GLI3/KIF21A or EHMT2/UBE2I) in separate families were modeled. We used a multi-electrode array, with patch-clamp recordings, to determine a reproducible synaptic phenotype in 25% of the individuals with ASD (other relevant data on the remaining lines was collected). Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. The biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.Induced pluripotent stem cell (iPSC)-derived neurons are increasingly used to model Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of penetrant and weaker polygenic risk variants to ASD, ‘isogenic’ iPSC-derived neurons are critical. We developed a set of procedures to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD. Heterozygous de novo and rare-inherited presumed-damaging variants were characterized in ASD risk genes/loci. Combinations of putative etiologic variants (Editorial note: This article has been through an editorial process in which authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (ddressed . The past two decades of research has determined autism spectrum disorders (ASD) to be clinically  and genebona fide autism-susceptibility (or risk) genes, although none of them show specificity for ASD alone  technology , followeCNTN5 or EHMT2—which could be responsible for ASD-related phenotypes.Here, we develop a resource of 53 different iPSC lines derived from 25 individuals with ASD carrying a wide-range of rare genetic variants, and from unaffected family members. We also used clustered regularly interspaced short palindromic repeats (CRISPR) editing  to creatParticipants were enrolled in the Autism Speaks MSSNG whole-genome sequencing (WGS) project . All ASDOCT4/POU5F1, SOX2, KLF4 and MYC, and a lentiviral vector that encoded the pluripotency reporter EOS-GFP/PuroR  nor for 36O-36P (AGBL4), thus they were not chosen for further study. When we compared their values to the pooled values recorded from all the different familial controls available, no difference was found for NRXN1 and a significantly lower wMFR was observed at weeks 5 and 7 for AGBL4 (The weighted MFR (wMFR), which represents the MFR per active electrode, was used as a primary read-out for all tested iPSC-derived neurons, at one-week intervals from week 4 to 8 post-NGN2-induction (PNI) . To iden neurons . EHMT2 h or GLI3 , suggestt week 4 , and thefor THRA . No unafor AGBL4 .SET), or 61I and 61K versus 62M and 62X (GLI3), at different timepoints (THRA) at week 4, or lines 75G and 75H (CAPRIN1) at week 8 (EHMT2) at weeks 4 and 5  and inter-individual  variability, we plotted all the values obtained from each single well, independent experiment, cell line and individual, at each of the five reading timepoints . Most limepoints . A few lt week 8 . We alsoCNTN5-mutant neuron lines 27H and 27N, we first showed a significantly higher network burst frequency at weeks 5, 6 and 8  is rescued by expressing a functional version of human UBE2I. Several missense variants in UBE2I have been accurately predicted as deleterious at conserved positions, or benign at other positions  sex-matched member. Simultaneous multi-line electrophysiological evaluation revealed hyperactivity of the simple-variant CNTN5  and biological (up to 4) replicates to best compensate for inter-well and inter-iPSC neuronal induction variations . It is plication . Since tlication . Line 19EHMT2-/+ lines could be due to a better capacity to survive, disperse or adhere than EHMT2+/+ cells, without affecting synaptic activity. However, excluding all inactive electrodes would then result in a comparable wMFR between mutant and control cells, which was not the case. Indeed, both MFR and wMFR were significantly higher in EHMT2-/+ cells. That does not exclude the possibility of a better survival, dispersion or adhesion, but it is likely not the only reason for the observed increase in spiking activity, suggesting greater synaptic activity, as supported by patch-clamp recordings. Indeed, we used patch-clamp recordings to show that sEPSC frequency was significantly increased in mutant line 38E compared to control line 37E. We believe these results directly support synaptic alteration as one of the possible causes for the increased neuronal activity measured by MEA. However, a detailed analysis of cell maturation will be required for each different cell line involved in this study to clarify this issue. It will be interesting in the future to investigate the possible mechanisms involved in the decreased activity observed in THRA-mutant neurons  is a glycoprotein of the extracellular matrix including four consecutive fibronectin type III domains. Loss-of-function variants in ANOS1 were shown to cause the Kallmann syndrome, which is characterized by congenital hypogonadotropic hypogonadism associated with anosmia, delayed puberty and infertility  is an immunoglobulin cell adhesion molecule, with four fibronectin type III domains, involved in neurite outgrowth and axon connection in cortical neurons, and was associated with ASD  is a histone methyltransferase (HMTase) that forms a complex with EHMT1 (GLP) to catalyze mono- and dimethylation of lysine nine on histone H3 (H3K9me1/2) (EHMT2 (K1164Nfs) on the protein function remains to be determined  . Of noteK9me1/2) . ActuallK9me1/2) . EHMT2 rK9me1/2)  and poteK9me1/2) . The impined see . Since tined see , we suspined see . Upon ouined see , we propCNTN5 or EHMT2 significantly intensifies excitatory neuron synaptic activity in vitro. Such phenotype offers the possibility to implement NGN2-based high-throughput drug screening strategies  at the Hospital for Sick Children, Toronto, Canada, iPSCs were generated from dermal fibroblasts or CD34 +blood cells. Three different informed consent forms for iPSC derivation and publication were obtained: i) Research Consent Form for Parent/Legal Guardians ; ii) Research Consent Form for Unaffected Individuals; iii) Assent form . These consent forms describe in details the purpose of the research, the description of the research, the potential harms, the potential benefits, confidentiality, storage of the research samples, participation, reimbursement, sponsorship, and declaration of conflict