Brain morphogenesis is an important process contributing to higher-order cognition, however our knowledge about its biological basis is largely incomplete. Here we analyze 118 neuroanatomical parameters in 1,566 mutant mouse lines and identify 198 genes whose disruptions yield NeuroAnatomical Phenotypes (NAPs), mostly affecting structures implicated in brain connectivity. Groups of functionally similar NAP genes participate in pathways involving the cytoskeleton, the cell cycle and the synapse, display distinct fetal and postnatal brain expression dynamics and importantly, their disruption can yield convergent phenotypic patterns. 17% of human unique orthologues of mouse NAP genes are known loci for cognitive dysfunction. The remaining 83% constitute a vast pool of genes newly implicated in brain architecture, providing the largest study of mouse NAP genes and pathways. This offers a complementary resource to human genetic studies and predict that many more genes could be involved in mammalian brain morphogenesis. Brain morphogenesis is an important process contributing to higher-order cognition, however our knowledge about its biological basis is largely incomplete. Here, authors analyzed 118 neuroanatomical parameters in 1,566 mutant mouse lines to identify 198 genes whose disruptions yield neuroanatomical phenotypes How many genes influence brain morphogenesis and what functional systems these genes operate in are important unsolved problems in developmental biology.Brain morphogenesis is a complex process requiring coordinated development of multi-scale structures and relies on a system of sophisticated cues, which together contribute to the proper formation of neural circuits and higher-order cognitive functions. Genetic inheritance has a significant role in brain morphogenesis as shown in twin studies3. However, malformations of brain development, affecting about 0.5% of children4, are due to rare mutations of large effect size that are frequent causes of intellectual disability (ID)6, autism spectrum disorders (ASDs)8, and schizophrenia9. These disorders originate from defects in neurogenesis, cell survival, neuronal migration, axon guidance, and synaptogenesis, and while our capacity to detect genetic variants has significantly improved10, distinguishing pathogenic mutations from normal variation remains an issue11. Their disabling nature together with the fact that as many as 60% of cases are of unknown genetic etiology6 also calls for ways to improve clinical identification and functional stratification, as both are key for better treatment.Several recent genome-wide association studies identified common genetic variants at five loci that together explained between 15% and 25% of the variance influencing intracranial volumeTUBA1A12 and EML113, demonstrating that mutations in the mouse translate to the clinic14. Here we utilize a reverse-genetic screen in mice and perform a systematic and accurate assessment of neuroanatomical defects in 1566 genetic mutant lines in order to identify key genes and functional pathways modulating brain morphogenesis. We then show how this resource can help gain insights into the biological mechanisms leading to neurodevelopmental and psychiatric disorders.Given that combined genetic and large-scale anatomical analysis of the human brain is technically challenging at high resolution, we turned to the mouse where both the environment and genetic background are controlled and where cell-level resolution is achievable using histology. Although the complex cortical folding of the human brain is absent in the naturally lissencephalic rodent brain, humans and rodents share neurodevelopmental principles. This view is supported by the identification of mutations in mice even before their implication in human cerebral developmental disorders, such as 15 , we obtained brain samples from 4796 adult male mice for heterozygous and/or homozygous or hemizygous mutations , as wellThis dataset represents 1566 alleles, each studied with three biological replicates, from 1446 unique genes , of whic17. The sagittal plane offers benefits we discussed elsewhere18, while essentially covering and enabling us to detect NAP variation in the same structures present on coronal sections19. Eighty-five co-variates , the necropsy date of the animal was computed as a random effect variable in order to account for temporal variation that may have occurred since the start of the study in 2009. This achieved the best fitting model (highest Maximum Likelihood Estimate score) against other models taking into account co-variates such as body or brain weights and total brain area  of <0.1, hereafter named as NAP genes. Brain abnormalities were annotated using existing or newly created Mammalian Phenotype Ontology (MPO) terms  of subcortical heterotopia with thinner homotopic cortex and abnormal fiber tracts surrounding it , while the ventricles and the height of the corpus callosum were increased ranging from +28% to +633% . Other examples of models with global impacts include Akap9+/−, Cbx6−/−, Daam2−/−, Mysm1+/−, Rnf10−/−, Ropn1l−/−, and Trappc10−/− . The remaining 49% caused specific abnormalities pertaining to a single category, of which 80% affected a single parameter. For example, Sik3−/− altered the anterior commissure , Anks1b−/− the dorsal hippocampal commissure    Fig. , and SgmMM) Fig. . In all,Smyd5, a lysine methyltransferase gene, increases the retrosplenial granular cortex by 17.5% . The remaining reproduced previous mouse reports  receptors, yield a smaller cortex and white matter structures as shown previously22 but also decreases hippocampal size .The vast majority of NAP genes (94%) have never been previously assessed and associated with brain anatomy in mice, for example, loss of 23 and identified only one gene reported previously (Trappc9) for which we did, however, detect a low-confidence neuroanatomical change. We demonstrated experimental reproducibility using technical repeats of 120 mutant lines engineered twice using various constructions, which showed consistent results, with the exception of Fundc1  database24 was used to impute missing data  of <0.1 , (iii) are more likely to be haploinsufficient (HIS), (iv) are depleted of genetic variation ), and (v) are predicted to be intolerant to protein-truncating mutations (pLI (probability of being Loss of function Intolerant))  induced by disruptions of the associated mouse orthologs . While not globally enriched in known human disease genes  25.To examine the relevance of genes associated with mouse neuroanatomical phenotypes to humans, we first compared the features of human unique orthologs of genes whose disruption in mouse models yielded the largest neuroanatomical variation (top 10%) to those study genes showing the least (bottom 10%) and evaluated differences to randomly permutated gene sets . The humt)) Fig. . More genes Fig. , we founst) Fig. . Althougst) Fig. , we obsehttps://sysid.cmbi.umcn.nl/; June 2018 download), 56% (627) of which account for ID without brain malformations and 43% (481) for ID co-occurring with brain malformations26, we identified an overlap of 30 NAP genes  are linked to highly or fully penetrant brain structural abnormalities  in many patients and for which 4 genes  had been previously reported using mouse models, while the remaining 15 are new in vivo mouse models exhibiting equivalent neuroanatomical defects to humans (with the exception of DONSON). The identification of finer-scale structural anomalies pertaining to the commissures in these models further supports brain connectivity perturbation as a common disease mechanism for ID. Eleven of the 30 genes  are primary ID genes annotated as not known to be associated with structural brain malformations, although when we used a different source (OMIM or Pubmed), low-penetrance structural brain anomalies were, however, reported for some of these genes , while in the Simons Simplex Collection31 we identified a de novo nonsense mutation affecting ARVCF in an ASD patient presenting with microcephaly. These observations suggest that the less-studied gene ARVCF may have a prominent etiological role in the 22q11.21 neurodevelopmental disease-associated phenotypes.Of the remaining 143 (83%) human unique orthologs of mouse NAP genes that did not overlap with primary ID-associated disease genes, our data may help in improving clinical interpretation especially if the identification of additional cases proves difficult or when the pathogenicity of variants is uncertain. For example, a novo missense mutation in Genomic data sources including gene expression, protein–protein interactions (PPIs) and functional mouse and human datasets were used to identify functional convergence among mouse NAP genes derived from our largest and most powerful dataset on coronal sections . Randomlp = 0.017 and p = 0.0035, respectively; Fig. Our 