This gene set was enriched for miR-34b/c binding sites, functional categories related to the central nervous system, such as axon guidance or neuron differentiation, and serotonin biosynthesis and signaling canonical pathways. Our results provide preliminary evidence for the contribution to ADHD of a functional variant in the pri-miR-34b/c promoter, possibly through dysregulation of the expression of mature forms of miR-34b and miR-34c and some target genes. These data highlight the importance of abnormal miRNA function as a potential epigenetic mechanism contributing to ADHD.Attention deficit and hyperactivity disorder (ADHD) is a prevalent neurodevelopmental disorder characterized by impairment to sustain attention and inability to control impulses and activity level. The etiology of ADHD is complex, with an estimated heritability of 70–80%. Under the hypothesis that alterations in the processing or target binding of microRNAs (miRNAs) may result in functional alterations predisposing to ADHD, we explored whether common polymorphisms potentially affecting miRNA-mediated regulation are involved in this psychiatric disorder. We performed a comprehensive association study focused on 134 miRNAs in 754 ADHD subjects and 766 controls and found association between the miR-34b/c locus and ADHD. Subsequently, we provided preliminary evidence for overexpression of the miR-34c-3p mature form in peripheral blood mononuclear cells of ADHD subjects. Next, we tested the effect on gene expression of single-nucleotide polymorphisms within the ADHD-associated region and found that rs4938923 in the promoter of the pri-miR-34b/c tags  Epidemiological studies report a worldwide ADHD prevalence of 5–12% for children and 3–8% for adults.5 Pathogenic models of ADHD have traditionally focused on the dysregulation of the dopaminergic, serotoninergic and noradrenergic neurotransmission systems, the regulation of neurotransmitter release (SNARE complex) and on neurotrophic factors.6 In addition to candidate–gene association studies, genome-wide association studies (GWAS) have also been carried out in nine independent data sets in ADHD, seven of which focused on childhood samples and two on adults.8 However, none of them reported genome-wide significant findings or highlighted previous candidate genes for ADHD or identified associations overlapping with those of other GWAS.Although association, linkage and animal model strategies have been followed to uncover the genetic background of ADHD, consistent genetic risk factors with a major role in its susceptibility have not yet been discovered.9 Thus, there is a growing interest into genetic variation affecting epigenetic mechanisms involved in the dysregulation of gene expression. Among them, microRNAs (miRNAs) have emerged as strong candidates. These small non-coding single-stranded RNA molecules have important gene-regulatory roles by pairing to the messenger RNA (mRNAs) of protein-coding genes to direct their posttranscriptional repression.10 Some observations, however, suggest that miRNAs can also target other genomic regions, such as 5′-UTR elements, promoters or coding sequences, and additionally they may even be able to stimulate gene expression. The miRNAs configure a complex network where individually they may regulate hundreds of genes and collectively they are predicted to modulate from 30 to 80% of the human genome.11 Thus, slight variations affecting this system may lead to altered miRNA function, have pleiotropic effects, and contribute to part of the genetic heterogeneity underlying complex diseases. Interestingly, around 70% of all known miRNAs are expressed in the brain and have key roles in cell-fate specification and survival, neurite projection and synaptic plasticity.12 Moreover, miRNAs are known to be implicated in psychiatric and neurological disorders. A GWAS in schizophrenia revealed that the strongest association corresponded to miR-137, and SNPs in multiple other miRNAs, such as miR-206, miR-198, miR-24 or miR-30e, have also been associated with this disorder.14 Abnormal miRNA biogenesis and expression levels have been observed in schizophrenia, bipolar or autism spectrum disorder (ASD). Moreover, variants within the miR-183-96-182 cluster locus or in the miR-641 binding sites at SNAP25 gene have been associated with ADHD and impulsivity, respectively.16 In addition, a recent study reported altered circulating levels of miRNAs in ADHD subjects and suggested that miR-107 levels below a certain threshold were highly predictive and specific for ADHD.17Research on ADHD has traditionally focused on protein-coding sequences, but some data suggest that most of the common phenotypic variation would stem from variants in regulatory elements rather than in protein-coding sequences.cis and trans in the peripheral blood mononuclear cells (PBMCs) from subjects with ADHD. Top hits from the trans-eQTL results were considered for functional and canonical pathway overrepresentation and gene-network construction.Considering this background altogether, we attempted to unravel novel susceptibility factors for ADHD by conducting a case–control association study focused on miRNAs and their target genes. We selected miRNAs based on the previous association of their validated targets to psychiatric illness, giving priority to those linked to ADHD. Subsequently, we conducted a case–control association study in 754 adult ADHD subjects and 766 healthy controls. The impact of the associated variants on gene expression was evaluated through analysis of Quantitative Trait Loci (eQTLs) in 18 The severity of ADHD symptoms was assessed with the long version of the Conners' ADHD Rating Scale (self-report [CAARS-S:L] and observer [CAARS-O:L]),19 the ADHD rating scale,20 the ADHD screening checklist21 and the Wender Utah Rating Scale for retrospective symptomatology.22 The level of impairment was measured with the Clinical Global Impression and the Sheehan Disability Inventory.24 Additional tests used for clinical assessment are available in Ribasés et al.25 The control sample included 766 unrelated Caucasoid Spanish healthy individuals matched for sex with the clinical group. Sixty-seven percent of the subjects were male with an average age of 57 years (s.d.=18.4). ADHD symptomatology was excluded retrospectively under the following criteria: (1) not having been diagnosed with ADHD previously and (2) answering negatively to the life-time presence of the following ADHD symptoms: (a) often has trouble in keeping attention on tasks; (b) usually loses things needed for tasks; (c) often fidgets with hands or feet or squirms in seat and (d) often gets up from seat when remaining in seat is expected. All the subjects were evaluated and recruited at the Hospital Universitari Vall d'Hebron of Barcelona (Spain) and diagnosis was blind to genotype. The study was approved by the ethics committee of our institution and written informed consent was obtained from all the subjects before the inclusion into the study.The clinical sample consisted of 754 Caucasoid Spanish adults with ADHD . Sixty-seven percent of the individuals were male and the average age at assessment was 33 years (s.d.=12.7). The evaluation of the ADHD diagnosis was carried out with the Structured Clinical Interview for DSM-IV Axis I and II Disorders (SCID-I and SCID-II) and with the Conners' Adult ADHD Diagnostic Interview for DSM-IV (CAADID parts I and II).Genomic DNA was isolated from peripheral blood leukocytes by the salting-out procedure.26 MiRNA clusters were defined as genomic regions containing at least two contiguous miRNAs with an interdistance of <10 kb. Candidate regions spanned 5 kb upstream and 3 kb downstream from each miRNA or from the first and last miRNA of each cluster. When miRNAs were located in a transcriptional unit, some additional 5 kb upstream from the host gene were also included.MiRNA and miRNA clusters: On the basis of literature data, 134 miRNAs distributed in 53 loci with at least one confirmed target gene involved in ADHD or other psychiatric disorders, including schizophrenia, bipolar disorder, major depression and/or ASD, were selected from a total of 5201 records of experimentally validated miRNA–target interactions available at the miRecords database (release 2012).29 Considering only those mRNAs that are targeted by the miRNA found to be associated with ADHD in this study, a total of 12 candidate genes were initially selected on the basis of experimental data supporting each miRNA–mRNA interaction , Diana-TarBase (version 7.2), miRTarBase , miRBase (release 21) and miRecords (release 2012), or through literature searches .26, 27, r2⩽0.85) considering SNPs with a minor allele frequency (MAF) ⩾0.10 from the CEPH population provided by the HapMap Project Database  and using the LDSelect Software . Under these conditions, we selected 214 tagSNPs covering, in terms of linkage disequilibrium (LD), genomic regions containing 134 miRNAs. We also selected 20 SNPs that tag the 3′-UTR regions of genes encoding mRNAs that are targeted by the miRNA found to be associated with ADHD in this study. From the 12 target genes initially selected, no SNPs at NOTCH4 or MYC fulfilled the aforementioned criteria and, therefore, only 10 candidate genes were finally considered . An additional variant, rs4525537, reported in the 1000 Genomes database , was also included owing to its close location to the miR-34c target site in the CRHR1 3′-UTR (14 nucleotides upstream from the miRNA–mRNA binding site). From the 235 SNPs that were initially selected, a proper design was not achieved for four of them, and in some additional 10, the genotyping assay failed (genotyping rate of 97.5% for the miRNAs assay and 100% for the target genes assay). All SNPs were genotyped using the PCR-based KASP platform .Tagged-SNP selection was based on genetic coverage criteria , assuming an odds ratio (OR) of 1.2, a disease prevalence of 0.05, a significance level (α) of 0.05, the lowest MAF observed in our control sample (0.10), and an additive model of inheritance. Potential genetic stratification in our sample was previously discarded.30The minimal statistical power of the sample was estimated PHWE<0.01) and the comparison of genotype frequencies between cases and control subjects under a log-additive model of inheritance were performed using the SNPassoc R Library.31 Dominant (11+12 vs 22) and recessive (11 vs 12+22) models of inheritance were only considered for those SNPs displaying nominal association under a log-additive model. Genotype frequencies of SNPs located in chromosome X were only studied in females. All the tests were adjusted by gender. To measure and control the proportion of false positives incurred in the study, we used the false discovery rate (FDR) method, an approach to adjust for multiple comparisons that provides a good balance between discovery of statistically significant findings and controlling type I errors (α). The q-value was computed using the qvalue R package32 considering all the association tests performed for miRNAs and target genes jointly. We considered all the P-values from each SNP tested under an additive model of inheritance, as well as the P-values from the dominant and recessive models when considered (when the association under the additive model was significant (P<0.05)). Under these conditions, a total of 232 P-values were included and a unique FDR of 20% that corresponded to P<1.58e−03 was considered.The analysis of Hardy–Weinberg equilibrium (HWE) in the control sample (https://vegas2.qimrberghofer.edu.au/).33 We considered all genomic regions containing miRNA or target genes with more than one genotyped SNP per region. All the genotyped SNPs available from each locus were included in the analysis considering the nominal P-value of each association test from the single-marker analysis. Regions were defined spanning 10 kb upstream and downstream from each locus, and European samples from 1000 Genomes were used to assess their LD structure.Gene-based association analysis was performed using the Versatile Gene-based Association Study (VEGAS) 2 software , considering the overall, the combined or the inattentive samples as dependent variables and the ADHD-risk variants as independent variables. The hyperactive-impulsive sample was not considered, owing to its limited sample size.Total RNA was extracted from PBMCs of a medication-naive subset of 45 ADHD individuals from the sample included in the case–control association study ) and from 32 additional control subjects ) using the Ficoll density gradient method and the RNeasy Midi kit . RNA and miRNA quality assessment was assayed by 2100 Bioanalyzer .CT) was defined as the fractional cycle number at which the fluorescence exceeded the threshold of 0.2. The relative quantification of miRNA expression was calculated by the 2−ΔΔCT method,34 considering U6 as endogenous control gene after checking its stability and linearity across all the samples. Batch, gender