of interest; REB approval file 1000012015.3 pieces with disposable scalpel in a 60 mm dish. 5 ml of collagenase 1 mg/ml  was added and the dish was placed in 37°C incubator for 1:45 hr. Skin pieces and collagenase were then transferred to a 15 ml tube, and centrifuged at 300 g for 10 min. Supernatant was removed, 5 ml of trypsin 0.05%/EDTA 0.53 mM (Wisent Bioproducts) was added, and the mix was pipetted up and down several times to break up tissue and placed in 37°C incubator for 30 min. After incubation, the mix was centrifuged at 300 g for 10 min, and supernatant was removed leaving 1 ml. The pellet was pipetted up and down vigorously to break to the pieces without creating bubbles. The mix was transferred in a T-12.5 flask along with 5 ml of Alpha-MEM, 15% Fetal Bovine Serum , penicillin 100 Units/ml and streptomycin 100 μg/ml (ThermoFisher), and placed in 37°C incubator for about a week until 100% confluence. Cultured cells were fed every 5–7 days if not confluent. Once confluent, cells were passed into three 100 mm dishes to expand, and frozen in liquid nitrogen.Skin-punch biopsies were obtained from the upper back area by a clinician at The Hospital for Sick Children. Samples were immersed in 14 ml of ice-cold Alpha-MEM (Wisent Bioproducts) supplemented with penicillin 100 Units/ml and streptomycin 100 μg/ml (ThermoFisher), and transferred immediately to the laboratory at The Centre for Applied Genomics (TCAG). Each biopsy was cut into ~1 mmPOU5F1, SOX2, KLF4, MYC, and lentiviral vectors encoding the pluripotency reporter EOS-GFP/PuroR were used and obtained as described  using CytoTune-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher). Fibroblasts were cultured in fibroblast expansion media . The desired number of wells for reprogramming from a 24-well plate was coated with 0.1% gelatin. Fibroblasts were dissociated using Trypsin (ThermoFisher) and allowed to settle overnight. Virus multiplicity of infection (MOI) was calculated and viruses combined according to number of cells available for reprogramming and manufacturer’s protocol. 24 hr after transduction, media was changed to wash away viruses. Media was additionally changed on day 3 and 5 after transduction. 6 days after transduction, 6-well plates were coated with Matrigel(Corning). Cells were removed from the 24-well plate using Accutase (ThermoFisher) and plated on Matrigel in expansion media. 24 hr later, media was replaced with E7 media (StemCellTechnologies). Cells were monitored and fed daily with E7. Once colonies were of an adequate size and morphology to pick, individual colonies were picked and plated into E8 media (StemCellTechnologies). Clones growing well were further expanded and characterized using standard assays for pluripotency, karyotyping, genotyping and mycoplasma testing. Directed differentiation was performed using kits for definitive endoderm, neural and cardiac lineages .Whole peripheral blood was processed at CCRM using Lymphoprep (StemCellTechnologies) in a SepMate tube (StemCellTechnologies) according to manufacturer’s instructions. The sample was centrifuged (10 min at 1200 g). The top layer containing PBMCs was collected and mixed with 10 mL of the PBS/FBS mixture and centrifuged (8 min at 300 g). The PBMC’s collected at the bottom of the tube were washed, counted and resuspended in PBS/FBS mixture. CD34 +cells were then isolated using the Human Whole Blood/Buffy Coat CD34 +Selection kit according to manufacturer’s instructions (StemCellTechnologies). Isolated cells were expanded in StemSpan SFEM II media (StemCellTechnologies) and StemSpan CD34 +Expansion Supplements (StemCellTechnologies) prior to reprogramming.Reprogramming of CD34 +PBMCs was performed at CCRM using CytoTune-iPS 2.0 Sendai Reprogramming Kit. Expanded cells were spun down and resuspended in StemSpan SFEM II media and StemSpan CD34 +Expansion Supplements, and placed in a single well of a 24-well dish. Virus MOI was calculated and viruses combined according to number of cells available for reprogramming and manufacturer’s protocol. The virus mixture was added to cells, and washed off 24 hr after infection. 48 hr after viral delivery, cells were plated in 6-well plates in SFII and transitioned to ReproTESR for the duration of reprogramming. Once colonies were of an adequate size and morphology to pick, individual colonies were picked and plated into E8. Clones growing well were further expanded and characterized as explained above.All iPSC lines were maintained on matrigel (Corning) coating, with complete media change every day in mTeSR (StemCellTechnologies). ReLeSR (StemCellTechnologies) was used for passaging. Accutase (InnovativeCellTechnologies) and 10 μM Rho-associated kinase (ROCK) inhibitor  were used for single-cell dissociation purposes.http://crispr.mit.edu/. We designed a HDR-based method using a synthesized single-stranded oligonucleotide (ssODN) template to replace the point mutation with the reference nucleotide. To prevent damage to the correct sequence, a silent mutation was introduced in the ssODN close to the proto-adjacent motif (PAM) of the reverse gRNA (gRNA-), which commands Cas9D10A to nick the plus strand, given that ssODN was synthesized as plus strand. All the CRISPR machinery was introduced into iPSC by nucleofection. Screening for correction of the appropriate base pair was based on absolute quantification of allele frequency using droplet digital PCR (ddPCR). Enrichment of corrected cells was obtained through sib-selection step cultures in 96-well plate format, as adapted from  system with two guide RNA (gRNAs) to reduce off-target activity. We devised the gRNA sequences using tools available at ted from , until ated from .6 HEK293T cells were seeded in a T-75 flask, grown in 10% fetal bovine serum in DMEM (Gibco). The next day, cells were transfected using Lipofectamine 2000 with plasmids for gag-pol (10 μg), rev (10 μg), VSV-G (5 μg), and the target constructs FUW-TetO-Ng2-P2A-EGFP-T2A-puromycin or FUW-rtTA  (7.5 × 10oratory) . Next da5 iPSCs/well were seeded in a matrigel-coated 6-well plate in 2 ml of mTeSR supplemented with 10 μM Y-27632. Next day, media in each well was replaced with 