158 mouse coronal NAP genes showed a strong interconnectedness within a large-scale functional gene network Fig. . Roles ip < 0.001; Fig. 32. These results are replicated using a PPI network and high-coverage unbiased body-wide and brain developmental gene co-expression networks  orthologs were identified for 147 of the 158 mouse NAP genes  and human orthologs of FMRP target genes  and the hippocampus    Fig. , while tFinally, we assessed whether mouse NAP genes are associated with whole-body traits. Among 26 categories of traits tested (MGI), 3 showed excess in behavioral/neurological, growth, and adipose traits   of functionally similar genes participating in shared cellular pathways involving the actin and microtubule cytoskeleton, and the synapse, and the disruption of genes within the same module can yield a similar pattern of neuroanatomical abnormalities. While synapse biology is implicated in ASDKptn, Donson, or Aff3) were subsequently associated with causative mutations in recent human studies48, suggesting that other genes identified in this study may be validated in future studies. Also, it is noteworthy that the rate at which new cases are diagnosed remains low despite most cases being genetic in nature29. Ultimately, our models offer a tangible chance of improving this discovery rate, addressing the problem of missing pathogenicity that most large-scale sequencing studies encounter and helping with disease stratification.Third, our in vivo mouse models show a great capacity for novel disease gene validation with 83% of NAP genes not yet linked to any cognitive disorders, constituting a new pool of candidate genes testable against human diseases. During the course of our study, several of our mouse models for previously unknown NAP genes  have been associated so far with congenital hydrocephalus, a pathology affecting 1:1000 live births50. Our top candidate gene for congenital hydrocephalus is Atad3a, associated with severe enlargement of the ventricles in the heterozygous state and previously reported to be lethal when homozygously deleted51. Interestingly, Harel and colleagues have recently reported a biallelic deletion of ATAD3A associated with infantile lethality52.Importantly, our study justifies the use of brain anatomy as a common denominator of cognition, offering endophenotypes for neurological or psychiatric disorders. For example, mutations of In conclusion, our study represents the largest atlas of the causal link between gene mutation and its associated neuroanatomical features to date. It contributes a breadth of new knowledge on the genetics of brain morphogenesis and their underlying networks, providing evidence as to the importance of the cytoskeleton and the synapse in how the brain forms its structures in a highly specific spatiotemporal manner, and adequately complements human genetic studies of neurodevelopmental and neuropsychiatric disorders.We provide a summary of our main methods below. A detailed description and any associated references are available in 15. The strategy relies on the identification of an exon common to all transcript variants, upstream of which a LacZ cassette was inserted to make a constitutive knockout. A subset of mutants were generated using CRISPR/Cas9 methodology, similar to previously reported method53. Genes were selected for entry into the pipeline from requests by WTSI faculty and collaborators working on a wide variety of fields, including cancer, metabolism, infection, immunology, behavior, and human evolution, but without a priori interest in the data generated from the brain histopathology screen. In addition, genes without prior characterization were assigned by the IMPC to WTSI for phenotyping. Assessed genes were distributed on all chromosomes except Y with no bias on their genomic distribution. One hundred and five genes were studied multiple times for validation purposes  were double-stained (Luxol Fast Blue for myelin and Cresyl violet for neurons) and scanned at cell-level resolution using the Nanozoomer whole-slide scanner 2.0HT C9600 series . A total of 85 co-variates, for example, sample processing dates and usernames, were collected at every step of the procedure   suitability for analysis, (2) adequacy of the intensity and contrast of the staining to properly delineate brain structures, (3) symmetry, and (4) sectioning precision. By contrast to more conventional histopathological screens that often rely on qualitative assessment, we used a quantitative approach where each section had to pass well-defined stereotaxic coordinates defined according to the Mouse Brain Atlas20. Supplementary Data http://www.informatics.jax.org/) , a package developed for statistical analysis of large-scale phenotypic data from the IMPC24), based on a Bayesian multiple-phenotype mixed model method, was used to overcome the problem of missing data in downstream analyses that relied on full datasets. This resulted in a new imputed dataset made of 1380 alleles from 1306 unique genes, totaling 5281 mouse samples . We formed 1000 permuted sets of top 10% genes by randomly drawing the same number of genes from human mutant orthologs (n = 96). Consistent results were found using 1%, 5 and 15% top and bottom, and when defining them using cumulative absolute z-scores across brain parameters, although the signals were weaker.Goodall3 index55. Given the low incidence of significant abnormalities post BH correction , we examined the shared presence of abnormalities between gene pairs, weighted by their overall frequency. This simplification generated a more conservative index, whereby strong similarity was not driven by genes lacking neuroanatomical defects. The new similarity index was given by:Sk is the Goodall3 measure between genes i and j for brain feature k and fk is the proportion of NAP orthologs associated with abnormalities in k. The overall phenotypic similarity of two human NAP orthologs was estimated as the average similarity across Bregma +0.98 mm and −1.34 mm.Considering 1:1 human orthologs of NAP genes, we re-scored significant abnormalities (BH-p ≤ 0.05) as 1 (presence) or otherwise 0  for each gene and brain parameter. The similarity of defects caused by the disruption of pairwise gene combinations was calculated using a simplified version of the 56. We constructed human body-wide and brain spatiotemporal gene co-expression networks, using GTEx57 and BrainSpan58 RNA sequencing data across 51 bodily tissues and across 27 brain tissues spanning 12 developmental stages, respectively. Genes with FPKM (Fragments Per Kilobase of transcript per Million mapped reads) or RPKM (Reads Per Kilobase of transcript per Million mapped reads) values <1 in >95% of samples were excluded and gene co-expression networks were derived by estimating the correlation of expression patterns of pairs of protein-coding gene across all associated samples, using Pearson’s coefficients59. Human PPI networks were downloaded from String60. Lastly, by combining various human genomic datasets , we constructed an integrated gene network, termed PLN, wherein pairs of gene are assigned scores, reflecting their likelihood of functional interaction, based on multiple lines of evidence32.The mouse and human gene networks downloaded or constructed (and associated references) are summarized in Supplementary Data 32. By running 1000 permutations, we derived an empirical p value, reflecting the fraction of randomized gene sets, which are more interconnected in the evaluated network, than the set of interest. We identified modules of strongly interconnected genes by partitioning gene networks using the Louvain algorithm61. This greedy optimization method attempts to maximize the modularity of a network division (strength of connections inside modules as compared to between modules). The algorithm was implemented in Gephi using a resolution parameter of 1.To examine the network interconnectedness of a set of genes, we compared the sum of their network links to that of permuted gene sets, constructed by randomly sampling the same number of human genes, matched for CDS and network connectivityThe care and use of mice in the Wellcome Sanger Institute study was carried out in accordance with UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986 under two UK Home Office licenses (80/2485 and P77453634) that approved this work, which were reviewed regularly by the Wellcome Sanger Institute Animal Welfare and Ethical Review Body.Further information on research design is available in the Supplementary InformationPeer ReviewReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14Supplementary Data 15Supplementary Data 16Supplementary Data 17Supplementary Data 18Supplementary Data 19Supplementary Data 20Supplementary Data 21