and age were considered as covariates when a significant association with the study outcome was observed (P>0.20) and were kept in the statistical model if the corresponding equation parameter was significant in the fitted model (P<0.05). Logistic regression models were applied to compare expression differences between ADHD cases and controls. All the statistical tests were two-sided and the significance threshold was set at P<0.05.In 31 out of the 45 cases mentioned above, the total RNA isolation protocol from PBMCs was performed with further enrichment of miRNAs. Considering only the miRNA found significantly associated with ADHD in this study, the expression levels of the mature forms miR-34b-3p, miR-34b-5p, miR-34c-3p and miR-34c-5p were assessed in these 31 cases and in 32 additional control subjects by reverse transcription reaction using the Taqman MicroRNA Reverse Transcription Kit . Quantitative real-time reverse transcription PCR (qRT-PCR) for each assay was run in triplicate using Taqman MicroRNA Assays (Applied Biosystems) and measured in Applied Biosystems 7900HT fast real-time PCR system. The threshold cycle . All the statistical tests were two-sided and the significance threshold was set at P<0.05.The trans-eQTL analyses in 45 ADHD subjects (60% overlap with the ADHD sample included in the qRT-PCR assays). RNA was reverse transcribed using the Ambion WT Expression Kit . The cRNA was subsequently fragmented, labeled and hybridized with the GeneChip WT Terminal Labeling and Hybridization Kit . The samples were hybridized to the Genechip Human Gene 1.1 ST 96-Array plate (Affymetrix), covering a total of 36 079 transcripts that correspond to 21 014 genes. The array processing and data generation were assessed using the Gene Titan Affymetrix microarray platform. The raw data were pre-processed with the Robust Multichip Analysis method  using the R environment and the Oligo library.35 Transcript probes that did not correspond to known genes or matched with more than one gene in the GRCh37/hg19 human genome build (release 32) were discarded, and the study was finally restricted to 19 263 probes corresponding to 18 475 unique genes. Trans-eQTL analyses considering rs4938723 were performed with the R environment and the MatrixEQTL Package,36 including gender and batch as covariates. FDR correction was applied for multiple comparisons and statistical significance was set at P<0.05.Genome-wide gene expression data from microarray assays was used for the http://bioinfo.vanderbilt.edu/webgestalt/).37 Functional and cluster overrepresentation analyses were performed considering Biological Process category from Gene Ontology using the Database for Annotation, Visualization and Integrated Discovery resource (http://david.abcc.ncifcrf.gov/).38 Evaluation of overall enrichment in direct and indirect interactions between encoded proteins from our gene set was addressed with Disease Association Protein-Protein Link Evaluator software (http://www.broadinstitute.org/mpg/dapple/dapple.php).39 Enrichment analysis of canonical pathways and gene networks were performed using the Ingenuity Pathway Analysis software . Gene networks were considered of true relevance when the network score ) was over 3 (P=10−3) and the total number of focus molecules was above 20 out of 35. Benjamini–Hochberg's correction was used to adjust for multiple comparisons and significance threshold was set at P<0.05.MiRNA binding-site enrichment was explored using the WEB-based GEne SeT AnaLysis  Toolkit  and three had significant departure from HWE in the control sample . Of the 21 SNPs initially selected, three did not pass through the design pipeline, one was monomorphic (MAF<0.10) and another had significant departure from HWE  and P=0.017, OR=1.19 (1.03−1.39), respectively), rs699779 in the NOTCH2 gene ) and rs1175982 in the HMGA2 gene ; MET and HMGA2 genes , we further tested potential additive and epistatic effects among them. The combined effect of all ADHD risk variants was estimated to account for 3.2 and 4.5% of the variance of the overall (P=1.2e−06) and combined ADHD (P=3.4e−07), respectively, which resulted in sensibility and specificity values over 55%. No significant interactions between miR-34b/c and any of the targets genes were detected.As ADHD was associated with SNPs in the miR-34b/c cluster and in the 3′-UTRs of three validated target genes =4.19; CI=1.25–14.05), with 1.4-fold higher levels in patients as compared with controls. MiR-34b-3p also showed a trend towards increased expression in ADHD when compared with controls =2,70; CI=0.89–8.20) and no differences were observed for miR-34b-5p or miR-34c-5p.Expression levels of the mature forms of miR-34b and miR-34c were evaluated by qRT-PCR in PBMCs from 31 medication-naive ADHD individuals and 32 controls. Significant overexpression of miR-34c-3p was identified in ADHD subjects  and expression levels of miR-34b-3p and miR-34c-3p was observed  were identified considering the 681 differentially expressed transcripts from the trans-eQTL analyses (data not shown). Functional enrichment analyses revealed 36 categories nominally associated with our gene set, mainly related to lipid metabolism, and neuron dynamics and axonogenesis (P=0.032), ‘refractory schizophrenia' (P=0.032), major affective disorder (P=0.032), ‘susceptibility to anorexia nervosa type II' (P=0.032) or ‘susceptibility to bulimia nervosa type II' (P=0.032), as well as for neurologic diseases such as ‘Huntington disease grade II' . In addition, 12 canonical pathways were overrepresented in the gene set, among which we highlight ‘Serotonin and Melatonin Biosynthesis' (P= 9.33e−03) and ‘Serotonin Receptor Signaling' , ‘release of neurotransmitter' (P=8.43e−03), ‘influx of dopamine' (P=0.032), ‘release of GABA' (P=0.032), ‘synthesis of 5-hydroxytryptamine' (P=9.32e−03), ‘recycling of synaptic vesicles' (P=0.032) or ‘declarative memory' . We also identified 20 significant networks, three of which had more than 20 focus molecules. The best generated network was highly scored , had follicle stimulating hormone as a central node and displayed 312 specific overrepresented functions and diseases  injuries and psychiatric disorders including ADHD.59 Among the top three genes, we found Synaptotagmin 2 (SYT2), an essential component of the calcium-triggering machinery for neurotransmitter release that was found associated with ADHD.60 As expected, among the trans-eQTLs, we also found experimentally validated miR-34b/c target genes, such as NOTCH3, enrichment for miR-34b/c binding sites, including predicted targets in ADHD-associated genes such as HTR2C and VAMP2, and genes related to different psychiatric disorders including schizophrenia, major affective disorder or eating disorders.5 Taken together, these data reinforce a possible scheme for miR-34b/c having a role in adult ADHD etiopathogenesis through a multifactorial mechanism integrating miRNAs as well as genes previously associated with ADHD and/or other psychiatric conditions.The transcriptome-wide P=0.56 and P=0.33, respectively). Heterogeneity between populations, methodologies and statistical tests may explain discordant results across studies, making it difficult to reproduce the same findings and to establish direct comparisons between reports. Our study included only adult ADHD patients from Spain, whereas the Psychiatric Genomics Consortium sample consists mainly of children and adolescent Caucasians of European ancestry. These age differences as well as differential proportion of remitting and persisting ADHD subjects between samples may account for inconsistencies. Other explanations could also contribute to these discordant results, such as sample size, comorbid disorders as well as the proportions of clinical subtypes or genders. In addition, our findings regarding miR-34b and mirR-34c expression levels also require replication in a larger and independent cohort as the limited sample size considered may have led to imprecise estimates of the magnitude of the miRNA expression differences and could have prevented us from detecting significant differences for miR-34b, which only displayed a trend towards significance. (2) The modest sample size of the association study may have prevented us from detecting susceptibility loci with very small effects in ADHD. In addition, although the study was designed to assure a full genetic coverage in terms of LD, the minimum MAF threshold was set at 0.10, which may also have led to an underestimation of the contribution of less common sequence variants. (3) Our inability to detect, in the single-marker analysis, the association between ADHD and rs4938723 that tags eQTLs for miR-34b and miR-34c, may be attributed to the complex genetic background of ADHD as well as potential gene-by-environment interactions, which might be particularly relevant in ADHD. (4) As one of the tagSNPs in the miR-34b/c locus could not be tested owing to experimental constraints, it is still possible that additional sequence variants in the region may contribute to the dysregulation of miR-34b and/or miR-34c. (5) The selection of the FDR approach as the multiple-testing correction method rather than other more conservative strategies, such as Bonferroni, was based on the fact that the latter may be too stringent to identify subtle genetic factors involved in the etiology of a complex disease. In addition, the Bonferroni correction requires independence between all performed tests and, therefore, may be too restrictive in our study as some of the selected SNPs show some degree of LD (LD threshold: r2=0.85), and the different inheritance models tested for some of the SNPs are not fully independent. (6) Although SNPs in 3′-UTR regions that are targeted by miR-34b or miR-34c were selected solely on the basis of genetic coverage criteria, prediction of the functional significance of the ADHD risk variants in MET, NOTCH2 and HMGA2 genes supports their impact on miRNA target site recognition but none of them involved miR-34. (7) As we limited the gene expression analyses to miRNAs and SNPs showing significant evidence for association with ADHD, we cannot rule out the contribution to ADHD of other variants affecting the processing or target binding of miRNAs. (8) All the participants were recruited by a single clinical group in a restricted geographic area (Barcelona), and the diagnosis of ADHD was based on structured interviews with no variability in measurements across data. In addition, expression patterns were assessed in medication-naive subjects with no comorbid disorders, which may facilitate the identification of ADHD-related transcriptional signatures that might be neglected by a broader recruitment strategy. (9) The expression experiments were performed in PBMCs and, therefore, further evidence in brain tissues is required to disentangle the involvement of miR-34b and miR-34c in ADHD. It is worth pointing out, however, that the expression profiling of miRNAs shows correlation between brain and PBMCs, where both tissues cluster together based on the pattern of miRNA expression.61 A recent meta-analysis also showed that cis-eQTLs in the peripheral blood and brain display strong overlap and that there is solid evidence supporting PBMCs as a proxy to study transcriptional and epigenetic biosignature in different psychiatric disorders.62 Consistently, at the transcriptome level, whole blood also shares significant gene expression similarities with multiple CNS tissues, suggesting that the use of peripheral gene expression may be a useful surrogate for gene expression in the CNS.63 Furthermore, similarities between the mechanisms of transduction and receptor expression in cells of the CNS and blood lymphocytes have also been described.64 In this sense, disturbances in cellular functions and metabolism in the CNS, as well as alterations in the major neurotransmitter and hormonal systems, have been identified in neuropsychiatric disorders concomitant with altered functioning and metabolism of lymphocytes, which also support that the latter could reflect the metabolism in brain cells.65There are several methodological considerations, however, that should be discussed. (1) The results of the present association study should be considered preliminary until further replication in additional cohorts. In this regard, we explored results of the ADHD GWAS meta-analysis from the Psychiatric Genomics Consortium and observed no significant association between rs28690953 or rs4938723 and ADHD (In conclusion, to our knowledge, this is the first comprehensive attempt to evaluate whether miRNAs are involved in ADHD and we identified sequence variants in the pri-miR-34b/c associated with ADHD and with expression levels of the mature forms of miR-34b and miR-34c in ADHD subjects. This work increases the understanding of biological mechanisms by which epigenetic functions may contribute to ADHD and highlights the importance of miRNAs in the etiology of the disorder.