2 ml fresh media plus 10 μM Y-27632, 0.8 μg/ml polybrene (Sigma), and the minimal amount of NGN2 and rtTA lentiviruses necessary to generate 100% GFP +cells upon doxycycline induction, depending on prior titration of a given virus batch. The day after, virus-containing media were replaced with fresh mTeSR, and cells were expanded until near-confluency. Newly generated ‘NGN2-iPSCs’ were detached using accutase, and seeded in a new matrigel-coated 6-well plate at a density of 5 × 105 cells per well in 2 ml of mTeSR supplemented with 10 μM Y-27632 (day 0 of differentiation). Next day (day 1), media in each well was changed for 2 ml of CM1  supplemented with fresh doxycycline hyclate (2 μg/ml) and puromycin . Media was replaced with CM2 supplemented with fresh doxycycline hyclate (2 μg/ml). The same media change was repeated at day 4. At day 6, media was replaced with CM2 supplemented with fresh doxycycline hyclate (2 μg/ml) and araC . Two days later, these day eight post-NGN2-induction (PNI) neurons were detached using accutase and ready to seed for subsequent experiments, as described below.5 × 1048-well opaque-bottom MEA plates , 16 electrodes per well, were coated with filter-sterilized 0.1% polyethyleneimine solution in borate buffer pH 8.4 for 1 hr at room temperature, washed four times with water, and dried overnight. 120,000 ‘day8-dox’ neurons/well were seeded in a 5 ul drop of CM2 media at the centre of each well, then covered with 250 μl CM2 media after one hour in the incubator. The day after, 5,000 mouse astrocytes/well were seeded on top of neurons in 50 μl/well CM2 media. Astrocytes were prepared from postnatal day 1 CD-1 mice as described . Media wA receptor antagonist picrotoxin  at 100 μM, AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dion  at 60 μM, and sodium ion channel antagonist tetrodotoxin  at 1 μM. The plates were recorded consecutively, 5–10 min after addition of the antagonists. A 60 min recovery period was allowed in the incubator at 37°C between each antagonist treatment and plate recording.Offline advanced metrics were re-recorded and analysed using Axion Biosystems Neural Metric Tool. An electrode was considered active if at least five spikes were detected per minute. Single electrode bursts were identified as a minimum of five spikes with a maximum interspike interval (ISI) of 100 milliseconds. Network bursts were identified as a minimum of 10 spikes with a maximum ISI of 100 milliseconds covered by at least 25% of electrodes in each well. No non-active well was excluded in the analysis. After the last reading, each well was treated with three synaptic antagonists: GABA2, 0.1 CaCl2, 1 Mg-ATP, and 0.2 Na4GTP (pH 7.2). 0.06% sulpharhodamine dye was added to select neurons for visual confirmation of multipolar neurons. Composition of extracellular solution was (in mM): 140 NaCl, 2.5 KCl, 1 1.25 NaH2PO4, 1 MgCl2, 10 glucose, and 2 CaCl2 (pH 7.4). Whole cell recordings were clamped at −70 mV using Clampex 10.6 (Molecular Devices), corrected for a calculated −10 mV junction potential and analyzed using the Template Search function from Clampfit 10.6 (Molecular Devices). Following initial breakthrough and current stabilization in voltage clamp, the cell was switched to current clamp to monitor initial spiking activity and record the membrane potential . Bias current was applied to bring the cell to ~70 mV whereby increasing 5 pA current steps were applied (starting at −20 pA) to generate the whole cell resistance and to elicit action potentials. Data were digitized at 10 kHz and low-pass filtered at 2 kHz. Inward and outward currents were recorded in whole-cell voltage clamp in response to consecutive 10 mV steps from −90 mV to +40 mV.Day 3 PNI neurons were replated at a density of 100,000/well of a poly-ornithin/laminin coated coverslips in a 24-well plate with CM2 media. On day 4, 50,000 mouse astrocytes were added to the plates and cultured until day 21–28 PNI for recording. At day 10, CM2 was supplemented with 2.5% FBS in accordance with . Whole-cThe method for the yeast complementation assay was described previously .Cells were washed in ice-cold PBS and total protein was extracted in RIPA supplemented with proteinase inhibitor cocktail, and homogenized. Equivalent protein mass was loaded on gradient SDS-PAGE (4–12%) and transferred to Nitrocellulose membrane Hybond ECL . Primary antibodies used were rabbit anti-CNTN5  and rabbit anti-EHMT2/G9A . HRP-conjugated secondary antibodies (Invitrogen) were used and the membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce). Images acquired using ChemiDoc MP (BioRad) and quantified using software Imagelab v4.1 (BioRad). Western Blots were repeated at least twice for each biological replicate.All cell lines were regularly tested for presence of mycoplasma using a standard method . In the interests of transparency, eLife includes the editorial decision letter, peer reviews, and accompanying author responses.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed.]The revised manuscript has been evaluated by a previous referee, who found the majority of the concerns have been adequately addressed by reorganizing the data and presenting the findings in a consistent manner. The Reviewing Editor's assessment is that several minor issues require attention. There is a need to acknowledge alternative explanations for the results and for further clarification of the MEA phenotypes. The editors feel that these issues can be dealt with by small revisions and additions to the text. Please make all the edits as outlined below.Reviewer #1:eLife.The authors have done a good job responding to my criticisms. The manuscript now more transparently details the variability between hiPSCs/experiments. The authors present phenotypic MEA and electrophysiological differences in CNTN5+/- and EHMT2+/- hiPSC-neurons from ASD. While they now clarify in the abstract that they identified synaptic phenotypes in 25% of the individuals tested, they should more clearly note that they cannot rule out synaptic phenotypes in the other 75% of ASD cases. For example, secondary differences in cellular replication, survival or patterning could