Dear Editor,2 Elucidating the genetic basis of their complex etiology has eluded our best efforts to date. Although there are more than 400 genes capable of producing an NTD phenotype when mutated in the mouse,4 studies of human candidate genes based on mouse NTD genes have not been informative, except for genes in the planar cell polarity pathway.5 Recently, an omnigenic model of inheritance was proposed for complex traits, suggesting that the associated signals tend to spread across almost the entire genome.6 In light of this new perspective on the genomic architecture of complex traits, we re-evaluated whole-genome sequencing (WGS) data from three different NTD cohorts .Neural tube defects (NTDs) are a class of major structural malformations affecting the brain and spinal cord. They are among the most common congenital anomalies with a worldwide prevalence of 0.1%.7 as controls (208 Chinese Han). Due to the limited sample size of the NTD cohort and uneven coverage of 1KGP sequences (higher coverage in coding regions), only rare (MAF < 0.01) protein-coding variants in NTDs and controls were selected for functional prediction. The selected deleterious missense (D-mis) and loss-of-function (LoF) variants were further compared with 1KGP and ExAC databases (MAF1KGP < 0.001 and MAFExAC < 0.001). Although D-mis and LoF variants are considered more likely to be causative variants, we failed to observe more rare D-mis variants in our Chinese NTD cases compared to the 1KGP controls .We wanted to validate our observations in different cohorts; however, when we examined WGS data from the 1KGP, we found that the number of rare LoF variants varies among different populations. However, the number of singleton LoF variants (SLoFVs), which are those LoF variants that appeared only once when compared to entire 1KGP data, is very similar among different populations.P = 1.7 × 10–13; Fig. Further comparison between the two major subtypes of NTDs, anencephaly (from Chinese cohort) vs. spina bifida (from US cohort) demonstrated that anencephalic cases carried more SLoFVs than spina bifida cases (15 vs. 9, two-sided Wilcoxon test, 8 (Supplementary information, Fig. 8 which suggests that most of the genome is transcriptionally active during neural tube development.Both gene and pathway distributions of those SLoFVs identified in NTD cases from Chinese Han and US CEU cohorts were analyzed to determine whether there is any specific enrichment. Each SLoFV was mapped to KEGG pathways; the frequency of pathways with mutations, based on the percentage of individual NTD cases carrying mutations in each pathway, was calculated. There are 14 pathways containing SLoFVs in > 10% of the NTD samples (Supplementary information, Fig. 6 This enhances our understanding of the genomic architecture underlying susceptibility to these severe congenital malformations.We have now demonstrated that the genetic basis for NTD risk can be measured by the accumulation of SLoFVs in one’s genome. Approximately nine SLoFVs is a genomic threshold for NTD risk, regardless of their genetic background or ethnicity. Together with the finding that no single pathway was enriched for SLoFVs in NTD cases, our study suggests that almost all genes have some minor impact on the etiology of NTDs. In summary, our data demonstrated how a complex reproductive disadvantage disease like NTDs can benefit from the omnigenic model.Materials and Methods are available in Supplementary information, Data Supplementary information, Data S1Supplementary information, Tables S1-S4Supplementary information, Figures S1 and S2


With each ASL scan measurement, suicidal ideation was assessed with the Beck Scale for Suicidal Ideation (BSI) and depression severity with the Beck Depression Inventory (BDI). Compared with active stimulation, the attenuation of suicidal ideation after 4 days of placebo aiTBS was related to significant frontopolar prefrontal perfusion decreases. These findings were unrelated to changes in depression severity scores. Although both active and sham aiTBS resulted in prompt decreases in suicidal ideation, specifically sham aiTBS significantly attenuated frontopolar perfusion in relation to reductions in BSI scores. Our findings show that in accelerated neurostimulation paradigms, placebo responses are related to perfusion decreases in brain areas associated with higher cognitive processes, resulting in suicidal ideation attenuation.The application of repetitive transcranial magnetic stimulation has been shown to rapidly decrease suicidal ideation in major depressive disorder (MDD). However, the neural working mechanisms behind this prompt attenuation of suicidal thoughts remains to be determined. Here, we examined how placebo-accelerated intermittent theta burst stimulation (aiTBS) may influence brain perfusion and suicidal thoughts using arterial spin labeling (ASL). In a randomized double-blind sham-controlled crossover trial, 45 MDD patients received aiTBS applied to the left dorsolateral prefrontal cortex (Trial registration:  To increase response and remission rates, intensified rTMS treatment paradigms have been introduced more recently4. Instead of spreading rTMS sessions over several weeks, a similar amount of sessions is applied within only a few days, usually involving several sessions per day. Such accelerated rTMS treatment protocols result not only in decreases in depressive symptoms but also in prompt decreases of suicidal ideation5. For instance, George et al.5 applied high-frequency rTMS (10 Hz) to the left prefrontal cortex delivering 6.000 pulses/session, three times daily for 3 days . Inpatients, who were admitted because of suicidal ideation or a suicide attempt, were randomized to active or sham rTMS. Trained and blinded raters administered the Beck Suicidal Scale Inventory (SSI6) at baseline and at the end of each day during the 3 days of treatment. After the first day of stimulation, active and sham rTMS were associated with a 50% and a 25% reduction in SSI scores, respectively. After completion of the intensified protocol, almost all subjects showed substantial reductions in suicidal ideation. These findings demonstrate that, although active stimulation in the first day was superior to sham, at the end of the protocol suicidal ideation decreased comparably in both study groups, indicating some form of placebo response. Our recent randomized controlled trial of accelerated intermittent theta burst stimulation  showed similar rapid decreases in suicidal ideation in MDD patients after both active and sham stimulation7. Importantly, the attenuation of suicidal ideation scores was not related to changes in the severity of depression symptoms, suggesting that aiTBS treatment reduces suicidal ideation independently of an improvement in depressive symptoms. These findings thus also suggest placebo effects of TMS on suicidal ideation, independently of its effects on the severity of depressive symptoms.Over the last decades, repetitive transcranial magnetic stimulation (rTMS) has been successfully used to treat unipolar and non-psychotic major depressive disorder (MDD), particularly when combined with antidepressant medication9. A placebo may consist of any form of treatment or application that is used for its ameliorative effect on a symptom or disease, but that is ineffective or not specifically effective for the condition being treated10. A placebo thus is not an inert substance or intervention, but it is rather the administration of this substance or intervention within a set of sensory and social stimuli that inform the individual that a beneficial treatment is given11. The psychophysiological responses elicited by placebos can be very specific depending on the provided information, resulting in mind/body interactions that are guided by subjective factors  and relational parameters8. Placebo effects of rTMS on depressive symptoms may appear large, but in fact are of a similar magnitude as those of psychopharmacotherapy12. In addition, a recent meta-analysis of rTMS studies in depressed patients showed that the magnitude of placebo response is associated to the effect size in the actively treated group, suggesting that the placebo response is a component of the therapeutic response to rTMS13.Placebo (and nocebo) effects are well known in medicine as they have been reported following many kinds of treatments14.) are not limited to neuropsychiatric disorders, but they have been particularly documented in a variety of such disorders, including chronic pain, addiction, Alzheimer’s and Parkinson’s disease. Such responses are usually, but not unequivocally, associated with decreases of neural activity involved in brain regions15. In general, it has been suggested that placebo responses correlate with changes in a core network of brain regions associated with the default mode network (DMN) that is involved in self-generated emotion, self-evaluation, thinking about the future, social cognition, and valuation of rewards and