Neuropsychological research on adults with ADHD showed deficits in various aspects of attention. However, the majority of studies failed to explore the change of performance over time, so-called time-on-task effects. As a consequence, little is known about sustained attention performance of adults with ADHD. The aim of the present study was therefore to test the hypothesis of sustained attention deficits of adults with ADHD. Twenty-nine adults with ADHD and 30 healthy individuals were assessed on four 20-min tests of sustained attention, measuring alertness, selective attention, divided attention and flexibility. The deterioration of performance over time (time-on-task effects) was compared between patients with ADHD and healthy individuals to conclude on sustained attention performance. Compared to healthy individuals, patients with ADHD showed significant deficits of medium size in selective attention and divided attention. Furthermore, medium sustained attention deficits was observed in measures of alertness, selective attention and divided attention. This study supports the notion of sustained attention deficits of adults with ADHD. Symptoms of inattention, such as distractibility and concentration difficulty, are core features of attention deficit hyperactivity disorder (ADHD)  effects , anxiety disorders (n = 3), posttraumatic stress disorders (n = 2), eating disorders (n = 2), and obsessive–compulsive disorder (n = 1). Three patients with ADHD reported to have been treated with stimulant medication in the past; however, none of the participants was treated with stimulant medication at the time of the study. Moreover, two patients were treated with antidepressant medication for a prolonged period of time because of affective disorders. Further, none of the participants reported having a history of substance abuse disorder during the previous 6 months and none reported a history of neurological disorder including head injury. Characteristics of patients with ADHD are presented in Table Twenty-nine adults with ADHD participated in the study. Patients were self-referred or referred from local psychiatrists or neurologists to the Department of Psychiatry and Psychotherapy, SRH Group, Karlsbad-Langensteinbach, Germany. A diagnostic assessment for ADHD in adulthood as well as participation in the research project was offered to all participants. Diagnostic assessments were performed by experienced clinicians associated to the Department of Psychiatry and Psychotherapy and involved a clinical psychiatric interview according to DSM-IV criteria for ADHD as devised by Barkley and Murphey (Barkley and Murphy rder n = . Three pχ2(1) = 0.015; p = 0.902). However, healthy individuals had a significantly higher school education than patients with ADHD. As expected, healthy individuals scored lower on both questionnaires for ADHD symptoms.Furthermore, 30 healthy individuals were assessed. Healthy participants were recruited from the local community via public announcements, word-of-mouth and through contacts of the researchers involved. None of the healthy individuals reported having a history of neurological or psychiatric diseases and none were taking any medication known to affect the central nervous system at the day of the assessment. All healthy individuals completed the same self-rated questionnaires for current and retrospective ADHD symptoms prior to the assessment  were measured using the Multiple Choice Vocabulary Test Lehrl . This teSustained attention was assessed with the Vienna Test System (VTS) . In the present study, maintained intrinsic alertness was measured with an adaptation of the test for perception and attention functions called ‘alertness’     and color (black or grey). Participants were instructed to respond as quickly as possible either to shape or color of the stimulus presented. Responses were given by pressing one of two predefined buttons on a response panel . When a response to the shape of the stimulus was requested (independent from the color), a triangle required a response on the left button, whereas a circle required a response on the right button. When a response to the color of the stimulus was requested (independent from the shape), a grey stimulus required a response on the left button whereas a black stimulus required a response on the right button. Each stimulus was presented for a maximum of 5000 ms but disappeared as soon as a response was given . The time between a response in the present trial and the presentation of the stimulus in the subsequent trial [response–stimulus interval (RSI)] was 750 ms. Participants were instructed to alternately switch their focus between shape and color in every other trial. That means participants were requested to respond to the shape in two consecutive trials, subsequently respond to the color in two consecutive trials, switch back to the shape for another two trials, etc. . In case of incorrect responses of any type, participants were corrected by the test and prompted to give the correct response. This was achieved by a message automatically appearing on the screen referring to the incorrect response made in the respective trial and indicating the correct response the participant was supposed to give. The test continued as soon as the participant gave the prompted correct response. In total, 560 stimuli were presented throughout the task in pseudorandomized presentation order. Two variables were calculated as depended variables indicating switching costs, i.e., speed costs and accuracy costs. For the calculation of speed costs, the mean reaction time was calculated for the correct trials requiring responses to the other type of stimulus (shape or color) as the preceding trial . In addition, the mean reaction time was calculated for the correct trials requiring responses to the same type of stimulus (shape or color) as the preceding trial . The calculation of reaction times was based on the trials with correct responses, while trials with incorrect or omitted responses were not taken into account. The variable speed costs were calculated by subtracting the mean reaction time for switching trials from the mean reaction time for repetitive trials. Accordingly, for the calculation of accuracy costs, the percentage of the correct trials was calculated that required responses to the same type of stimulus (shape or color) as the preceding trial . In addition, the percentage of correct trials was calculated that required responses to the other type of stimulus (shape or color) as the preceding trial . The variable accuracy costs were calculated by subtracting the percentage of correct repetitive trials from the percentage of correct switching trials. Speed costs and accuracy costs were calculated for each time block.All participants were invited to take part in the study on a voluntary basis and received no reward for participation. Before the start of the assessment, all participants were informed about the aim of the study and it was pointed out that all data collected in the research project will be analyzed anonymously and will not affect clinical assessment and treatment. It was also emphasized that participants had the right to withdraw from the study at any time. At the beginning of the experiment, descriptive and anamnestic information  was obtained. Subsequently, four tests of sustained attention were performed, each taking about 20 min. Participants were free to use either their right or left hand to perform the tests on the VTS. A break followed the execution of each test. Short breaks (1–2 min) were allowed between the first test and the second test as well as between the third and the fourth test. However, a longer break (10–15 min) was taken between the second and the third test. The order of the four tests  was counterbalanced across participants. A complete counterbalanced design resulted in 24 different test orders which were each allocated to patients and healthy controls. Remaining participants were each randomly allocated to one of the 24 test orders. The total duration of the assessment was about 120 min.The study was conducted in compliance with the Helsinki Declaration. Ethical approval was obtained by the ethics committee of the medical faculty of the University of Heidelberg, Germany. All participants gave written informed consent prior to the assessment and were debriefed at the end of the assessment.U tests for independent samples. These analyses were performed in order to explore group differences in attention within short test periods which place only limited requirements on sustained attention. Mann–Whitney U tests were also applied to compare attention performance of participants in the last time block of each test in order to compare group differences in attention after a prolonged period of testing. Moreover, performance of sustained attention was analyzed (TOT effects). For this purpose, attention performance between the first time block and the last time block was compared separately for healthy individuals and individuals with ADHD by applying Wilcoxon signed-rank test for dependent samples. Furthermore, ipsative scores were calculated for each individual and each measure of attention by subtracting attention performance of the last time block from the attention performance of the first time block. Ipsative scores were then compared between healthy individuals and individuals with ADHD by applying Mann–Whitney U tests. An alpha level was set to 0.05 for all tests. Furthermore, effect sizes were calculated for all comparisons. Whereas the significance criterion represents the standard measure for analyzing whether a phenomenon exists, the effect size refers to the magnitude or the importance of effects (Cohen r < 0.1), small effects (0.1 ≤ r < 0.3), medium effects (0.3 ≤ r < 0.5) and large effects (r ≥ 0.5) were distinguished . Attention performance during the first time block of each test was compared between healthy individuals and patients with ADHD by applying Mann–Whitney ts Cohen . For paied Cohen . Statistr < 0.1), small effects (0.1 ≤ r < 0.3), medium effects (0.3 ≤ r < 0.5) and large effects (r ≥ 0.5) were distinguished.Furthermore, explorative analyses were carried out in order to examine the association between TOT effects of various measures of attention. This was done by correlating (Spearman rank correlation) ipsative scores with each other for a collapsed group of healthy individuals and individuals with ADHD. According to Cohen , negligiGroup comparisons between healthy individuals and individuals with ADHD revealed a significantly decreased performance of patients with ADHD in reaction times of the selective attention task as well as in the number of omissions of the divided attention task Table . The calreaction times in the alertness task, a significant group difference of small size was found for the last time block . Both healthy individuals  as well patients with ADHD  displayed a significant and medium decline of performance over time (TOT effects); however, the groups did not differ significantly in this decline and the difference was only small  . While patients with ADHD showed a significant and large deterioration of performance over time (TOT effects) , such an effect was not observed in healthy individuals as indicated by a non-significant and small effect size . Consequently, a comparison of TOT effects demonstrated a significantly greater decrease of performance over time in patients with ADHD than in healthy individuals . The effect was of medium size . However, neither healthy individuals  nor patients with ADHD  showed a significant change of performance over time (TOT effects), as noted by negligible to small effect sizes. Consequently, only a non-significant small difference was observed between groups with respect to their deterioration of performance over time (TOT effects)  . Whereas healthy individuals did not change significantly in reaction times between the first and the last time block , patients with ADHD showed a significant increase in reaction times over time  (TOT effects). Effects were of small and medium size, respectively. The increase in reaction times, however, did not differ considerably  between groups  . Neither healthy individuals  nor patients with ADHD  showed a significant change of performance over time, resulting in a non-significant difference of TOT effects between groups . Effects were of negligible to small size . Whereas a non-significant and small change of performance over time was observed for healthy individuals , patients with ADHD showed a significant and medium decline of performance over time . Significant and medium differences in TOT effects were found between healthy individuals and patients with ADHD  . Whereas healthy individuals showed a small and non-significant TOT effects in reaction times , patients with ADHD displayed a significant and medium decline in performance over time . The difference between groups in the decline of reaction times was significant and of medium size  . Neither healthy individuals  nor patients with ADHD  demonstrated significant TOT effects. Effects were of negligible to small effects. A non-significant difference of small size was also found when comparing TOT effects between groups  . Whereas healthy individuals exhibited non-significant and negligible TOT effects , patients with ADHD showed a significant and medium deterioration of performance over time . The TOT effects observed in patients with ADHD were significantly greater than TOT effects of healthy individuals . The effect was