mask activity differences by MEA. Moreover, the authors should consider in the discussion that MEA phenotypes need not predict electrophysiological deficits and vice versa, as evidenced for ASTN2 in their recent Stem Cell Reports publication. Finally, the authors still fail to resolve whether deficits in CNTN5 and EHMT2 result from impaired maturation rather than impaired synaptic function. With modest and mostly textual revisions, this report will be suitable for publication at Minor Comments:The nomenclature used in Figure 2–supplement 1 is very unclear. Can the authors add the relevant family ID and gene name such that the reader does not need to constantly refer back to Table 1? Reviewer #1:[…] Major detail:-/+ and EHMT2-/+. The findings (even if null) should be aggregated and clearly presented as a main figure. Additionally, the authors should make all raw MEA data and all hiPSC lines widely available upon publication.1) ASD neuronal phenotypes – The authors conducted MEA across all 25 hiPSC lines (Supplementary file 3) but do not seem to comprehensively present this data anywhere, focusing instead on CNTN5We thank the reviewer for these helpful suggestions. Accordingly, we have aggregated all the null data  along with the positive data into a single new Figure 3. All raw MEA data and all iPSC lines will be publicly available upon request at the time of publication. We are also open to submitting the iPSC cell lines to an appropriate repository if one is available. We are in discussions with Autism Speaks who have expressed interest in supporting this. As is the case for all of our bioresources described in publications, they will be available upon request. We adhere closely to standard academic publishing principles.2) Inter-individual and intra-individual variability – The authors have generated perhaps the largest systematic MEA dataset across unrelated controls and ASD patients with different rare mutations. While they aggregate all control data in SI Figure 2 and 3, it would be tremendously valuable to the field if they might plot their results by donor as a main figure. Being able to show how MEA activity changes over time, and between donors, would greatly inform our understanding of inter-individual variation in MEA. Similarly, in those cases where the same line was tested on multiple MEA plates, again, it would be useful to present variability.We agree that plotting all the results by donor as a main figure would help to appreciate the inter-individual and intra-individual variability. Hence, we have aggregated all the data in a new Figure 3 showing how the activity changes over time, and between donors, and even between different biological replicates for each donor. Correspondingly, we have added the following (framed) paragraphs to the section “Multi-Electrode Array Analysis of iPSC-Derived Neurons” of the Results:CNTN5 and EHMT2, a significant higher wMFR was recorded in ASD variant neurons at week 6 compared with their corresponding familial control neurons . EHMT2 had a strikingly increased wMFR at all timepoints, whereas CNTN5 at other timepoints was equivalent to its controls. We therefore ranked these two genes as high priorities for further study. In contrast, no significant differences were observed for DLGAP2, CAPRIN1, SET or GLI3 , suggesting that these variants do not differ from control neuronal activity in our MEA assays, and therefore were not studied further. Different dynamics of altered wMFR were observed for ANOS1 and VIP at week 4 , and the ANOS1 nonsense variant was ranked as an example of a candidate for further study. Conversely, a significant lower wMFR was recorded at weeks 7 and 8 for THRA . No unaffected family members were available as controls for the single NR3 line (NRXN1) nor for 36O-36P (AGBL4), thus they were not chosen for further study. When we compared their values to the pooled values recorded from all the different familial controls available, no difference was found for NRXN1 and a significantly lower wMFR was observed at weeks 5 and 7 for AGBL4 .“The weighted MFR (wMFR), which represents the MFR per active electrode, was used as a primary read-out for all tested iPSC-derived neurons, at one-week intervals from week 4 to 8 post-NGN2-induction (PNI) . To identify a preferred timepoint for this screen, we first pooled the data of all the independent control lines. Since the highest wMFR value for this pool of ‘all controls’ (~1.8 Hz) was detected at week 6 , we initially used that timepoint to compare the activity of ASD variant and control lines for each family. In two different families, i.e., EHMT2) at weeks 4 and 5 . Note that similar profiles were monitored in terms of MFR , indicating that these differences were not due to having more or less active electrodes in different lines. While consistent activity across lines was generally observed, the presence of variability prompted us to interrogate independent variants created by genome editing of CNTN5, ANOS1 and EHMT2.”To explore intra-individual  and inter-individual  variability, we plotted all the values obtained from each single well, independent experiment, cell line and individual, at each of the five reading timepoints . Most lines from an individual were not significantly different from each other, and reassuringly low inter-individual variability was observed with different siblings bearing the same mutation(s), e.g., 48K and 48N versus 49G and 49H (SET), or 61I and 61K versus 62M and 62X (GLI3), at different timepoints . A few lines showed a significant intra-individual variability, e.g., lines 52A and 52C (THRA) at week 4, or lines 75G and 75H (CAPRIN1) at week 8 . We also noted some inter-independent experiment variability for a given line, e.g., line 38E (3) Inter-well and inter-hiPSC neuronal induction efficiency – It is incredibly difficult to assess and adjust for differences in total cell number and cell type composition in MEA experiments, both of which are expected to impact MEA recordings. What was the authors' strategy? It would be helpful if in the discussion they might inform the readers how best to learn from and extend their analyses.The third and fourth