punishment16. Placebo responses in antidepressant medication trials appear to correlate with functional changes in ventromedial prefrontal and in posterior midline structures (both part of the DMN) and in striatal regions17.Placebo responses  and provides reliable absolute CBF quantifications20. At every ASL, fMRI measurement suicidal ideation and depressive symptoms were assessed using the 21-item Beck Scale for Suicidal Ideation (BSI21) and the 21-item Beck Depression Inventory (BDI-I22).No studies have yet addressed placebo effects of non-invasive neurostimulation on brain perfusion and suicidal ideation in MDD patients. The current study therefore aimed at assessing suicidal ideation and correlating brain perfusion changes using arterial spin labeling (ASL) fMRI before and after neurostimulation treatment in a randomized sham-controlled trial in MDD patients. All patients were at least stage I treatment-resistant according to the Rush et al.Although active and sham accelerated paradigms both have been shown to result in prompt attenuation of suicidal ideation, we hypothesized that perfusion pattern changes after sham aiTBS would be distinctly different compared with those after active aiTBS treatment. We expected brain perfusion reductions after sham aiTBS to be present particularly in brain areas related to higher cognitive processes, such as the DMN.http://clinicaltrials.gov/show/NCT01832805). All subjects gave written informed consent. Inclusion criteria for the study were (1) a major depressive disorder selected with the Mini-International Neuropsychiatric Interview (MINI23), (2) right-handedness, (3) at least stage I treatment-resistance according to the Rush et al. criteria18, (4) at least 2 weeks free from psychotropic agents .The ethics committee of the Ghent University Hospital approved this monocentric randomized double-blind sham-controlled crossover study. The study was registered in the Clinical Trials.gov database  with the complete set of three ASL scans were included in the analyses see Fig. . The ful1, T2, T3, and T4). The BDI assesses the severity of symptoms of depression in terms of how patients felt over the previous week, while the BSI measures the current intensity of suicidal ideations, intentions, and plans.For the behavioral analyses, patients were assessed with the self-rating Beck Scale for Suicidal Ideation (BSI) and the Beck Depression Inventory (BDI-I) at each time point during the study (Thttps://www.magstim.com/product/43/70mm-double-air-film-sham-coil). To accurately target the left DLPFC, we used Brainsight neuronavigation  to locate the center part of the midprefrontal gyrus. The 20 iTBS sessions were spread over 4 days at five sessions per day, mounting a total of 32.400 stimuli . During each session, patients received 1620 pulses per session in 54 triplet bursts with a train duration of 2 s, and an intertrain interval of 8 s (from start to end and including the 2 s train duration). Between two sessions, there was a pause of ~15 min. Throughout the whole aiTBS treatment (active and sham), patients were blindfolded, fitted with earplugs, and were kept unaware of the type of stimulation they received. A stimulation intensity of 110% of the subject’s resting motor threshold (rMT) was maintained throughout the treatment.We applied accelerated intermittent TBS stimulation using a Magstim Rapid2 Plus1 magnetic stimulator  with an active and a sham 70-mm Double Air Film figure-of-eight shaped cooled coil. The Magstim 70-mm Double Air Film sham coil is identical to its active variant, in that it is identical in all aspects but without stimulation output. By stimulating the peripheral nerves of the face and scalp, the Air Film sham coil looks, sounds, and feels the same as an active coil, both to the subject and operator but it does not deliver active stimulation of deep nerves  of the brain. For every individual, we located the left DLPFC visually on the 3D surface rendering of the brain based on the known gyral morphology and marked the center part of the midprefrontal gyrus as the left DLPFC target (Brodmann 9/46).The scans were performed on a Siemens 3-T TrioTim MRI scanner  with a 32 channel SENSE head coil. To obtain individual anatomical information, all subjects underwent a first T1-weighted MRI  images with a 3D GRASE readout were obtained with the following parameters: TR = 3.4 s, TE = 14.46 ms, labeling duration = 1400 ms, post-labeling delay changing from 300 to 3000 ms in steps of 300 ms, resulting in 12 pairs of slice-selective (SS) and non-selective (NS) images, scan duration = 5.26 min. This scanning procedure was repeated three times: ASLp<0.05, two-tailed, for all analyses.All behavioral data were analyzed using SPSS 24 . The significance level was set at 27 (https://github.com/GGonEsc/petpve12). Finally, the generated CBF maps were spatially normalized into MNI space and smoothed with Gaussian kernel .The pASL and anatomical images were pre-processed and analyzed using FSL  and SPM12 . The individual structural images were segmented into gray matter, white matter, and cerebrospinal fluid with SPM12. All NS and SS images were realigned to the mean image to correct for motion (using 4th-degree B-spline interpolation in SPM12). The mean image was affine-registered to the anatomical image, and the resulting warps were applied to the realigned images with SPM12. Then, 12 perfusion-weighted images were generated by surround subtraction, i.e., the differences between the paired SS and NS images. The perfusion-weighted images were submitted for CBF estimation using Oxford ASL (oxford_asl) tool in FSL. The partial volume effects in generated CBF maps were corrected by a regression algorithm in PETPVE12 toolbox1) were used in a multiple regression analysis, with the baseline ASL scans (ASL1), and the baseline BSI scores at T1 as the regressors, while correcting for age and gender. Because we were interested in the placebo effects of aiTBS on suicidal ideation, the significant surviving clusters were used as mask for the following regression analyses.First, to evaluate the influence of the initial severities of suicidal ideation on whole-brain perfusion, the baseline ASL maps (ASL1–T2) separately for the group having received first active or sham treatment, we calculated the changes in perfusion between the baseline ASL scan and the second ASL scan, 3 days after the last aiTBS session (delta ASL = ASL1–ASL2). Gender, age, and the changes in depression severity (delta BDI = BDI1–BDI2) and suicidal ideation (delta BSI = BSI1–BSI2) were the covariates, while the change in perfusion (delta ASL = ASL1–ASL2) was the dependent variable. The change in depression severity was added as a co-variate because of the documented placebo responses in rTMS studies, including active stimulation13.Second, to examine the relation between the change in perfusion and the change in BSI scores after the first week of aiTBS stimulation (T1–ASL2). Gender, age, and the changes in depression severity (delta BDI = BDI1–BDI2) and suicidal ideation (delta BSI = BSI1–BSI2) were the covariates, while the change in perfusion (delta ASL = ASL1–ASL2) was the dependent variable.Third, to directly contrast the relation between the change in perfusion and the change in BSI scores between the group receiving sham versus active aiTBS in the first week , we calculated the changes in perfusion between the baseline ASL scan and the second ASL scan, 3 days after the last aiTBS session (delta ASL = ASL2–ASL3) in the group of 24 patients receiving first sham aiTBS in the first week and active aiTBS in the second week (within subjects). Gender, age, and the changes in depression severity (delta BDI = BDI2–BDI3), and suicidal ideation (delta BSI = BSI2–BSI3) were the covariates. The change in perfusion (delta ASL = ASL2–ASL3) was the dependent variable.Fourth, to examine the question whether the change in perfusion in relation to changes in BSI scores changed after the crossover from sham to active aiTBS, we also calculated the changes in perfusion between the second ASL scan and the third and last ASL scan (delta ASL = ASLp<0.05 as implemented in the REST toolbox 1.8 (restfmri.net/forum/), smoothness was estimated for all statistical maps, 1000 iterations). Corresponding brain regions were identified with the Talairach Daemon (search range: nearest gray matter) implemented in WFU PickAtlas28.For all ASL analyses, we applied the AlphaSim correction of χ2(45) = 0.16, p = 0.69), age , the number of comorbidities , baseline BSI , and baseline BDI . Baseline BDI and BSI scores correlated significantly .Twenty-one MDD patients received active iTBS treatment in the first week and sham stimulation in the second week. Twenty-four patients followed the reverse order , while 4 days of active aiTBS resulted in a borderline-significant decrease in BSI scores . The change in BSI scores (delta BSI week 1 = BSI2–BSI1) after 1 week of active stimulation  was not significantly differed from the change after 1 week of sham aiTBS . The change in BSI scores (delta BSI week 2 = BSI3–BSI2) during the second week after active stimulation  was not significantly different from the change after sham stimulation .Wilcoxon-paired 1) and the individual BSI (T1) scores in the superior frontal gyrus (Brodmann area (BA) 10: k = 1933 voxels: MNI coordinates: x = −6, y = 72, z = −3) and in the caudate . This means that the higher the individual BSI scores at baseline the higher the perfusion in these areas. The minimum k was 850 voxels after AlphaSim correction at p<0.05; see Fig. First, the whole-brain regression analysis revealed two large positive regression clusters between baseline perfusion (ASL1 and T2) showed a significant positive association between the change in suicidal ideation (delta BSI = BSI1–BSI2) and the change in perfusion (delta ASL = ASL1–ASL2) in the bilateral frontal cortices (BA 10). This means that a decrease in perfusion after the first week of sham stimulation was associated with a decrease in suicidal ideation, while perfusion increases correlated with a worsening of suicidal ideation. The 21 TRD who received active aiTBS during the first week  showed no significant positive or negative associations between perfusion changes (delta ASL = ASL1–ASL2) and BSI changes (delta BSI = BSI1–BSI2) , the group having received 1 week of placebo aiTBS (n = 24)  showed significant decreases in brain perfusion (delta ASL = ASL1–ASL2) in the superior frontal gyrus after placebo stimulation  associated with significant decreases in suicidal ideation (delta BSI = BSI1–BSI2). The minimum k was 39 voxels after AlphaSim correction at p<0.05 , showed no significant surviving regression clusters within the regions of interest . This suggests that the placebo effect on perfusion patterns is especially present in the first week  and not any more in the second week of aiTBS treatment , also in patients who do not respond to this intervention in terms of a decrease in depressive symptoms. Brain imaging shows that higher baseline levels of suicidal ideation are associated with higher perfusion patterns in brain areas overlapping with the default mode network (DMN), particularly in the right hemisphere, and in the caudate. After 4 days of stimulation with sham, aiTBS perfusion decreases are found in the bilateral frontopolar cortices (BA 10), part of the DMN, and this is associated with a decrease in suicidal ideation. Importantly, these changes in suicidal ideation and perfusion are not related to a change in the severity of symptoms of depression.This study shows that aiTBS treatment decreases suicidal ideation within 4 days of stimulation, whether active or sham. Our findings also indicate that suicidal ideation is primarily attenuated during the first week and not significantly further during the second week of aiTBS treatment. Remarkably, and as reported in Desmyter et al.1 and T2), in the second week were only those patients who had received first sham treatment, in their second week of active aiTBS (between T2 and T3), carry-over effects or blinding issues may have interfered with the perfusion findings.Preceding a discussion of these findings in terms of their contribution to the understanding and prevention of suicide, a few methodological issues need to be addressed. Major advantages of the current study include the neuronavigated coil localization of the left DLPFC and the use of a sham 70-mm Double Air Film figure-of-eight shaped cooled coil that is identical to its active variant but without stimulation output. Nevertheless, all interpretations should be limited to antidepressant-free treatment-resistant depressed patients. Furthermore, we only examined one aspect of possible placebo aiTBS effects on the brain, namely cerebral perfusion. In addition, we can only conclude a decrease on suicidal ideation in a depressed but not highly suicidal group of patients, where suicide attempts within the last 6 months were an exclusion criterion. Lastly, although patients were naive to the iTBS procedure in their first week of stimulation  and deep brain stimulation targeting the nucleus accumbens, have also been found to result in frontopolar glucose metabolism decreases in treatment-resistant depressed patients42. Brain lesions, located in the ventromedial parts, are associated with markedly low levels of depression and lower scores on cognitive/affective symptoms of depression , and possibly suicidal ideation43. While beneficial placebo effects of antidepressant pharmacotherapy on depressive symptoms are well established12, such effects on suicidal ideation are much less clear. A large clinical study among depressed patients, comparing psychotherapy and medication treatments to placebo, showed no differences after adjustment for change in symptoms of depression44. Diminished severity of depression may thus drive reductions in suicidal ideation during antidepressant treatment, whether active or placebo. To study the placebo effect of aiTBS, the change in severity of depression was therefore added as a co-variate in the brain perfusion analyses in the current study.The involvement of the frontopolar cortex, commonly referred to as “Brodmann area 10”, is of particular interest. This part of the prefrontal cortex is proportionally larger in volume relative to the rest of the human brain15. The involvement of BA 10 in response to painful events suggests that this frontopolar cortical area plays a critical role in the collation, integration, and high-level processing of nociception and pain45. As reductions in BA 10 perfusion were coupled with decreases in suicidal ideation in the current study, these findings may indicate changes in higher cognitive processes involving the formation of expectations46, but also self-referential thinking and appraisal, such as value processing16. Here, “value” is defined as an appraisal of the gain or cost  for current and future well-being, made about the self and in consideration of one’s goals. Frontopolar perfusion decreases after sham aiTBS suggest that MDD patients re-appraise their current situation more positively and consequently feel less suicidal. Of interest, the stimulated area (the left DLPFC) has also repeatedly been shown to be involved in the processing of placebo effects9. Indeed, the major direct synaptic projections to the frontopolar cortex originate in higher-order association areas, such as the DLPFC48. The DLPFC may also be involved in maintaining and updating expectancies that drive the placebo effect by modulating cortico-subcortical and corticocortical pathways49. On the other hand, sham-induced placebo effects on perfusion patterns as seen after the initial sham aiTBS were not observed after the crossover from active to sham aiTBS treatment. This may be due to carry-over effects, but also suggests that placebo responses to accelerated rTMS paradigms already occur at an early stage in this treatment protocol.The reduced frontopolar perfusion after sham aiTBS after 4 days of stimulation in the current study is in line with placebo-induced brain responses found in other pathologies, i.e., reduced pain-related brain activations during placebo analgesia, often correlated with psychophysical pain measures26.The lack of significant brain perfusion alterations in relation to suicidal ideation changes after active aiTBS is surprising. This could suggest a lack of effect of active aiTBS on suicidal ideation, but it could also indicate that the underlying neurobiological mechanisms of action in promptly reducing negative thinking patterns after active neurostimulation could be quite different from mechanisms involved in sham responses. This latter interpretation is supported by findings from a resting state fMRI study in the same sample as used in the current study: the decrease in the severity of symptoms of depression and the levels of hopelessness after active aiTBS, but not after sham stimulation, was associated with stronger functional connectivity between the subgenual anterior cingulate cortex and the medial orbitofrontal cortex. These cortical areas both are involved in cognitive inhibition and depressive ruminationIn conclusion, the current study findings indicate that sham aiTBS rapidly attenuates suicidal ideation in treatment-resistant depressed patients, while significantly reducing brain perfusion in the frontopolar cortex. Sham aiTBS may lead to a more positive appraisal of the current situation and thus to a reduction in suicidal ideation. It remains to be demonstrated whether or not these placebo effects are limited to accelerated rTMS paradigms. Future rTMS studies in depression should consider targeting the frontopolar cortical regions more directly, especially when suicidal ideation is present.