of small size . Neither healthy individuals  nor patients with ADHD  showed significant TOT effects. Effects were of small size. A non-significant negligible effect was also observed when comparing TOT effects between groups  . Again, neither healthy individuals  nor individuals with ADHD  showed significant TOT effects. Effects were of small size. A comparison of TOT effects between groups yielded a non-significant negligible effect  , measures of the selective attention task , between omissions occurring during the divided attention task and omissions in the selective attention task , between the standard deviation of reaction times in the alertness task and reaction times in the divided attention task , as well as between the accuracy costs in the flexibility task and omissions in the alertness task   Table .Table 2SThe present study examined the hypothesis of sustained attention deficits of adults with ADHD. Initial data analysis of attention performance in the first time block (5-min testing) demonstrated medium deficits of adults with ADHD in selective attention (increased reaction times) and divided attention (increased number of omissions). These results are in accordance with a large body of previous research which found deficits of adults with ADHD in various aspects of attention, including selective and divided attention .With respect to sustained attention, patients with ADHD showed a greater decline of performance over time as compared to healthy individuals (group differences of TOT effects). This was shown by mainly medium effects in several functions, i.e., alertness (variability of reaction times), selective attention (number of omissions) as well as divided attention (reaction times and number of omissions). Analysis of reaction times in the test of divided attention, however see Fig. , revealsFurthermore, a comprehensive analysis of attention performance requires a differentiation between different types of test variables. In this respect, the present assessment of attention considered the speed of responses (mean reaction time), the accuracy of responses (number of omissions) as well as the intra-individual variability of responses (standard deviation of reaction times). Reaction times of responses represent a general response readiness and arousal, whereas the number of omissions indicates a careless response style or distractibility. Furthermore, the intra-individual variability of responses  is more likely associated with the striking clinical characteristic of frequent lapses of attention seen in patients with ADHD, as it is clinically described by a moment-to-moment variability and inconsistency of performance  and subjective complaints of sustained attention (as obtained by self-reports), as this would provide valuable information about the predictive validity of different assessment strategies , comorbidity  and medication status (two patients were currently treated with antidepressant medication). Neuropsychological functions of adults with ADHD have been shown to be associated with various factors, including subtype . Sustained abilities in inhibition is worthwhile studying in adults with ADHD, e.g., by means of TOT effects in Go/No-go tests or Stop-Signal tests. Furthermore, a more fine-grained analysis of sustained attention deficits of adults with ADHD might be achieved by the analysis of intra-individual variability (IIV) of reaction times. Studies analyzing IIV of reaction times showed a greater proportion of extremely long reaction times in both children and adults with ADHD than in typically developing individuals  or objective measures of functioning in major life activities .


Regarding American Psychiatric Association, Attention-Deficit/Hyperactivity Disorder (ADHD) is one of the most common neurodevelopmental disorders in early childhood. The symptoms of ADHD include inattention, impulsivity, and hyperactivity. Such disorders lead to disruption in the educational, social and individual relationships areas and in other life aspects. Studies have highlighted the side effects of medications in the rehabilitation of individuals with ADHD and supported that many of children with ADHD despite pharmacological treatments experience cognitive dysfunctions ,2. In reThe goals of mHealth apps as one of the main subsets of e-health, are behavior change, education and diagnostic evaluations, collecting and reporting data, direct recording of health status, providing electronic decision supports, facilitating communication, planning and scheduling, alleviating the economic burden of diseases, enhancing the quality of health research and generally improving the effectiveness of healthcare services and health outcomes . mHealthThe interactive nature of mHealth apps along with their capability to be adapted and customized based on individuals’ needs, leads in improving care, promoting rehabilitation and enhancing the quality of life.


Objectives: While a group of children with ADHD may have normal behavioral responses to sensory stimuli, another group may be hyperreactive. The aim of this survey was studying association of tactile sensory responsivity with co-morbidity of oppositional defiant disorder (ODD) symptoms, subtypes of ADHD, and gender in children with ADHD.Methods: The subjects were 81 children with ADHD from a child psychiatry clinic. The diagnoses were made according to DSM-IV diagnostic criteria. Tactile dysfunction Checklist was used to assess the three types of tactile sensory dysfunction including Hypersensitivity, hyposensitivity, and poor tactile perception and discrimination (PTPD).Results: Their mean age was 8.4 (SD = 1.9) years. None of the gender, number of symptoms of ODD co-morbidity, and ADHD subtypes was as a predictor of scores of Hyposensitivity and PTPD subscales. Tactile defensiveness was not different between genders and different subtypes of ADHD.Conclusions: Number of ODD symptoms in children with ADHD is a predictor in association with hypersensitivity score of tactile sensory function. Girls are no more than the boys impaired in Hypersensitivity aspect. Different subtypes of ADHD are not distinct disorders regarding to tactile sensory function.


GIT1) has been proposed to be a risk gene for attention deficit hyperactivity disorder (ADHD), and it regulates the endocytosis of G protein‐coupled receptors like dopamine receptors. The purpose of this study was to investigate the interaction effects of GIT1 and dopamine receptor D4 (DRD4) gene variants on variables of the continuous performance test (CPT).The G protein‐coupled receptor kinase interacting protein 1 gene  who underwent CPT and genetic analyses. The genotypes were classified into two groups  and the genotype × genotype effects were examined using hierarchical multivariable linear regression analyses.This study recruited 255 GIT1 × DRD4 effects for commission errors on the CPT in the ADHD group (p = .006). In contrast, there were no significant GIT1 × DRD4 effects on any CPT variables in the HC.There were significant GIT1 and DRD4 gene variants on impulsivity in ADHD. Replication studies with larger sample sizes that include patients from various ethnic backgrounds are warranted to confirm these findings.The present findings demonstrated that there were significant interaction effects of the  DRD4 proteins are expressed in multiple brain regions that are thought to be involved in the etiology of ADHD  allele is associated with ADHD  between 6 and 17 years of age who were recruited between August 2010 and February 2015. For this study, participants from two studies that were conducted using the same protocol were combined into a single subject pool; detailed explanations of both study protocols and the combined protocol have been provided elsewhere  by board‐certified child and adolescent psychiatrists using the Kiddie Schedule for Affective Disorders and Schizophrenia‐Present and Lifetime version (K‐SADS‐PL) , 100 ng of gDNA, and 0.5 U Taq polymerase . The reaction mixture procedure consisted of incubation at 95°C for 15 min followed by 40 cycles of 95°C for 60 s, 55°C for 30 s, and 72°C for 30 s with a final extension at 72°C for 5 min. Following the PCR procedure, unincorporated dNTPs and primers were removed using ExoStar 1  with incubation at 37°C for 60 min followed by incubation at 85°C for 15 min for enzyme inactivation. The PCR products were directly sequenced using Big Dye Termination cycle sequencing  and the SNPs were manually determined via visual inspection.Genomic DNA was extracted from blood samples that had been frozen and stored using a G‐DEX II Genomic DNA Extraction Kit . SNPs were detected using Sanger Sequencing. For each SNP, a polymerase chain reaction (PCR) amplification was performed in 20 μl reactions with 1× PCR buffer, 200 μmol/L of deoxynucleotide triphosphates (dNTPs), 0.5 μmol/L forward and reverse primers , deoxythymidine triphosphate (dTTP), and deoxycytidine triphosphate (dCTP); 100 μmol/L of deoxyguanosine triphosphate (dGTP), 100 μmol/L of 7‐deaza‐dGTP , 0.5 μmol/L forward and reverse primers (5′‐ACC ACC ACC GGC AGG ACC CTC ATG GCC TTG CGC TC and 5′‐CTT CCT ACC CTG CCC GCT CAT GCT GCTGCT CTA CTG G‐3′), 1× Band Doctor , 100 ng of gDNA, and 2 U Pfu Taq polymerase (Solgent). The reaction mixture procedure consisted of incubation at 98°C for 5 min followed by 35 cycles at 98°C for 45 s, 57°C for 45 s, and 72°C for 60 s with a final extension at 72°C for 10 min. After amplification, the PCR products were resolved in 2% agarose gel.To analyze the VNTR of 2.3T‐scores adjusted for age relative to a normal population of 847 children between 5 and 15 years of age  group and in the ADHD and HC groups independently. Age and gender were included in Block 1, the GIT1 genotype and DRD4 genotype groups, we further conducted multivariable linear regression analyses with each CPT variables as independent variables, and the main effects, two‐way interactions and three‐way interactions as dependent variables ; a two‐tailed p‐value < .008 (0.05/[2 gene variants × 3 CPT variables]) was considered to indicate statistical significance.All statistical analyses were performed using SPSS ver. 22.0 software  . Of the CPT variables, there was a significant diagnosis effect for mean omission errors, commission errors, and response time variability . There were no significant differences in demographic and clinical characteristics according to the genotype group of the GIT1 or DRD4 variant in the ADHD and HC groups.The demographic and clinical characteristics of the ADHD and HC groups are presented in Table p > .05); no differences were observed in the allelic or genotypic frequencies of the GIT1 and DRD4 variants between ADHD and HC groups.The genotypic and allelic distributions of the two polymorphisms are shown in Table GIT1 and DRD4 variants on CPT are presented in Table DRD4 on commission errors (p = .003). In the ADHD group, there were significant effects of DRD4 and GIT1 × DRD4 on commission errors  and greater response time variability (p = .003) in the ADHD + HC group, as well as more omission errors and commission errors and greater response time variability  in the ADHD group , the results of studies comparing the effects of DRD4 4R/4R and other genotypes may differ according to the frequencies of each allele included in the other genotype groups .In the ADHD group, the GIT1 study by Won et al.  or interactions with other catecholamine‐related genes.After the first n et al. , subsequGIT1 variant on CPT variables in patients with ADHD. Won et al.  task, digit span task, flanker task, Sustained Attention to Response Task (SART), delay discounting task, and trail‐making task to investigate the behavioral phenotype of GIT1 in humans have all produced negative results ; this may have negatively affected the statistical power of the results. There were significant differences in age and gender between the ADHD and HC groups but we included these as covariates to compensate for this limitation. Additionally, all subtypes of ADHD were included in this study because the sample size was too small to perform independent analyses according to subtype. ADHD patients may exhibit differential CPT profiles according to subtype; further research using a more behaviorally homogeneous population is warranted. Furthermore, this study recruited only Korean participants, an ethnically homogenous group, which limits the generalizability of the results to other ethnic groups. Replication studies using participants with various genetic backgrounds are required. The GIT effect on ADHD seems to occur only in Koreans so far. Further studies exploring if this effect is due to something in the Korean genetic background or due to the low number of individuals with the minor allele are warranted. The genotypes of the DRD4 variant were divided into two groups without consideration for the functionality of these alleles. The DRD4 others group was a heterogeneous group; classifying the 2R–7R alleles into a single group may have obscured the effects of each allele. Finally, only one variant for each of the GIT1 and DRD4 genes was analyzed in this study.This study has several limitations that should be noted. First, the sample size was relatively small for genetic analyses, particularly for the 5GIT1 and DRD4 gene variants. Further studies that utilize larger sample sizes and multiple gene variants are needed to clarify and confirm the present findings.The present results suggest that impulsivity in ADHD patients was influenced by the interaction effects of the  Click here for additional data file.