paragraphs of the Discussion, as follow, now discuss this issue:“We assume that most NGN2-neurons are glutamatergic based on the data presented in the original publication establishing this technique , and on our previous publication using high-cell density RNAseq assessment . Moreover, we have treated several of our cultures at the end of the MEA experimentation with different neurotoxins  to show that most neurons are glutamatergic and not GABAergic, for different lines. In addition, our patch-clamp recordings have demonstrated that these neurons exhibit the properties of excitatory neurons.CNTN5 and EHMT2 because the phenotypes were confirmed in unrelated isogenic pairs, and by patch-clamp recordings.”Characterization of neuronal composition and survival when MEA is performed is difficult to achieve with high accuracy. Our strategy involved using several technical (3 to 12 per independent experiment) and biological (up to 4) replicates to best compensate for inter-well and inter-iPSC neuronal induction variations (Supplementary file 3). It is possible that some phenotypes were missed since we cannot exclude the possibility that differences in cell number or composition across individuals in a family actually masked potential phenotypes. However, this issue was ruled out for 4) CRISPR-engineering – How did the authors rule out off-target effects following CRISPR-editing to produce isogenic pairs?ANOS1 Rescues Defective Membrane Currents” section of the Results:In our recent publication, which is now out in the peer-reviewed literature , we have designed a new approach to detect any off-target mutations based on whole-genome sequencing of a series of isogenic KO lines. We have used the same strategy here and inserted the following (framed) sentence within the second paragraph of the “Repair of “No overt off-target mutations were detectable using our previously-described WGS strategy .”CNTN5 and 19-2-EHMT2, we have not investigated any potential off-target mutations since this method was previously described with very low probability of off-target mutations by several different groups. Moreover, finding similar significant phenotypes as those observed in the patient lines further dissociated any potential off-target mutations to such phenotypes.This was expected since we used the double-nicking approach. Regarding the isogenic pairs made using ribonucleoprotein (RNP) complexes in line 19-2, i.e., 19-2--/+ or EHMT2-/+, the authors should provide attempt to resolve this question (even a time course of transcriptomic changes over 6-weeks of NGN2-induction).5) Phenotypic characterization – Increased neuronal activity might indicate alterations in synaptic function and/or maturation. For the two genes focused on within the manuscript, CNTN5We agree that an increased neuronal activity might indicate alterations in synaptic function and/or maturation. We now discuss this in the fifth paragraph of the Discussion, as following:EHMT2-/+ lines could be due to a better capacity to survive, disperse or adhere than EHMT2+/+ cells, without affecting synaptic activity. However, excluding all inactive electrodes would then result in a comparable wMFR between mutant and control cells, which was not the case. Indeed, both MFR and wMFR were significantly higher in EHMT2-/+ cells. That does not exclude the possibility of a better survival, dispersion or adhesion, but it is likely not the only reason for the observed increase in spiking activity, suggesting greater synaptic activity, as supported by patch-clamp recordings. Indeed, we used patch-clamp recordings to show that sEPSC frequency was significantly increased in mutant line 38E compared to control line 37E. We believe these results directly support synaptic alteration as one of the possible causes for the increased neuronal activity measured by MEA. However, a detailed analysis of cell maturation will be required for each different cell line involved in this study to clarify this issue.”“An increased neuronal activity, e.g., MFR in mutant lines 38B/E, might indicate alterations in synaptic function and/or maturation. We have presented the MFR for all tested cell lines in Figure 3—figure supplement 1, in support of the wMFR in Figure 3. The wMFR is defined as the MFR divided by the number of active electrodes per well. If there is significant failure of electrode activation in a well, for example due to differences in cell survival, dispersion or adhesion, those data are excluded. For example, the increased MFR observed in EHMT2 neurons at day 21-25 PNI. We did not detect any significant change in sEPSC frequency and amplitude at this earlier timepoint, similarly to the MEA experiment. However, intrinsic properties showed a significant increase in capacitance and decrease in input resistance in mutant cells . These observations suggest that the mutant neurons at 3-4 weeks PNI potentially have a faster maturation rate, however, this phenotype is most pronounced in the hyperactivity recorded by MEAs later at 9-11 weeks PNI. These results support the conclusion that the inactivation of one allele of EHMT2 significantly increases spontaneous network activity of excitatory neurons, with possible effects on the neuronal maturation process. We also agree that a time course of RNAseq on the isogenic pairs could be informative regarding synaptic function and/or maturation pathways in the future. However, the presence of mouse astrocytes in the electrophysiological experiments could potentially limit the full understanding of what is happening at the two relevant timepoints.Moreover, as explained in the last paragraph of the Results, we also performed patch-clamp recordings on 19-2--/+ or EHMT2-/+ increase neuronal activity? Possibly by linking their results to existing animal models? Perhaps RNAseq analyses might provide insights into the downstream changes in patient-derive neurons?6) Mechanism – Can the authors speculate as to the mechanism through which CNTN5EHMT2, for example, which is mostly located in the nucleus and could be impacting activity-dependent transcription of synaptic signaling genes.Speculation about potential mechanisms and linking to