ad libitum. Animals were sacrificed in a commercial slaughterhouse following national and institutional guidelines for the Good Experimental Practices and approved by the Ethical Committee of INRA . The experimentation registration number for the experimental farm on which the animals were raised is A-17-661.The reciprocal translocation was identified in a Large-White pig line produced on an INRA experimental facility . Metaphase spreads were obtained from classic lymphocyte cultures and subjected to GTG-banding as previously described . The chr6 lymphocytes of a homozygous t  translocation carrier with 20.106 recipient hamster cells from the permanent HPRT- Chinese hamster line Wg3hCl2. Fusion and selection were based on standard methods [Somatic cell hybrid clones were produced by fusing 20.10 methods . The selProbes for FISH experiments were generated using 50 ng of hybrid DNA as template for interSINE-PCR amplification as described previously . For eacPainted chromosomes or fragments of chromosomes were identified by comparison with G-banded metaphase chromosome pictures taken before hybridization .Sus scrofa (SSC) chromosomes SSC3 and SSC4. In a second step, the SNP sequences of the Illumina pig 60K SNP chip were used as STSs and pairs of primers were defined based on the sequences on either side of each SNP (http://www.ensembl.org/Sus_scrofa/Info/Index). Primers were selected using Primer3 software, and PCR conditions were determined for each STS (Microsatellite markers were chosen on the genetic maps of each SNP . In a theach STS .GeneAmp® XL PCR Kit (Life Technologies). Products from these reactions were purified by incubation with 10 units of Exonuclease I and 0.5 unit of SAP at 37 C during 45 min followed by heat inactivation (80 C during 30 min). They were then sequenced using the ABI PRISM® BigDye® Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM 3730 sequencer . Computational analysis of the resulting sequences allowed precise localization of the breakpoints on SSC3 and 4.Junction fragments across the t breakpoint were amplified by long-range PCR using the Trsl_SSC3.Up and Trsl_SSC3.Dn) and SSC4 (Trsl_SSC4.Up and Trsl_SSC4.Dn) to amplify the junction fragments across the t breakpoint. In order to genotype animals for the translocation (homozygous or heterozygous), only three primers were used: the unique forward primer Trsl_SSC4.Up was used with Trsl_SSC4.Dn (amplification of a 346-bp fragment in the absence of translocation) or with Trsl_SSC3.Dn (amplification of a fragment of 277 bp in the presence of the translocation) (2 for 35 cycles at 58 C.Two pairs of primers were selected respectively on SSC3 (ocation) . GenotypGTCTCCAACCGGATACTGGA) and 14 (Dn: TACCAGGGCCACAGGAAC), and ADAM_cDNA_control in exons 15 (Up: TGATGAAGTGAGCGAGCAAG) and 17 (Dn: CCAGTTTGGACACACAGATGA)  . PCR ampThe pooled genomic DNA of five Large White animals , one of which was heterozygous for the t translocation, was used to generate a mate-pair library following the Nextera protocol from Illumina. The target insert size of 6 kb was selected and the library was sequenced with read lengths of 100 bp for each mate. A second pool of five normal Duroc animals, which are assumed to harbor only normal chromosomes with respect to the translocation, was used as a reference normal sample. Sequencing data of the Duroc and Large-White pooled samples are publicly available in the Sequence Read Archive (SRA) database under accession numbers SRX2996560 and SRX2996558, respectively. Sequences were trimmed for adapters using cutadapt . The revThe balanced t  was originally detected by G-banded karyotype analysis in a LW pig with a small litter size . The comTo verify the presence of porcine DNA in the hybrids and select the clones containing the translocated chromosomes, a set of 37 microsatellites was defined and used for PCR experiments on the DNA of each clone. The set included 12 microsatellites that are located on 12 different chromosomes on the porcine genetic map , plus 12To confirm these PCR results, pIRS-PCR (porcine Interspersed Repetitive Sequence) products were prepared using the DNA of Hb1.8 (chosen among the seven clones carrying the der(4) chromosome) and Hb2.5 clones as probes in FISH experiments on porcine metaphase chromosomes as described previously . pIRS-PCThe first localizations of breakpoints were obtained by using the 25 microsatellites initially used to characterize the presence of SSC3 and SSC4 in the somatic hybrid clones . The fouFor the two other microsatellites that were not mapped on the draft, the positions of the closest microsatellites on the genetic map were used as reference: SW251 (next to SW2527) was localized at 21.948 Mb on SSC3 and SW2435 (next to SW524) was