We hypothesize that one possible component may lie in species-specific differences in the kinetics of transcriptional responses to differentiation signals. Using a liquid-handling robot, mouse and human pluripotent stem cells were exposed to identical neural differentiation conditions and sampled for RNA-sequencing at high frequency, every 4 or 10 minutes, for the first 10 hours of differentiation to test for differences in transcriptomic response rates. The majority of initial transcriptional responses occurred within a rapid window in the first minutes of differentiation for both human and mouse stem cells. Despite similarly early onsets of gene expression changes, we observed shortened and condensed gene expression patterns in mouse pluripotent stem cells compared to protracted trends in human pluripotent stem cells. Moreover, the speed at which individual genes were upregulated, as measured by the slopes of gene expression changes over time, was significantly faster in mouse compared to human cells. These results suggest that downstream transcriptomic response kinetics to signaling cues are faster in mouse versus human cells, and may offer a partial account for the vast differences in developmental rates across species.Pluripotent stem cells retain the developmental timing of their species of origin  For regenerative medicine, a slow developmental clock operating in human pluripotent stem cells imposes severe obstacles to many pluripotent stem cell therapies. Producing the desired cell types with the necessary level of physiological maturity in clinically-relevant time frames will be essential if the therapy is aimed at being patient-specific. Thus, understanding what controls species-specific rates of differentiation and identifying ways to accelerate it would have a broad impact on improving the therapeutic value of human pluripotent stem cells. In this paper, we measured the immediate-early gene expression responses to neural differentiation signals in mouse and human pluripotent stem cells. We quantitatively summarized the gene expression patterns using a flexible model that allowed us to identify shared trends and dynamic changes across species. We found that responses occurred equally early across species, but expression patterns were prolonged in human cells and the rate of upregulation in gene expression was significantly faster in mouse cells. Our results represent a significant contribution in furthering the knowledge of early developmental transcriptional programs. Pluripotent stem cells, due to their properties of self-renewal and potency, provide a unique model for studying early development and are of particular interest for their exciting potential in regenerative therapies . In ordeex utero, even in the absence of an intact embryo or the maternal environment [Indeed, human and mouse development occur on vastly different time scales. Humans develop over an approximately nine-month gestation, whereas mice require approximately three weeks. Between animals of different sizes, the interval between conserved developmental milestones scales with body size, allowing additional growth between key transitions ,8. For eironment –5,11–14.ironment .in vitro stem cell differentiation alike, signaling factors bind receptors on the plasma membrane to initiate complex signal transduction cascades that ultimately result in the regulation of genes that drive lineage commitment programs [During embryonic development and programs ,17. We tTo explore this question, here we used a custom automated liquid-handling cell culture robotic system to collect RNA-sequencing (RNA-seq) data at a scale of minutes during the initiation of neural differentiation of human and mouse pluripotent stem cells. We report surprisingly early, rapid, and dynamic gene expression patterns in both human and mouse stem cells within minutes of differentiation, identifying biological processes that would otherwise be concealed in daily or even hourly time courses. We find evidence that innate species-specific differences in transcriptional response rates to differentiation factors may therefore explain, at least in part, the differences in developmental rates observed across species.in utero and in vitro [We set out to directly compare initial response rates of mouse and human pluripotent stem cell differentiation by exposing them to identical neural differentiation conditions and monitoring changes in gene expression at high temporal frequency. Human embryonic stem (hES) cells and mouse epiblast stem (mEpiS) cells are widely considered to represent primed (post-implantation) stages of embryonic development –21 and win vitro  Fig 1A)in utero Dynamic gene expression patterns over the time course in the mouse and human datasets were identified by fitting a set of segmented regression models to the expression of each gene using the R package Trendy  Fig 1A)Fig 1A). 2) as a goodness of fit measure for the segmented regression model to eliminate genes not having a dynamic trend or those with noisy expression. There were 1436 genes having an adjusted R2 > 0.2 on their fitted gene expression trends in both human and mouse time courses  (Methods). We found that mouse cells retained their earlier breaktimes and had fewer monotonic genes compared to human cells  of them also had dynamic trends during pluripotent medium feeding . FurtherThe most common pattern among the set of 1436 dynamic ortholog genes was the peak trend (including both immediate and delayed) with 156 orthologs having a peak in both mouse and human cells . Here weMyc, Jun, Btg2 and Atf4 are transcription factors, although the others such as Sqstm1, Adm, and Ubc are all genes known have protein products involved in neuronal differentiation [Previous reports indicate that gene peak patterns tend to be enriched in genes identified as ‘immediate-early response’ genes (IEG) . IEGs cantiation –28  the “up” segment leading towards the peak begins earlier in mouse compared to humans, and/or (2) the slope of the segment leading towards the peak, or rate of change in gene expression, is steeper.As previously mentioned, there was no substantial difference between the percentage of genes that began their upward trends immediately versus later in the time course between mouse and human cells  . We expaWe then asked if this was true for all genes that were upregulated as a first response to differentiation (i.e. any gene that was immediately dynamic up or had delayed upregulation). We found no differences in the percentage of genes that go up immediately versus delayed across species, for either common dynamic orthologs or all genes that go up in either mouse and human cells . Again, In the situation of a delayed peak in human versus mouse cells, the explanation for earlier peak times is clear. However, the vast majority of genes and orthologs showed no species-specific difference in the onset of upregulation, thus we hypothesized that the component responsible for the earlier peak times in mouse compared to human genes must be the rate at which genes are upregulated. The rate of change in gene expression was assessed by measuring the slopes of changes in gene expression over time in mouse and human cells.To ensure that slopes were comparable across species and not biased by any species-specific differences in dynamic range of expression, we scaled each gene’s expression to be between 0 and 1 prior to running Trendy  model approach implemented here is both powerful and flexible by estimating gene expression changes and trends without a priori defining trend categories. The method implemented in the Trendy R package output the best fit breakpoint model for each gene, summarizing quantitative measures of breakpoint times and slopes of the connecting segments which we were able to compare across species in our statistical analysis. We uncovered (1) a previously unidentified highly dynamic gene expression landscape within the first twenty minutes of mouse and thirty minutes of human stem cell differentiation, (2) gene expression patterns were protracted in human compared to mouse cells, (3) the time of initial gene upregulation off of baseline was not different between human and mouse genes, and, most interestingly, (4) changes in gene expression occurred significantly faster in mouse compared to human pluripotent stem cells as measured by the slopes of gene upregulation. Taken together, these results identify species-specific differences in the kinetics of gene expression responses to differentiation cues from the earliest stages of development.Time courses of pluripotent stem cell differentiation have typically involved daily sampling frequencies, and rarely as frequently as every 6 hours . While tWe also found enrichment of immediate-early response genes in our peak genes corresponding to species-specific differences in response to neural induction media. We consider the peak genes identified in our study that are not yet classified as IEG in the literature as IEG candidates and contend that the segmented regression framework here could easily allow identification