existing animal models are part of the Discussion. We have used RNAseq analyses in a previous publication  to provide insights into the possible downstream changes in several different isogenic pairs involving KO of other ASD genes, and revealed interesting convergence of DEGs and networks. In the future, this tool will certainly help to shed light into the downstream genes and pathways affected by the absence of 7) Data sharing – The authors have generated a great resource that is likely to be of great interest to the community. The value of such a collection would be enhanced by making publically available the inclusion of SNP and exome genotype data from each donor and clarification of the specific hiPSC lines available (at which repository). How many validated hiPSCs per donor are being made available? Through which repository? Will the MEA data be made available?All raw MEA data, as well as all iPSC lines, will be publicly available upon request. Genotype data, in many different forms, from each donor and the relevant controls described in the paper is already available through the Autism Speaks MSSNG WGS project . Of course, as our group always does, we will follow standard academic publishing rules and provide any information/resource needed for others to replicate our experiments.Minor detail:1) Clarification of terminology – NGN2-overexpression is best described as "induction" not "differentiation." The authors are cautioned against describing the neurons as "cortical" without clear evidence of patterning.We agree with this comment and we have made appropriate modifications throughout the manuscript where applicable.2) Can the authors clarify in the main text  that astrocyte co-culture was used with NGN2-neurons to improve maturation for MEA and electrophysiological.We have modified the main text accordingly.Additional data files and statistical comments:Please make available all MEA raw data to the community.All raw MEA data will be publicly available upon request at the time of publication. We are also actively searching for a public repository to accept it .Reviewer #2:[…] Specific comments follow:Abstract: It seems to this reviewer that the Abstract communicates a more positive result than was actually achieved, as regards the "standardized set of procedures," that "used a large-scale multi-electrode array (MEA).…" These statements seem to suggest the value of this approach to the general field. Yet, of the 12 families with genetic variants, only 3 of the 12 MEA studies revealed any differences, which does not necessarily recommend this as a mainstay approach. The alternative approach, using single cell electrophysiology, defined only one additional pathology. The Abstract should be modified to change the emphasis, and consider stating the success rate of 33%.We have modified the Abstract accordingly:“We used a multi-electrode array, with patch-clamp recordings, to determine a synaptic phenotype in 25% of the individuals with ASD.”Materials and methods:The authors do not mention performing a power analysis in designing the study, and power analysis is not discussed in the statistical methods. There is concern that the inherent variability in the MEA data is not well appreciated nor well controlled, and without this analysis, it cannot be transparently reported.We agree that variability in the MEA data could not have been well appreciated in the previous version of the manuscript. In a two-sample comparison setting, a power analysis estimates the sample size required to likely detect an effect of a given size. In a screening approach, the sensitivity and specificity of the screen are also influenced by the sample size. Here, we elected to use MEA as a screening tool because we had previously evaluated the sensitivity of our protocol. For example, MEA was used to confirm a hypoactive neuronal phenotype, previously detected using patch-clamp recordings, in 4 out of 5 KO lines, in which 6-8 MEA wells were recorded per line per experiment, in mostly 4 independent experiments . With such a high level of sensitivity, plus testing usually 2 different lines per individual, we assumed that aiming at 6 technical and 3 biological replicates per line would support enough sensitivity for our screen. In addition, we have aggregated all the data in a new Figure 3 showing how the activity changes over time, and between donors, and even between different biological replicates for each donor. Correspondingly, we added the following (framed) paragraphs to the section “Multi-Electrode Array Analysis of iPSC-Derived Neurons” of the Results:CNTN5 and EHMT2, a significant higher wMFR was recorded in ASD variant neurons at week 6 compared with their corresponding familial control neurons . EHMT2 had a strikingly increased wMFR at all timepoints, whereas CNTN5 at other timepoints was equivalent to its controls. We therefore ranked these two genes as high priorities for further study. In contrast, no significant differences were observed for DLGAP2, CAPRIN1, SET or GLI3 , suggesting that these variants do not differ from control neuronal activity in our MEA assays, and therefore were not studied further. Different dynamics of altered wMFR were observed for ANOS1 and VIP at week 4 , and the ANOS1 nonsense variant was ranked as an example of a candidate for further study. Conversely, a significant lower wMFR was recorded at weeks 7 and 8 for THRA . No unaffected family members were available as controls for the single NR3 line (NRXN1) nor for 36O-36P (AGBL4), thus they were not chosen for further study. When we compared their values to the pooled values recorded from all the different familial controls available, no difference was found for NRXN1 and a significantly lower wMFR was observed at weeks 5 and 7 for AGBL4 .