localized at 120.509 Mb on SSC4 . Using tIn order to control the quality of the pig draft sequences in the two intervals of interest, the porcine sequences (pig Sscrofa10.2 assembly) were compared to the human draft genome sequence (human GRCh38/hg38 assembly). On SSC3, we observed a collinear organization between the 23.803–23.876 Mb interval on SSC3 and the 22.764–22.831 Mb interval on HSA16. Seven new STS markers were designed from the pig draft sequence and PCR were performed with Hb1.8 DNA as template . The minECM1 gene located in the missing sequence  was selected on the pig BAC clone CH242-160A10 (accession number CU655899), which contains ADAMTSL4 and was used to reduce the breakpoint interval on SSC4 to 20 kb , and six from porcine EST sequences corresponding to genes located in the orthologous human interval, including the sequence . The locto 20 kb .ADAMTSL4 on SSC4. In order to bridge the breakpoint region, the four possible crossed-combinations of oligonucleotides (combining one primer for the SSC3_Tsloq_frag29 locus and one primer for ADAMTSL4) were tested on Hb1.8 DNA. One primer combination yielded an amplification product of approximately 1 kb. This product was purified, directly sequenced, and the sequences were compared to identify precisely the junction between the two chromosomal segments. The der(4) breakpoint was precisely mapped on SSC3 to position 48,007 Mb within the sequence of clone CH242-117J1 (GenBank accession number CU928801) and on SSC4, to position 27,558 Mb within the sequence of clone CH242-160A10 (GenBank accession number CU655899). To validate this result, a pair of primers was selected from the two BAC sequences on both sides of the breakpoint to amplify the junction der(3) using Hyb25.1 DNA as template. The resulting PCR product was sequenced and compared to the BAC DNA sequence. The breakpoint sequence obtained matched to the previously obtained alignment except for a small deleted sequence of 5 bp (TACAC)  . Using t (TACAC) . Product (TACAC) .We used data from a parallel study on the detection of structural variations (SVs) in pigs to confirm the translocation breakpoint. In that study, samples from different animals were pooled to detect breed-specific SV. A heterozygous carrier of t was included in a pool of five Large White animals as a test case for the detection of SVs. Three SV-finding computational tools were tested on the generated sequencing data see . The trahttp://www.ensembl.org/Sus_scrofa/blastview website. On SSC3, an intergenic small interspersed nucleotide element (SINE) retrotransposon spans the breakpoint and on SSC4, the breakpoint lies in intron 15 of the ADAMTSL4 gene, resulting in the loss of the four last exons of the gene  homozygous carrier.The two translocated sequences were annotated using the the gene . In ordethe gene . Since tthe gene , confirmADAMTSL4 gene.One central goal of genome analyses is the comprehensive identification of all the genes together with their functions in the relevant biological processes. Studying chromosomal translocation breakpoints can provide insight into the function of genes when the translocation event disrupts their sequence. Here, we report the delineation and sequencing of a chromosomal breakpoint associated with a t reciprocal translocation that was detected in the Large-White pig line. We chose to characterize as finely as possible the breakpoints using somatic cell hybrids. While these are without doubt a powerful tool, constructing different panels for each translocation that occurs in pig populations is not feasible. Thus, we assessed the relevance of a high-throughput sequencing strategy using a pool of individuals including one translocation carrier. Our findings underline the fact that genome-wide screening for translocations, and more generally structural variants, using mate-pair sequencing on pools of individuals is not straightforward for various reasons. First, the status of the current pig genome reference assembly (SusScrofa 10.2) is certainly responsible for a large number of predicted structural variants that have no biological basis as attested by the significant reduction in the number of predicted SVs when using a differential case/control approach. A second reason arises from the fact that the genome that harbors the structural variant is pooled with “normal” genomes that hinder the detection of the translocation signal. Moreover, the signal associated with structural variants is known to be affected by high background noise due to false-positive discordant mate-pairs  Fig 5)..Fig 5). ADAMTSL4 are clearly causative for ectopia lentis (EL) and congenital abnormalities of the iris [ADAMTS  and ADAMTSL  proteins are essential for the regulation of the development