of novel IEGs when applied to additional early time course experiments by identifying immediately dynamic genes and calculating the fold-change of the response as the amplitude of the expression trend. Given the short 10-hour time-frame focus in our study we focused mainly on peak trends, however this framework is also able to identify cyclic trends and could be expanded to allow for non-linear segmented modelling.Our data in this study does not support a model of delayed signal delivery to the nucleus (model 1), but rather a difference in the velocity of transcriptional upregulation across species model 2). First, we did not observe an overall increase in percentage of genes with a delayed onset of up- or downregulation changes in human compared to mouse genes , 1 X non-essential amino acids , 2 mM L-glutamine , 7.5 ng/mL activin A , and 5 ng/mL bFGF ).EGFP-positive H1 and mEpiS cells were maintained as previously described . H1 cell5 or 5.28X105 cells were seeded into 12- or 6-well matrigel-coated plates, respectively, and were shuttled to 37°C incubators and attached under pluripotent conditions  until neural induction.Cell suspensions were seeded in pluripotent media prior to neural induction. H1 cells were lifted in 0.5uM EDTA and cells were transferred to the robotic system at 4°C under sterile conditions in E8 medium. mEpi3S cells were dissociated using TryPLE, however were first depleted of MEFs by MACS separation using feeder cell removal beads according to the manufacturer’s instructions . Cells were then resuspended in TSS medium and transferred to the robotic system under sterile conditions at 4°C. Mouse and human cells were kept at 4°C until either 2.5X10A Freedom Evo200 liquid handling robot was located in a BigNeat custom controlled environmental enclosure. The time course was automated using a custom-built automated platform . The platform was programmed using TECAN Evoware Plus software, and various custom software drivers to control peripheral devices were used. The enclosure maintained an environment of 30°C, with 5% CO2 and 80–90% relative humidity (RH) during all liquid handling steps.Integrated peripheral devices were utilized to perform automated tissue culture and generate cellular lysates. These devices included a Liconic STX500 incubator , a Liconic STX200-DF freezer (for sample storage after cell lysis), a Tecan Te-Shake shaker, and a Tecan Tilt Carrier for media removal.At time 0, pluripotent media were removed from the wells of the previously seeded 12- or 6-well matrigel plates, cells were washed with 1 X PBS , 37°C pre-warmed neural induction medium was added  supplemented with 1 X N2 supplement , 1 X B27 supplement , and 100 ng/μL of mNoggin ), as previously described , and celRNA-seq samples were processed from cell lysates as previously described . BrieflyTo map intron reads, we adapted our previous alignment pipelines as follows. We first built an expanded transcriptome reference using hg38 (rather than hg19), and incorporated both NM (mRNA) and NR (non-coding RNA) transcripts. Then we built an ‘intronic’ transcriptome reference, with a single entry for each gene in the “mature” transcriptome reference. Each entry contained the full genomic sequence from the most 5’ TSS through the most 3’ UTR of all annotated transcripts for that gene. We applied our usual filters to trim low quality base calls and adapter sequences. We ran RSEM-1.3.0, using bowtie2 (v2.3.5.1) against the new mature-transcript reference . From thi,jY represents the normalized expression of gene i in sample j.After quality control the mouse and human RNA-seq datasets had 145 and 61 samples, respectively. Each dataset was normalized using median ratio normalization proposed by Anders and Huber 2010  using thi,jX as a function of time t. For the mouse experiment t =   and for the human dataset t = . For notation, let t =  for either experiment with N = 145 in mouse and N = 61 in human. The segmented regression model for gene i having a single breakpoint is:The segmented models were fit with expression bi,1 is the breakpoint and βi,1 is the slope of the first segment prior to the breakpoints and βi,2 is the slope of the second segments after the breakpoint. The number of segments is always one plus the number of breakpoints. For each gene we fit a model having no breakpoints (standard linear regression) and then extended the model above to accommodate fitting five additional models each having the number of breakpoints from 1 to 5. For a breakpoint model fit to be valid, each segment was required to contain a minimum of five or three consecutive time-points representing 20 minutes in the mouse time course and 30 minutes in the human, respectively . While every gene has a model fit, here we focused only on those considered having a ‘good’ fit, which we defined as genes having a fit with an adjusted RPeak genes were classified as those having either a consecutive “up” and “down” segment along the time course experiment, including cyclic genes, or those having an “up”, “no-change”, “down” pattern. Genes displaying distinct patterns shown in All analyses were performed in R version 3.6.0 . The TreAnalyses to determine functional enrichment of orthologs having a peak and genes having dynamic expression in both the human differentiation and control experiment were performed using the GSEA algorithm on the GO biological processes collection in MSigDB  ,67. For S1 Text1 –Evaluating the appropriateness of piece-wise linear model fits2 –Choice of threshold for dynamic Trendy fit.3 –Choice of minimum segment length parameter.4 –Detectable resolution of dynamic expression by Trendy.5 –Scaling data to obtain comparable slope magnitudes.(DOCX)Click here for additional data file.S1 TableSheets 3 contains the lists of genes having each pattern in mouse cells sampled every four minutes. Sheet 4 contains the lists of genes having each pattern in human cells sampled every 10 minutes.(XLSX)Click here for additional data file.S2 TableThe information includes: segment slopes, segment direction, segment p-value, time of breakpoint, and trend direction for each time-point.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(TXT)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S1 Figrd mouse sample in the time course to effectively simulate a reduced sampling rate to levels comparable to the human time course (lengthened from 4 minutes to every 12 minutes in mouse compared to 10 minutes in human cells). Using this modified sampling frequency, we compared the time of initial onset of Up/Down trends in gene expression (A), numbers of monotonic genes (B), and time to first breakpoint (C) of common ortholog genes between mouse and human cells. The same analysis was carried out on all dynamic human and mouse genes even if the same gene was not fitted with a dynamic trend in the other species . Similarly, gene expression peak times (G) and slopes Up (H) and Down (I) from the peak were measured in ortholog genes having a peak in both mouse and human cells, as well as for genes with a peak in at least one species .We re-ran Trendy and our entire analysis pipeline using every 3(TIF)Click here for additional data file.S2 Fig2 > 0.2) in the feeding control RNA-seq time course. Similarity of trends was based on genes initial response direction; genes starting in the same direction were considered similar and genes with trends in the opposite direction were considered opposite. B) The breakpoint distribution of the differentiation and control experiments with times where multiple genes had changes in expression highlighted. C.) Top GO terms from a gene-overlap enrichment analysis for the genes indicated in the sets from B are shown. None of the synchronized breaktimes in the control were enriched for differentiation processes and instead had strong cell cycle signals. Given that those cells are not responding to differentiation media, cell cycle is the dominant biological process we detect.A) Among all 4332 human genes identified by Trendy, 1634 are also dynamic (adjusted R(TIF)Click here for additional data file.S3 FigFor all orthologs having at least one peak in both mouse and human cells, the time of the first (or only) peak is shown with the Mouse peak time on the x-axis and the Human peak time on the y-axis.(TIF)Click here for additional data file.S4 FigA) Treating the first and last ten time-points as a ‘condition’, the mean change in exon reads versus mean change in intron reads is shown for the human and mouse time courses. The high correlation indicates transcriptional activity is detectable. B) The intronic read slopes between mouse and human cells for genes that also were upregulated in either mouse or human exonic reads.(TIF)Click here for additional data file.S5 FigA) For all ortholog peak genes, the slopes of the up-trend (top) and down trend (bottom) are shown in a boxplot for the 10 hour robot time course. B) The same plots are shown for ortholog peaks in mouse and human time courses of three and six weeks, respectively, in Barry et al., 2017.(TIF)Click here for additional data file.