“The weighted MFR (wMFR), which represents the MFR per active electrode, was used as a primary read-out for all tested iPSC-derived neurons, at one-week intervals from week 4 to 8 post-NGN2-induction (PNI) . To identify a preferred timepoint for this screen, we first pooled the data of all the independent control lines. Since the highest wMFR value for this pool of ‘all controls’ (~1.8 Hz) was detected at week 6 , we initially used that timepoint to compare the activity of ASD variant and control lines for each family. In two different families, i.e., EHMT2) at weeks 4 and 5 . Note that similar profiles were monitored in terms of MFR , indicating that these differences were not due to having more or less active electrodes in different lines. While consistent activity across lines was generally observed, the presence of variability prompted us to interrogate independent variants created by genome editing of CNTN5, ANOS1 and EHMT2”To explore intra-individual  and inter-individual  variability, we plotted all the values obtained from each single well, independent experiment, cell line and individual, at each of the five reading timepoints . Most lines from an individual were not significantly different from each other, and reassuringly low inter-individual variability was observed with different siblings bearing the same mutation(s), e.g., 48K and 48N versus 49G and 49H (SET), or 61I and 61K versus 62M and 62X (GLI3), at different timepoints . A few lines showed a significant intra-individual variability, e.g., lines 52A and 52C (THRA) at week 4, or lines 75G and 75H (CAPRIN1) at week 8 . We also noted some inter-independent experiment variability for a given line, e.g., line 38E ;" that reference is to a manuscript submitted to bioRxiv that has not undergone peer review, and for the current article to be published, analysis of the NEUROG2 induction procedure needs to be presented. The statement that "differentiation levels" were uniform between cell lines, is quite a claim, and it is not at all clear what this means. For example what endpoints were assessed among multiple lines to indicate uniformity? One can imagine, for example, that failure to observe a specific differentiation feature, could reflect that the marker is not expressed by a cell, versus, that it is expressed but at a time point not examined. Such an outcome could reflect changes in i) cell fate, ii) induction of differentiation, iii) survival of a specific cell class, or simply, iv) a delay in its expression in suboptimal culture conditions. There is no characterization of the surviving cell population when MEA analysis of the cultures is performed, so that specific experimental outcomes cannot be related to the cell population under study. Even though the culture bottoms are opaque, immunocytochemistry using fluorescent labeling would allow visualization of cell numbers (DAPI) and cell type  by use of the epifluorescence microscope to understand how cell composition at the time of MEA recording impacted the outcomes. This seems a reasonable request since the majority of MEA studies did not reveal group genetic differences, with only 3 of the 12 families demonstrating changes in firing rate and network burst frequencies.We have used the NEUROG2 overexpression strategy for its high consistency in differentiation levels between several different lines. The reference to our bioRxiv paper was made for expediency and openness, and the paper has now undergone stringent and full peer-review and is published . The third and fourth paragraphs of the Discussion, as followed, now discuss this issue:“We assume that most NGN2-neurons are glutamatergic based on the data presented in the original publication establishing this technique , and on our previous publication using high-cell density RNAseq assessment . Moreover, we have treated several of our cultures at the end of the MEA experimentation with different neurotoxins  to show that most neurons are glutamatergic and not GABAergic, for different lines. In addition, our patch-clamp recordings have demonstrated that these neurons exhibit the properties of excitatory neurons.CNTN5 and EHMT2 because the phenotypes were confirmed in unrelated isogenic pairs, and by patch-clamp recordings.”Characterization of neuronal composition and survival when MEA is performed is difficult to achieve with high accuracy. Our strategy involved using several technical (3 to 12 per independent experiment) and biological (up to 4) replicates to best compensate for inter-well and inter-iPSC neuronal induction variations (Supplementary file 3). It is possible that some phenotypes were missed since we cannot exclude the possibility that differences in cell number or composition across individuals in a family actually masked potential phenotypes. However, this issue was ruled out for The MEA methods are incomplete as reported, and extensive review of Supplementary file 3 still leaves uncertainties for this reviewer. The authors employ MEA as the single major measure of outcome, but they do not reference their previous use of this technology. It is not adequate to cite other laboratories. The authors should describe more fully the nature of the MEA technology, how the cultures appear in them, the data that is generated, and how it was analyzed. To start, the authors indicate that 48-well opaque-bottom MEA plates (Axion Biosystems) were used. But going to the website there are four (4) "Classic" varieties of this plate, as well as many other formats. Please provide exact catalogue number or numbers. How many electrodes are there in each well? It may be helpful to show a layout/picture for the electrode array, and also pictures of fixed and stained neuronal cultures to define the variability in cell contact to multiple electrodes, and perhaps the extremes of electrode contact they observe. Doing a brief PubMed search, I found two recent 2018 publications that provide images that show large variability of cell growth around fixed electrode arrays . Please state the range of individual MEA wells used in these types of studies: Supplementary file 3 seems to indicate a range from 3 wells to as many as 12. How many independent MEA assay experiments are performed for each line? Supplementary file 3 is confusing, specifically the column labeled "Total nb independent plates." This number coincides with the reported number of different MEA plates . Is an independent plate a separate experiment? Usually in iPSC studies, within a single clone (as reported here), independent experiments imply different