and maintenance of the extracellular matrix (ECM) as revealed by the clinical manifestations of Mendelian disorders resulting from mutations in each gene of these two families ,30. ADAMthe iris –35. In vthe iris . Microfithe iris . Disruptthe iris . In our a posteriori. The prolificacy of sires, which are heterozygous for the translocation, was 26% (P<2.10−16) lower than the performances of non-affected sires. Animals carrying the translocation in a homozygous state should not be impacted regarding their prolificacy or the segregation of chromosomes at meiosis since they produce only balanced gametes. Nevertheless, the number of homozygous animals is too small to test this hypothesis. With the current available data, it is not possible to determine whether the change in frequency is only due to genetic drift. An alternative hypothesis could be that the translocation itself or a quantitative locus localized near the translocation on SSC3 or 4 could have a favorable effect on a quantitative trait that is taken into account in the selection of this line. To test this hypothesis, it would be interesting to perform an association study between the genotypes obtained by diagnostic testing and the phenotypic traits recorded for the animals of the linen. A significant association would indicate that over the generations, the frequency of this translocation increased as a result of selection.Among the animals of the line analyzed here, sows and boars that were pre-selected as future founders for the next generation were systematically analyzed by G-band karyotyping. Among the 110 candidates of the current generation, 27 heterozygous or two homozygous individuals were detected. Based on the precise identification of the translocation from this study, we developed a simple PCR diagnostic test to determine the genotype of the animals for this translocation. PCR amplifications were performed for all sires and dams from all generations since the creation of the line. Among the 636 reproducers, 42 heterozygous and one homozygous individuals were identified. The founder animal, which is responsible for the dissemination of the translocation, is one of the G0 founder boars. At each generation, six to 13 carrier individuals were selected, which gradually led to an increase in the frequency of the translocation in the line and to the occurrence of the first homozygous animal at generation 4.The effect of the translocation on litter size was confirmed In conclusion, here we describe the fine characterization, at the base pair level, of a reciprocal translocation identified in a pig line. This work was carried out using a positional cloning  strategy in somatic cell hybrids generated from one animal carrying the translocation. This approach was successful but it is too time-consuming to be generalized for mapping other reciprocal translocations in pigs. The parallel mated-pair approach is the most rapid and powerful tool for characterizing and mapping translocations at the nucleotide level. In the future, systematic characterization of translocation points associated with the phenotypic analysis of carrier animals could contribute, as in humans, to a better understanding of the function of the genes affected by chromosomal rearrangements.S1 TablePositions of the primers on the reference sequence and PCR conditions are reported.(PDF)Click here for additional data file.S1 FigAlignment of the der(3) translocated sequence obtained from Hb25.1 (in blue) and the der(4) translocated sequence obtained from Hb1.8 (in yellow) with the SSC3 genomic reference sequences (a) and the SSC4 genomic reference sequence (b). On SSC3 alignment, the 5-bp missing motif is colored in yellow. Positions of the four primers selected to perform the validation and the genotyping of the translocation are indicated in boxes.(PDF)Click here for additional data file.S2 Fig(a) PCR validation on genomic DNA, according to the genotype of the individuals for the translocation. Primers were selected on each side of the translocation point to generate PCR products of different sizes depending on the amplified copy (Translocated vs Not-translocated).(b) Amplification results obtained with two pairs of primers selected in exons 13–14 and exons 15–17 on lung and heart cDNA samples from three animals with different genotypes for the translocation. No amplification was observed with the Ex15_17 pair, which overlaps the translocation point, in animals that are homozygous for the translocation.(PDF)Click here for additional data file.S3 FigADAMTSL4 gene, based on data reported in [ADAMTSL4 (black arrows).Schematic representation of the intron-exon and protein structures of the orted in  The diff(PDF)Click here for additional data file.