Until recently, cervical mediastinoscopy was considered to be the reference standard for mediastinal staging for Non-Small Cell Lung Carcinoma (NSCLC). In the absence of metastases, mediastinal lymph node involvement is the most important prognostic factor and as such it determines therapeutic strategies. In this study we evaluated the adequacy of cervical mediastinoscopy in NSCLC lymph node staging in a large university hospital over more than a decade. In addition, we determined the influence of: (1) surgeon’s experience (2) video-assisted mediastinoscopy (VAM) and (3) patient-related restrictions (PRR) on the adequacy of lymph node sampling.Between January 2001 and December 2014, 225 patients underwent cervical mediastinoscopy for lymph node staging. Surgical and histological data were reviewed. Thirty-day follow-up was available for all patients. Lymph node sampling was considered adequate when stations 4 L, 4R and 7 were sampled (ESTS guidelines). A surgeon was considered to be experienced when he or she performed at least 40 procedures during the study-period.Intraoperative mortality was 0%. Thirty-day mortality was 1.3%. Overall adequacy of lymph node sampling was 56%. Univariate and multivariate logistic regression analyses of lymph node sampling adequacy revealed level of surgical experience and PRR as independent predictors of lymph node sampling adequacy.Surgical experience and PRR independently predict the adequacy of cervical mediastinoscopy in NSCLC lymph node staging. VAM does not independently predict the adequacy of mediastinal lymph node sampling. In light of the expected further decline in mediastinoscopy numbers, we recommend to limit this procedure exclusively to the armamentarium of the experienced thoracic surgeon. Lung cancer is the leading cause of cancer death in developed countries and accounts for an estimated 20% of all cancer deaths . Five-yeFluoroDeoxyGlucose - Positron Emission Tomography – Computed Tomography (FDG-PET-CT), Endoscopic UltraSound guided-Fine Needle Aspiration and EndoBronchial UltraSound guided-TransBronchial Needle Aspiration (EUS-FNA/EBUS-TBNA) have become the most important techniques in mediastinal lymph node assessment in recent years , 9. CervMediastinoscopy provides access to the upper paratracheal lymph nodes (stations 2R and 2 L), the lower paratracheal lymph nodes (stations 4R and 4 L) and subcarinal lymph nodes (station 7) , 14, 15.A well-executed cervical mediastinoscopy has a sensitivity of 76–85% and a negative predictive value of 82–92%  with an In this study we evaluated the adequacy of mediastinal lymph node sampling at our center over more than a decade. In addition, we analyzed the influence of: (1) surgeon’s experience, (2) the use of VAM and (3) patient-related restrictions (PRR) on the adequacy of lymph node sampling (based on the ESTS guidelines).This study was conducted in accordance with the guidelines of the University Medical Center Groningen Institutional Review Board.Between January 2001 and December 2014, 225 patients underwent cervical mediastinoscopy for NSCLC lymph node staging. VAM was introduced at our center in September 2008 and from then on used in each mediastinoscopy case. Patient characteristics are summarized in Table Based on the ESTS guidelines the minimal requirement for adequate lymph node sampling during cervical mediastinoscopy was defined as histologically proven samples from at least the left and right lower paratracheal lymph nodes (station 4 L and 4R) and the subcarinal lymph nodes (station 7) .The level of surgical experience was based on the number of cervical mediastinoscopies performed by individual surgeons. For surgeons who performed at least 40 mediastinoscopies during the study-period the adequacy of lymph node sampling was > 70%. Therefore experienced surgeons were defined as those who performed at least 40 mediastinoscopies during the study-period. Based on these criteria two out of sixteen surgeons could be considered experienced. Both experienced surgeons in this study were trained as thoracic surgeons.PRR were defined as intraoperative conditions or findings, which complicated the adequacy of lymph node sampling. An overview of PRR is shown in Table Follow-up was obtained directly from outpatient visits or by telephone interview with the patient and/or the referring physician. Thirty-day follow-up was complete.2 test or Fisher’s exact test as appropriate for categorical variables and the independent samples t-test or Mann-Whitney U test, as appropriate for continuous variables. Univariate variables with P < 0.10 were included in the multivariate analysis. Age and gender were forced in the multivariate model. Multivariate logistic regression analyses by means of a forward stepwise algorithm were performed to identify independent predictors of lymph node sampling adequacy. Odds ratios were reported with 95% confidence intervals (CI). Goodness-of-fit of the final logistic regression models was assessed with the Hosmer-Lemeshow statistic.Continuous variables were expressed as mean ± SD. Categorical variables were expressed as percentages. Comparisons between groups were performed using Pearson’s XP < 0.05.All calculations were performed using a commercially available statistical package . Statistically significant differences were defined as . Univariate and multivariate logistic regression analyses of lymph node sampling adequacy are shown in Table 2 = 0.24, df = 1, P = 0.878).The overall adequacy of lymph node sampling was 56%. In patients who underwent cervical mediastinoscopy by an experienced surgeon, adequacy of lymph node sampling was 64%, versus 47% when operated by a less experienced surgeon P = 0.01, Table 3n = 3). All deaths were unrelated to cervical mediastinoscopy. Causes of death included cerebrovascular accident and respiratory insufficiency after partial mandibular resection for a second primary tumour, respiratory insufficiency after thoracotomy and rib resection, and multi-organ failure after early bronchial fistula formation following right-sided pneumonectomy.An overview of thirty-day mortality and post-operative complications is provided in Table This study demonstrates that surgical experience as well as PRR are independent and powerful predictors of the adequacy of cervical mediastinoscopy in NSCLC lymph node staging. When an experienced surgeons performs the mediastinoscopy adequate lymph node sampling is almost 2 times more likely than when a less experienced surgeon performs the mediastinoscopy (OR 1.96) and when PRR are not present adequate lymph node sampling is almost 6 times more likely than when PRR are present (OR 5.94). Other studies have also shown that mediastinoscopy yield depends strongly on operator skills , 19 and One of the drawbacks of conventional mediastinoscopy is the uncomfortable position for the surgeon. The surgeon has only a narrow view through the instrument and has to find a way among anatomical entities such as; trachea, esophagus, azygos vein, right pulmonary artery, recurrent nerve and pleural space/lung, and depending on patient anatomy; the carotid and innominate arteries. As such, conventional mediastinoscopy is a complex procedure and teaching can also be difficult because of the risk of ‘collateral damage’. These events strongly depend on the experience and teaching skills of the surgeon. VAM, with its superior visualization and teaching possibilities, has made the procedure safer and easier to adopt for surgeons in training . In thisSuccessful treatment of patients with NSCLC strongly depends on strict and reliable staging. The mediastinal lymph node status determines the sequence of treatment modalities. Until recently, mediastinoscopy was the gold standard for invasive mediastinal lymph node staging in NSCLC. Mediastinoscopy provides access to upper paratracheal lymph nodes (stations 2R and 2 L), lower paratracheal lymph nodes (stations 4R and 4 L) and subcarinal lymph nodes (station 7) , and hasLimitations of our study include the long time frame and the retrospective design.Both mediastinoscopy and endosonography are complex technical procedures and depend strongly on operator skills and experience. The complexity of a procedure is inversely related to the adoptability of a procedure . ComplexSurgical experience and PRR are powerful and independent predictors of the adequacy of cervical mediastinoscopy in NSCLC lymph node staging. Experience and skills vary with the training of the operator. Therefore, a solid training is required in educational programs and every center has to look at its own diagnostic yield and negative predictive value. VAM with its superior visualization and teaching possibilities, makes the procedure safer and easier to adopt for surgeons in training, but does not independently predict the adequacy of lymph node sampling. Since mediastinal lymph node staging is crucial in patient treatment and outcome, we urge that cervical mediastinoscopy should be performed and taught by experienced thoracic surgeons only.