Candida milleri, Lactobacillus brevis and Lactobacillus plantarum using non-targeted metabolic profiling utilizing LC–QTOF–MS. The aim was to determine the fermentation-induced changes in metabolites potentially contributing to the health-promoting properties of whole-grain wheat and rye. Overall, we identified 118 compounds with significantly increased levels in sourdough, including branched-chain amino acids (BCAAs) and their metabolites, small peptides with high proportion of BCAAs, microbial metabolites of phenolic acids and several other potentially bioactive compounds. We also identified 69 compounds with significantly decreased levels, including phenolic acid precursors, nucleosides, and nucleobases. Intensive sourdough fermentation had a higher impact on the metabolite profile of whole-grain rye compared to milder whole-grain wheat sourdough fermentation. We hypothesize that the increased amount of BCAAs and potentially bioactive small peptides may contribute to the insulin response of rye bread, and in more general, the overall protective effect against T2DM and CVD.Sourdough fermentation by lactic acid bacteria is commonly used in bread baking, affecting several attributes of the final product. We analyzed whole-grain wheat and rye breads and doughs prepared with baker’s yeast or a sourdough starter including  This has been attributed to cereal dietary fiber and the array of phytochemicals within the fiber matrix5, both of which interact with gastrointestinal microbiota and undergo transformations, possibly mediating physiological changes6. However, the metabolic pathways leading to these effects are still mostly unknown. Among the phytochemical classes, phenolic acids and alkylresorcinols are abundant in the bran section of whole grains8. These compounds have shown antioxidative, antimicrobial and anticancer effects in vitro.Increasing evidence is supporting the protective effect of whole-grain cereal consumption against several noncommunicable diseases, such as type 2 diabetes mellitus, cardiovascular disease and colorectal cancer, as well as overall mortality9. The intake of bread baked from whole-grain rye  has been shown to cause a lowered postprandial insulin response, known as the rye factor11, via unknown mechanisms. Common wheat (Triticum aestivum L.) is one of the primary sources of dietary fiber in the United States and several other industrialized countries, whereas rye provides an important dietary fiber source in parts of Northern and Eastern Europe. In the baking of nearly all rye breads and several artisanal wheat breads, such as the San Francisco bread, sourdough fermentation by lactic acid bacteria (LAB) and yeasts is used to improve the texture, sensory properties and shelf life of the bread product12. Whereas the emergence of industrial baking caused yeast fermentation to become the dominant practice in bread baking, sourdough has gained interest during the last years, not only from the technical and gastronomical perspective but also because of the increased nutritional value and potential health benefits offered by the ancient biotechnological process13. Sourdough fermentation is known to cause transformations of lipids and macromolecules14 and several phytochemicals, such as phenolic acids, folates and sugar-conjugated bioactive compounds15. However, the overall metabolism of small molecules during sourdough fermentation has not been studied with a non-targeted method or any other comprehensive chemical analysis.Bread is one of the most important staple foods consumed worldwide and thus serves as a major source of whole-grain cerealsCandida milleri, Lactobacillus brevis and Lactobacillus plantarum, were selected for both types of doughs to represent a typical microbial distribution in sourdough. The fermentation conditions for wheat and rye were chosen to represent a typical sourdough process for each cereal. Liquid chromatography–mass spectrometry (LC–MS) -based non-targeted metabolomics was used to detect and identify the discriminatory compounds for each comparison. The proportion of identified compounds was maximized by combining the use of a standard library, database searches, in silico generated mass spectra, and MS/MS fragment motifs associated with certain molecular moieties.The aim of this study was to elucidate the changes in the metabolite profile caused by sourdough fermentation as compared to yeast fermentation and to indirectly compare the differences between sourdough-fermented wholegrain wheat and rye breads. Emphasis was given to bioactive compounds, which potentially contribute to the health benefits of whole-grain products. Similar starters, containing Principal component analysis (PCA) of the raw data, including the two first components and all the detected molecular features, showed a clear separation of the metabolic profiles of the flours from the rest of the sample groups (doughs and breads) along the first component of the PCA Fig. . It alsop < 0.01, fold change (FC) ≥ 2). Correspondingly, 212 features fulfilled the same criteria in the sourdough fermented wheat bread. Out of these, 79 features had significantly increased levels in both cereal type sourdough breads , six sugars or sugar derivatives, two phosphatidylcholines [PC(18:2/18:2) and PC(18:2/18:3)], and two fatty acids were among the increased compounds. Other identified potentially bioactive compounds with significantly increased levels were 2-benzoxazolinone (benzoxazinoid), 2-hydroxyvaleric acid, isorhamnetin (flavonoid), N-acetylspermidine, and phenylethanolamine. The identified and unidentified compounds were plotted based on their retention time, fold change and average signal intensity to highlight the most important differential compounds found in the analysis  , 29 small peptides and six amino acid metabolites (the same as in rye sourdough) had significantly increased levels ( 2) Fig. . In cont 2) Fig. . No phosp-coumaric and sinapic acid), five nucleosides involved in nucleic acid synthesis , nucleoside derivatives and nucleobases , amino acid–derived betaines , myo-inositol, two phosphatidylcholines [LysoPC(15:0) and PC(16:0/18:3)], mono- and polysaccharides, and oxidized fatty acids  increased or decreased in their levels based on the chosen thresholds in sourdough fermented breads. Nine alkylresorcinols  were significantly (p < 0.05) increased in the sourdough rye bread  in rye sourdough and significant minor increase in levels  in wheat sourdough.We identified several compounds belonging to the major phytochemical classes known to exist in whole-grain cereals that were not significantly . Clear differences were seen between wheat and rye, between whole-grain and processed samples, and between rye sourdoughs and straight doughs, highlighting the significance of food processing in the final biochemical composition of the products. The effect of sourdough alone was considerable, especially in rye, where more than 700 molecular features had increased levels with the chosen inclusion criteria and over 350 decreased levels with high significance. These changes are likely contributing to the differences in the sensory properties and the potential metabolic effects of sourdough bread. Many of the observed changes in the metabolite levels were specific to rye sourdough; overall, sourdough fermentation of whole-grain wheat produced considerably less significantly changed metabolites and a less separated metabolic profile from yeast-fermented wheat dough and bread in the PCA. The main reason behind this is likely the different conditions chosen for the two sourdoughs, selected to represent widely consumed breads, with the higher temperature and longer fermentation time of rye sourdough allowing a more extensive metabolism. In contrast to rye, only mild acidity is accepted by consumers in wheat sourdough bread, thus requiring differences in the fermentation process17. BCAAs are known to activate the mTORC1 signaling pathway in skeletal muscle in a similar manner to exercise, and this in turn leads to uncoupling of the insulin signaling18. This may contribute to the decreased insulin response seen in vivo. However, the role of BCAAs in insulin metabolism is more complex than this, as their increased circulating levels are associated with obesity-related insulin resistance, possibly because of an overload of BCAA catabolism19. In a study by Moazzami et al., higher fasting concentrations of leucine and isoleucine were correlating with a higher insulin response after the intake of all types of study breads20. In contrast, phenylalanine and methionine, which were also among the significantly increased amino acids in sourdough rye in the current study, were the main metabolites associated with a lowered insulin response after 60 minutes of sourdough rye bread intake21. Few clinical studies on cereal products and their glycemic responses have included a comparison of fermented and unfermented bread. In a randomized cross-over trial, Johansson et al.22 found a lower insulin response for unfermented whole-grain rye crisp bread compared with yeast fermented bread and hypothesized that the effect could be partly explained by the BCAA levels increased by the yeast fermentation (up to 23%). The discrepancy in the effects of BCAAs in insulin response may be due to the difference in the role of moderate intake of dietary BCAAs and pathogenic metabolic pathways related to insulin resistance, manifesting as increased circulating BCAA levels. Leucic acid, a leucine metabolite, is known for its anti-catabolic effects on muscle tissue23; it remains unclear whether this compound or the other BCAA-related metabolites identified in the current study  could exert their own impact on the insulin response. A clinical study on the effect of sourdough fermented rye bread in insulin response, having yeast fermented rye as comparison, could further elucidate whether sourdough fermentation is one of the main contributors to the observed rye factor.Sourdough fermentation considerably increased the levels of branched-chain amino acids leucine and isoleucine, BCAA metabolites as well as several small peptides containing BCAAs. This effect was more prominent in rye than wheat sourdough most likely due to intensive proteolysis in the acidic rye sourdough utilized in this study. As has been shown previously, the consumption of rye bread decreases the postprandial insulin response without a decrease in glucose response due to an unknown mechanism independent from the dietary fiber content of the bread25. These characteristics seem to be governed by the presence of certain amino acids in the peptide sequence; e.g. leucine may increase both the radical scavenging activity and ACE inhibition of the peptide26. Specifically, 28 peptides that had increased levels in sourdough , likely linoleic acid and alpha- or gammalinolenic acid. The dietary intake of ω − 3 phosphatidylcholines has been shown to improve fatty acid and glucose metabolism in rats37, but on the other hand, the colonic microbial metabolism of the choline group in phosphatidylcholines may result in adverse cardiovascular effects38. We could not determine in the current study whether the identified phosphatidylcholine species contain ω − 3 or ω − 6 PUFAs; in addition, more research is needed to determine the association of polyunsaturated phosphatidylcholine intake with the risk of CVD and diabetes.Interestingly, nucleobases, nucleosides and their derivatives had lower levels in sourdough wheat and rye compared to yeast fermented samples. Since their relative levels were found to be low in flours as well, they are likely metabolites of yeast fermentation. Little is known about the biological significance of these compounds regarding dietary intake; among the few reported effects is that they may act as immunomodulators in infants receiving nucleosides in breast milkWhile non-targeted metabolomics is ideal for a wide-scale investigation of the metabolic profile of any biological sample, it is limited by the incomplete availability of spectral references for the reliable annotation of all statistically significant compounds, and this holds true especially for metabolite-rich sample matrices, such as the ones studied in this work. The high relative amount of unknowns may cause bias towards more well-known types of compounds, for which more reference data is available. Therefore, future targeted studies are warranted to further investigate the changes occurring in these metabolites as well as providing confirmation to the identifications. There is some uncertainty in annotating small peptides due to limitations in separating the signals originating from leucine and isoleucine and deducing the correct order of the amino acid residues in the peptide. Peptides larger than three amino acid residues have limited reference spectra available. The different fermentation conditions in wheat and rye sourdoughs inevitably affect the metabolic profiles observed in the current study; however, the differences in the profiles are relevant regarding the potential health implications, since the studied breads represent similar types of breads as normally consumed. The selection of LAB strains may affect the metabolic profiles or sourdough fermentation depending on the array of enzymes present in each strain. Here, we aimed to use one representative sourdough starter; studying the effect of the selection of strains on the same metabolic profiles is warranted for the future.The non-targeted metabolomics approach provided wide insight into the metabolic profile of sourdough fermentation. We hypothesize that sourdough fermentation contributes to the beneficial health effects of whole grains by increasing the amount of several bioactive compounds, such as BCAAs, small peptides, and microbial phenolic acid metabolites, in the baked products. The effect is more profound in rye sourdough compared to wheat, likely attributable to the more extensive metabolism occurring in a typical rye sourdough and suggesting a potential contributor to the rye factor. The current study indicates the potential for future research to reveal the molecular basis of physiological signals caused by wholegrain bread intake, and in more general, of the observed protective effect of whole grains against non-communicable diseases.Four different types of flours were used in the baking: endosperm wheat flour (manufacturer’s product code: V500P), wholegrain wheat flour V1700), endosperm rye flour (R700) and wholegrain rye flour, coarse (R1800KA). All flours were obtained from Fazer Mill & Mixes, Finland. The nutritional composition of the flours is presented in Table 700, endoCandida milleri) and lactic acid bacteria (Lactobacillus brevis and Lactobacillus plantarum) are commonly found in both rye and wheat sourdoughs39. For the rye sourdough, a high proportion of starter and intensive fermentation conditions were selected to produce a high level of acidity, which is desirable for obtaining a good crumb structure in rye bread40. For the wheat sourdough, a lower level of starter and milder fermentation conditions were selected to produce a moderate acidity level, which has been related to improved wheat sourdough bread flavour and volume16. The microbial strains were obtained from the culture collection of VTT. The starters were prepared by refreshing the microbial strains twice in succession in general edible medium41 for 24 h at 30 °C. The cells were collected from the cultures by centrifugation and suspended in sterile water to obtain desired cell concentrations. Sourdoughs were prepared by mixing 1 kg of flour with 1.5 l of water and starter suspension by hand and incubating the mixture in a covered container. After the fermentation, a sample was taken from the sourdough for the analysis of microbial growth and a sample was frozen for a subsequent analysis of acidity. The viable counts of lactic acid bacteria were determined using plate count technique on MRS agar . Yeasts were enumerated on YM agar . Acidity of the sourdoughs  was analysed according to a standard method42. The fresh sourdoughs were immediately used for bread baking.The microbial starters, their levels and fermentation conditions used in the preparation of sourdoughs are presented in Table Six different breads were prepared by straight dough or by sourdough baking process at VTT Technical Research Centre of Finland Ltd., Espoo Table . For eacn = 6), dough (n = 6) and flour (n = 4). The frozen samples (−80 °C) were cryoground into fine powder using a tissue homogenizer . The grinding frequency was set at 20 s−1 and the duration at 40 s. The ground sample was inspected visually, and the procedure was repeated in the case of any unground pieces. Approximately 100 mg of each sample was weighed into Eppendorf tubes; at this stage, four technical replicates were taken from the flour samples and three replicates from the dough and bread samples into separate tubes. The metabolite extraction solvent was produced by mixing HPLC gradient grade methanol, Milli-Q water and 80% formic acid using a MeOH:H2O:HCOOH ratio of 80:19.9:0.1 v/v/v. The solution had a pH of 3.6. The extraction solvent was added into the tubes in a ratio of 300 µl to 100 mg of sample. The tubes were vortexed, sonicated for 15 min, vortexed again, and centrifuged for 10 min at 13,000 rpm. The supernatant was collected and filtered (Acrodisc CR 13 mm syringe filter with 0.2 µm PTFE membrane). The filtrate was then transferred to HPLC sample vials.Samples were obtained from each type of bread  for the HILIC separation. After each chromatographic separation, the ionization was carried out using jet stream electrospray ionization (ESI) in the positive and negative mode, yielding four data files per sample. The collision energies for the MS/MS analysis were chosen as 10, 20 and 40 V, for compatibility with the spectral databases.The liquid chromatography–mass spectrometry was performed on a 1290 Infinity Binary UPLC coupled with a 6540 UHD Accurate-Mass Q-TOF  as described previously by Hanhineva 34. The raw values of the peak areas were used in determining the relative levels of the compounds in different samples. The p values were calculated using one-way ANOVA and the fold changes as the ratio of the average raw peak areas of sourdough fermented samples to yeast fermented samples, using a negative inverse value in case of a ratio below 1 (level decrease). After the statistical analysis, filtering was performed in Microsoft Excel to extract the list of statistically significant peaks for each comparison. Fold change was set at ≥2 (overexpression in sourdough samples), p value at <0.01, RSD at ≤30% (variation within sample group), and average signal intensity (in the sample group being compared) at ≥200 000. Principal component analysis (PCA) was performed in SIMCA version 14 (Umetrics AB) using Pareto scaling. Hierarchical Clustering method in Multiple Array Viewer version 4.9.0 (TM4 Software Suite) was used to create the heat map of the significant identified compounds.The molecular features were extracted from the data by using MassHunter Qualitative Analysis version 7.0 (Agilent Technologies) and the biostatistical analysis was performed in Mass Profiler Professional version 2.2 (Agilent Technologies), as described previously44. For some of the compounds with no spectral database matches, tentative IDs were given based on in silico generated MS/MS spectra acquired from molecular database entries using MS-FINDER software45. For the identification of small peptides, fragment motifs specific for amino acid residues were determined from the identifications based on MS/MS data and used in the further identification process. The fragment motifs observed in at least two peptides and used in the identifications are presented in Table The accurate masses, retention times, and MS/MS fragmentation patterns of the detected statistically significant compounds were used in comparison with an in-house standard library, previously published literature, and freely available MS spectral databases. The tentative annotation of compounds based on spectral database searches was performed in MS-DIALThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.Supplementary information


Viperin mRNA is a substrate for endoribonucleolytic cleavage by RNase mitochondrial RNA processing (MRP) and mutations in the RNase MRP small nucleolar RNA (snoRNA) subunit of the RNase MRP complex cause cartilage-hair hypoplasia (CHH), a human developmental condition characterized by metaphyseal chondrodysplasia and severe dwarfism. It is unknown how CHH-pathogenic mutations in RNase MRP snoRNA interfere with skeletal development, and aberrant processing of RNase MRP substrate RNAs is thought to be involved. We hypothesized that viperin plays a role in chondrogenic differentiation. Using immunohistochemistry, real-time quantitative PCR, immunoblotting, ELISA, siRNA-mediated gene silencing, plasmid-mediated gene overexpression, label-free MS proteomics, and promoter reporter bioluminescence assays, we discovered here that viperin is expressed in differentiating chondrocytic cells and regulates their protein secretion and the outcome of chondrogenic differentiation by influencing transforming growth factor β (TGF-β)/SMAD family 2/3 (SMAD2/3) activity via C-X-C motif chemokine ligand 10 (CXCL10). Of note, we observed disturbances in this viperin–CXCL10–TGF-β/SMAD2/3 axis in CHH chondrocytic cells. Our results indicate that the antiviral protein viperin controls chondrogenic differentiation by influencing secretion of soluble proteins and identify a molecular route that may explain impaired chondrogenic differentiation of cells from individuals with CHH.Viperin ) is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses.  Viperin, also known as RSAD2, is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses. Viperin localizes to the cytosolic face of the endoplasmic reticulum (ER),7–A). Cells in the upper zone of the growth plate displayed dispersed viperin positivity; viperin expression was either barely detectable in individual cells, was weakly expressed, or was relatively high expressed in individual cells (B).To investigate whether viperin is expressed during early chondrogenic differentiation, tissue sections were prepared from embryonic day 15.5 mouse embryos. Expression of viperin was detected in growth plate chondrocytes throughout the developing growth plate, with the highest expression levels in proliferating and terminally differentiating hypertrophic chondrocytes A. Cells al cells B.A and B) and increasing levels of Runx2 and Col10a1 (C and D). During the first 3 days of ATDC5 chondrogenic differentiation, viperin mRNA expression was hardly detectable (E). From day 4 through day 14 (the latest time point tested), viperin mRNA expression was induced as compared with undifferentiated ATDC5 cells (t = 0) and with a prominent peak expression at days 5 and 6 in differentiation. Similar expression dynamics were observed for viperin protein expression in these chondrogenic cultures (F). During chondrogenic differentiation of hBMSCs (COL2A1 and COL10A1 mRNA expression in G and H), peak induction of viperin mRNA expression was detected at day 7 of differentiation (I), which is in agreement with viperin expression during ATDC5 chondrogenic differentiation. Collectively these data show that developing chondrocytes express viperin in vivo and in vitro, with undetectable to low expression in the early differentiation phase and a profoundly increased expression later in differentiation.We further interrogated potential viperin expression during chondrogenic differentiation of the ATDC5 cell line , 13 and  Col10a1 , C and Dtectable E. From dcultures F. Duringntiation I, which A), and ectopic expression of FLAG-viperin was confirmed at the same time points . Bioluminescent analyses showed that the amount of secreted Gaussia-luciferase was reduced following viperin knockdown and increased with viperin overexpression . This was the case at both days 5 and 7 of differentiation. These data indicate that overall cellular protein secretion responds to changing viperin levels during chondrogenic differentiation.Previous work by Hinson and Cresswell  showed tFig. 3A). Differentiation of ATDC5 cells in CM obtained from donor cultures in which viperin levels were reduced led to an overall increase in chondrogenic capacity, evidenced by increased expression of Sox9, Col2a1, Runx2, Col10a1, and Alpl . Reciprocally, CM obtained from differentiating ATDC5 donor cultures in which viperin levels were overexpressed displayed an inhibitory effect on the chondrogenic differentiation capacity, evidenced by reduced Sox9, Col2a1, Runx2, Col10a1, and Alpl expression . The stimulatory and inhibitory actions of these CM on chondrogenic differentiation were further confirmed by determination of the glycosaminoglycan (GAG) content of these cultures. Differentiation of ATDC5 in CM obtained from donor cultures with reduced viperin levels caused an increased GAG content, whereas differentiation of ATDC5 in CM obtained from donor cultures in which viperin was overexpressed led to a reduced GAG content . These results together indicate that alterations in viperin levels during chondrogenic differentiation change the cell's secretome with important functional consequences for differentiation.Considering the central role secreted signaling molecules play during chondrogenic differentiating , 16, we Fig. 4A), we identified six protein species that were differentially expressed in the CM (C). When comparing the conditioned culture supernatants of differentiating ATDC5 cultures in which an empty FLAG vector was transfected or in which viperin levels were overexpressed using the FLAG-viperin plasmid (B), we identified eight differentially expressed proteins in the CM (C). Interestingly, CXCL10  was the only protein species that was detected in both differential secretome proteomes. CXCL10 was decreased in CM obtained from cultures in which viperin levels were reduced and was increased in conditioned medium obtained from cultures in which viperin levels were overexpressed. Differential CXCL10 protein levels in these culture supernatants were independently confirmed by ELISA . Because expression of both viperin and CXCL10 are interferon-dependent , we next verified CXCL10 secretion dynamics during chondrogenic differentiation of ATDC5. Using an ELISA, supernatant samples from cultures in Fig. 5E) during ATDC5 chondrogenic differentiation mirrored viperin expression dynamics (E and F). The data together demonstrate that alterations in viperin levels during chondrogenic differentiation lead to specific differences in the protein composition of the chondrocyte's secretome, with CXCL10 a shared factor between the differential viperin-dependent secretomes.Because media obtained from donor cultures with reduced or overexpressed viperin levels enhance or inhibit chondrogenic differentiation (respectively), we postulated that this was caused by a differential protein composition of their secretomes. To determine the differential proteome of the conditioned culture supernatants obtained from differentiating ATDC5 cultures with reduced or overexpressed viperin levels, we undertook a label-free MS proteomics approach using LC-MS/MS. When comparing the conditioned culture supernatants of differentiating ATDC5 cultures in which a scrambled siRNA was transfected or in which viperin levels were reduced using the viperin siRNA A, we iden the CM C. When c plasmid B, we iden the CM C. Intereependent  . To determine whether CXCL10 inhibits chondrogenic differentiation, differentiating ATDC5 cultures were exposed to exogenously added mCXCL10 from day 5 of differentiation until day 7 of differentiation . Analysis showed that exogenously added mCXCL10 attenuated chondrogenic differentiation, evidenced by decreased expression of Sox9, Col2a1, Runx2, Col10a1, and Alpl (A). To verify the inhibitory action of CXCL10 in an independent model for chondrogenic differentiation, primary hBMSCs were differentiated into the chondrogenic lineage and exposed to increasing concentrations hCXCL10 from day 7  in chondrogenic differentiation onward. CXCL10 inhibited chondrogenic differentiation of hBMSCs, evidenced by a decline of COL2A1 and COL10A1 mRNA expression (B). These data indicate that viperin-dependent alterations in secreted CXCL10 levels influence chondrogenic differentiation capacity.Conditioned culture medium obtained from differentiating ATDC5 cultures in which viperin levels were overexpressed was found to inhibit chondrogenic differentiation. In addition to a limited number of other differentially expressed proteins, CXCL10 was the only shared protein species that was differentially present in the viperin-dependent secretomes and was increased in viperin overexpression culture supernatant. We therefore postulated that CXCL10 contributes to the inhibitory action on chondrogenic differentiation of the viperin overexpression culture supernatant C. To detand Alpl A. To verpression B. These A), and CMs were collected. Subsequently, proliferating ATDC5 cells were used as a bioassay for SMAD2/3-dependent TGF-β activity by transfection of a CAGA12-luciferase reporter plasmid (B). In contrast, CM obtained from differentiating ATDC5 cultures in which viperin levels were overexpressed displayed a TGF-β/SMAD2/3 activity–attenuating action (B). In concert with these findings, Pai1 and SMAD7 mRNA levels (TGF-β/SMAD2/3 target genes (B and C) from viperin knockdown or overexpression cultures, respectively (C and D). Subsequently, we determined whether differential CXCL10 levels directly alter TGF-β/SMAD2/3 activity during chondrogenic differentiation. ATDC5 cells were transfected with the CAGA12-luciferase reporter plasmid at day 5 in chondrogenic differentiation. Then starting at day 6, the cells were exposed to increasing concentrations of recombinant murine CXCL10 and sampled for bioluminescent analyses 24 h later. The data show that CXCL10 dose-dependently reduces TGF-β/SMAD2/3 reporter activity in these cultures (E). Immunoblotting for SMAD2 phosphorylation (pSMAD2C) further showed that CXCL10 inhibits SMAD2 phosphorylation during ATDC5 chondrogenic differentiation (F), which is in concert with the observed reduction of TGF-β/SMAD2/3 reporter activity. Together these results demonstrate that alterations in viperin expression levels and CXCL10 impact SMAD2/3-dependent TGF-β activity during chondrogenic differentiation. This is in concert with the observed impact of these conditions on chondrogenic differentiation capacity  weret genes , B and Cectively , C and Dcultures E. Immunontiation F, which A) indeed led to the induction of viperin expression , recapitulating the endoribonucleolytic relationship between RNase MRP and its viperin mRNA substrate. A transdifferentiation protocol was used that drives dermal fibroblasts toward a chondrocyte-like phenotype . In concert with a CHH-associated pathological defective RNase MRP endoribonucleolytic activity, we found that viperin expression in chondrogenic transdifferentiated CHH fibroblasts was increased . This was accompanied with increased levels of secreted CXCL10 protein in these culture supernatants  and decreased expression of the TGF-β target genes PAI1 and SMAD7 . These data suggest that defective chondrogenic transdifferentiation of CHH fibroblasts is caused by changes in TGF-β activity, induced by alterations in the viperin–CXCL10–TGF-β/SMAD2/3 axis.Mutations in the RMRP gene, encoding the essential RMRP snoRNA present in the RNase MRP macromolecular protein–RNA complex , are theFig. 8henotype . As deschenotype , RMRP exin vitro chondrogenic differentiation. Interestingly, a subpopulation of resting zone cells displayed high levels of viperin expression. Chondrogenic differentiation is synchronously initiated in cell culture, although this is not expected to be synchronous in the growth plate's resting zone. We speculate that high viperin–expressing cells in the resting zone are a representation of asynchronous initiation of chondrogenic differentiation and that these cells are in a similar differentiation phase as ATDC5 and hBMSC cells during their early differentiation, when peak expression of viperin was observed. At later stages in chondrogenic differentiation, viperin expression remained increased. This was also observed in growth plates, where proliferating and prehypertrophic chondrocytes are positive for viperin expression. Viperin expression is remarkably high in terminally differentiating growth plate chondrocytes. This was not observed in the late ATDC5 and hBMSC differentiation time points tested. ATDC5 and hBMSCs are excellent in vitro models for chondrogenic differentiation . Interferon-related signaling during chondrogenic differentiation is almost unexplored. Interferon-inducible proteins p202 . Interestingly, platelet-derived growth factor subunit A was also found differentially expressed in the viperin overexpression secretome and platelet-derived growth factor has been described to synergistically act with IFN-γ to induce CXCL10 expression in blood-derived macrophages . CXCL10 Because we utilized conditioned media as a means to study the secretomic consequences of alterations in viperin expression during chondrogenic differentiation, we cannot distinguish paracrine from autocrine secreted signals. However, it is currently not clear why chondrogenic differentiation is regulated via viperin. Control over the activity of differentiation signals in the early differentiation phase may be required to enable the cell to undertake chromatin remodeling and coordinated transcriptomic reprogramming, before it can adopt a fully differentiated phenotype. We expect that temporal para- and/or autocrine viperin-dependent CXCL10 secretion may aid in this by antagonizing TGF-β signaling and pacing cellular differentiation. Binding of CXCL10 with its CXCR3 receptor is known to activate PI3K in human airway epithelial cells , and recWith a number of varying RNase MRP substrate RNAs, it would not be likely that all aspects of CHH pathobiology are caused by defective processing of one specific substrate. Instead, defective processing of one or the other specific RNase MRP substrate RNA will have different implications for different tissue/cell types. CHH-related defective processing of viperin mRNA leads to aberrant viperin levels in chondrocytic cells as shown in this study. Viperin responses  and CXCLIn conclusion, our data link the antiviral protein viperin to chondrogenic development via a viperin–CXCL10–TGF-β/SMAD2/3 axis , and shom NaCl (Merck Millipore), 2.7 mm KCl (Merck Millipore), 9.0 mm Na2PO4.H2O (Merck Millipore), 1.8 mm KH2PO4 (Merck Millipore)). For antigen retrieval, the sections were incubated in hot citrate buffer (1.8 mm citric acid (Sigma–Aldrich) and 8.2 mm trisodium citrate ) for 30 min. Endogenous peroxidase activity was inactivated using peroxidase-blocking solution . Slides were blocked with 10% normal sheep serum in PBS-T . Primary antibody was incubated for 2 h at room temperature. Mouse monoclonal anti-Viperin  was used at a 1:200 dilution. IgG2a (Dako) was used as an isotype control. Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse secondary antibodies (Dako EnVision) for 30 min at room temperature. DAB chromogen substrate (Dako) was used for detection. Sections were counterstained with hematoxylin (Dako), dehydrated, and mounted with histomount . Microphotographs were acquired using a Zeiss Axioscope A1.5-μm-thick formalin-fixed paraffin-embedded tissue sections were prepared from embryonic day 15.5 NMRI mouse embryos. Use of the embryos was approved by the University of Freiburg (number X-14/10H), according to German law, and methods utilized to obtain the embryos were carried out in accordance with German law. Sections were deparaffinized in a xylene/ethanol series ending in PBS (136 m2 in proliferation medium (DMEM/F-12 (Invitrogen), 5% fetal calf serum (FCS) (Sigma–Aldrich), 1% antibiotic/antimycotic (Invitrogen), and 1% nonessential amino acids (NEAA) (Invitrogen)). The cells were tested negative for potential mycoplasma infection. To induce chondrogenic differentiation, the cells were plated at 6,400 cells/cm2. After 24 h, chondrogenic differentiation was initiated by changing the medium to differentiation medium (proliferation medium supplemented with 10 μg/ml insulin (Sigma–Aldrich), 10 μg/ml transferrin (Roche), and 30 nm sodium selenite (Sigma–Aldrich)). Differentiation medium was refreshed every 2 days for the first 10 days and each day after day 10. In addition, ATDC5 cells were differentiated for 5 days and then exposed to 0.5 or 5 ng/ml recombinant murine CXCL10  until day 7 in differentiation. When ATDC5 cells were differentiated using CM, the CM was refreshed every other day.ATDC5 cells  , 50 μg/ml l-ascorbic acid-2-phosphate (Sigma), and 1 ng/ml TGF-β3 (R&D)) . Proliferation medium consisted of DMEM high-glucose (Invitrogen), 10% FCS (ES grade), 1% antibiotic/antimycotic, and 1% NEAA. Passage 3 cells were plated at 30,000 cells/cm3 (R&D)) . Medium RNA was extracted from cells with TRIzol reagent (Invitrogen) and isolated by collecting the aqueous phase after phase separation. RNA was precipitated with isopropanol  and centrifuged for 30 min at 15,000 rpm, 4 °C. RNA pellets were washed with 80% ethanol, and potential DNA contamination was removed by DNase I (Roche) treatment . After subsequent ethanol precipitation, RNA was dissolved in 15 μl of DNase/RNase-free water . RNA quantity and purity were determined spectrophotometrically . Reverse transcription of RNA and RT-qPCR were performed as described previously . Housekem NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris pH 8.0, 5.0 mm EDTA, pH 8.0, 0.5 mm DTT, 1 mm phenylmethylsulfonyl fluoride, and phosphatase inhibitor ). Samples were homogenized by sonication (Soniprep 150 MSE) using the following protocol: 14 cycles of 1-s sonication followed by a 1-s interval, amplitude 10. Cell debris was removed by centrifugation. Total protein concentration was determined with a BCA assay (Sigma–Aldrich). Samples were separated by gel electrophoresis and transferred to nitrocellulose membranes by electroblotting. The following primary antibodies were used for immunodetection: mouse monoclonal anti-Viperin  and rabbit monoclonal anti-pSMAD2C . As controls, mouse monoclonal anti-Histone H3 , mouse monoclonal anti–α-Tubulin , and mouse monoclonal anti-GAPDH  were used. HRP-conjugated polyclonal rabbit anti-mouse (Dako) or HRP-conjugated swine anti-rabbit (Dako) was applied as a secondary antibody, and the bound antibodies were detected by ECL.The cells were washed three times with 0.9% NaCl. The cells were lysed in radioimmune precipitation assay buffer  with 100 nThe viperin coding sequence, flanked by NotI–XbaI restriction sites, was custom-made synthesized by GeneCust  and originally cloned in pUC57 vector and sequence verified. The insert was then subcloned into the p3xFLAG plasmid  using the NotI–XbaI restriction sites, yielding the viperin overexpression plasmid (FLAG-viperin). On day 4, 5, or 6 of ATDC5 chondrogenic differentiation, viperin expression was further induced in 6-well plates by transfection of 625 ng of FLAG-viperin plasmid or an empty plasmid (FLAG-empty) as a control, using polyethylenimine  transfection reagent. DNA and PEI were complexed for 10 min at room temperature in DMEM/F-12 (1.88 μl of PEI (1 μg/μl) per 625 ng construct per well) and added to the ATDC5 cells. To determine protein secretion 250 ng of CMV promoter-driven secreted Gaussia luciferase plasmid (pGluc-CMV) was co-m HCl or 0.1% (m/v) Crystal Violet (Sigma–Aldrich). The cells were washed six times with distilled water and allowed to air dry. Alcian Blue was extracted by incubation with 6 m guanidine-HCl (Sigma–Aldrich) for 2 h on a plate shaker . Crystal Violet was extracted by incubation with 10% acetic acid (VWR) for 15 min on a plate shaker (IKA). Extracted Alcian Blue and Crystal Violet were quantified spectrophotometrically at 645 and 590 nm, respectively, using a plate reader . Crystal Violet (DNA content) was used as normalization for Alcian Blue GAG content.The cells were washed twice with 0.9% NaCl and fixated with 4% paraformaldehyde in PBS for 10 min at room temperature. Fixated cells were washed six times with distilled water and air-dried. Dried fixated cells were incubated for 30 min at room temperature with respectively 1% (m/v) Alcian Blue  in 0.1 TM protein assay reagent , read at 660 nm. In solution tryptic digestion was undertaken as previously described  was used to accommodate MS analysis. To obtain sufficient amounts of CM, the experiment was simultaneously performed in triplicate for each condition. Triplicates were then pooled to obtain three times scrambled siRNA duplex, three times viperin siRNA duplex, three times empty FLAG, and three times FLAG-viperin overexpression CM samples. Two representative triplicates from each condition were utilized to confirm viperin knockdown and overexpression by means of immunoblotting. The CM was centrifuged for 5 min at 1200 rpm to remove dead cells and subsequently for 5 min at 13,000 rpm to remove remaining debris. Then Complete Ultra protease inhibitors  were added. The samples were stored at −80 °C prior to downstream analysis. Protein concentrations of CM were calculated using the Bradford assay with Coomassie Plusescribed . LC-MS/Mescribed . ProteinTM software  was used to identify fold changes in protein abundance between scrambled siRNA duplex and viperin knockdown siRNA duplex and between the FLAG-empty and FLAG-viperin overexpression constructs as described previously  and the human CXCL10/IP-10 Duoset ELISA  were utilized according to the manufacturer's protocol. Plates were colorimetrically measured on a ThermoScientific Multiskan FC plate reader .2) in aggrecan-coated  wells as previously described  from three CHH patients  and three healthy controls were hyperconfluently plated . A two-tailed independent samples The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository  with datM. M. F. S., M. M. J. C., B. Z., L. W. v. R., M. J. P., and T. J. M. W. conceptualization; M. M. F. S., M. M. J. C., D. A. M. S., G. G. H. v. d. A., P. J. v. D., F. F., M. J. P., and T. J. M. W. data curation; M. M. F. S., M. M. J. C., M. J. P., and T. J. M. W. software; M. M. F. S., M. M. J. C., D. A. M. S., G. G. H. v. d. A., M. J. P., and T. J. M. W. formal analysis; M. M. F. S., M. M. J. C., D. A. M. S., G. G. H. v. d. A., M. J. P., and T. J. M. W. validation; M. M. F. S., M. M. J. C., D. A. M. S., G. G. H. v. d. A., P. J. v. D., F. F., and M. J. P. investigation; M. M. F. S., M. M. J. C., G. G. H. v. d. A., and T. J. M. W. visualization; M. M. F. S., M. M. J. C., D. A. M. S., G. G. H. v. d. A., P. J. v. D., F. F., B. Z., M. J. P., and T. J. M. W. methodology; M. M. F. S., M. M. J. C., D. A. M. S., P. J. v. D., F. F., B. Z., L. W. v. R., M. J. P., and T. J. M. W. writing-original draft; M. M. F. S., M. M. J. C., G. G. H. v. d. A., M. J. P., and T. J. M. W. writing-review and editing; B. Z., L. W. v. R., M. J. P., and T. J. M. W. resources; B. Z., L. W. v. R., M. J. P., and T. J. M. W. supervision; B. Z., L. W. v. R., M. J. P., and T. J. M. W. funding acquisition; B. Z., L. W. v. R., M. J. P., and T. J. M. W. project administration.