passages of the same clone, analyzed in subsequent weeks, or after thawing an earlier passage and newly plated. But after NEUROG2 induction, it seems the cells by design are non-proliferative . But without a proliferative stem cell culture, how are independent experiments performed? Is the NEUROG2 induction performed multiple times on the same clone  to yield 3 independent plates? Or are glutamatergic-induced cells frozen and plated at multiple times, another valid approach? Or alternatively, is a single induction plated at the same time to multiple plates? The issue of reproducibility is critical in this kind of study, and it is surprising that there is no mention of these issues that are currently central to the field.We agree that the MEA technology is still in its infancy, but it is still important data to use, and in doing so we will help to move the field forward. We have now referenced our previous use of the MEA technology in the first sentence of the “Multi-Electrode Array Analysis of iPSC-Derived Neurons” section of the Results. Data generation and analysis is explained in Materials and methods. We have now provided the exact catalogue number of the MEA plates used, as well as the number of electrodes per well in Materials and methods. Since only opaque-bottom 48-well MEA plates were commercially available at the time this study was performed, it was not possible to take clear pictures of cell-electrode contact. We apologize for the confusion regarding Supplementary file 3, and have now modified it to reflect the reviewer’s requests. For example, we have replaced the column label "Total nb independent plates" for “Total nb independent experiments", as an independent plate meant independent experiment. Here, independent experiments imply independent infections with NGN2 viruses of iPSCs at different passages, entailing completely independent inductions. So, NGN2 induction was performed multiple times on the same clone  to yield 3 independent plates. We have clarified this issue in the legend of Supplementary file 3.Results:Line 19-2 is used as a control for one family and is also the line for all isogenic lines produced. It is the only line shown that was reprogrammed by retrovirus , so what is known about virus integration site mutations, and does this interfere or alternatively facilitate outcomes, and impact its use as a control? To make it easier for the reader to understand this report, the authors need to provide a description of line 19-2 here, rather than refer the reader to the un-reviewed manuscript in bioRxiv.To help us stay within length restrictions, the reader is now referred to our peer-reviewed and published paper , where line 19-2 was extensively characterized.Supplementary file 2 indicates that for some families, only a single line was assessed for pluripotency markers and surprisingly, this seems to include 2 important control lines, 19-2 and NR3. It seems a concern that a control line used for many experiments was analyzed using only a single clone; please address.The purpose of using the line 19-2 here was to validate any finding in an independent genetic background while providing an isogenic control via CRISPR editing. Since two different clones, e.g., 38B and 38E, have replicated an increased neuronal activity , and that properly editing one clone requires extensive time and costs, we thought that one genetically-independent and isogenic clone would be sufficient to support our primary findings. We have tried to accompany all our findings by using conservative wording throughout the manuscript.The authors indicate they "sought to determine if any selected ASD-risk variants would interfere with spontaneous spiking and synchronized bursting activity in a whole network of interconnected glutamatergic neurons." To determine when to make MEA comparative analysis they pooled the N=341 control line wells from the 12 families, and observed that the mean firing rate (MFR) was the highest (~1.7 Hz) at week six (6) post-NEUROG2-induction (PNI). Thus they used this time point to compare all cell lines for the 1) weighted mean firing rate (MFR) and the 2) network burst frequency. This approach seems reasonable and seems to establish a standard set of conditions and protocols, one of their goals, as stated in the Abstract.+/- revealed differences in MEA at 6 weeks, they created an isogenic line 19-2 control line by inserting the CNTN5 Stop-Tag. However they report they did not find any effects on MEA parameters at 6 weeks. Thus they extended analysis to 11 weeks, and now found statistically significant differences at 10 weeks. However, it seems that at week 7, the outcome appears to be the opposite, with StopTag showing 50% less activity . It seems the authors cannot have it both ways, we use 6 weeks, but if we do not find an expected outcome, we wait longer. Further, maybe many of the other negative MEA results would appear different if assessed at other time points.However, to this reviewer, the choice of a single time to make assessments seems to diminish the likelihood of finding differences between control and disease related genetic variants. I think it is not obvious nor expected that control lines from each family would exhibit the same maturation profile, and while the authors do perform intra-familial comparisons, the advantages of this type of analysis seem to be thwarted in this design. Given genetic heterogeneity, one could easily imagine different time frames. With all the expenses in cost, media, and time to set up these complex models, why not make recording every week for example? The current protocol as presented seems to be an inefficient use of resources. And perhaps in support of this criticism, the authors themselves seem to agree, as shown in Figure 3! Because the Family D mutant line #27 that carries CNTN5et al., Stem Cell Reports, 2018).We thank the reviewer for the constructive critique of our design. These are early days in performing this type of analysis and with standards of data presentation in place, we try to present the most relevant data to the experiments at hand. In the previous version of the manuscript, we assumed that it would be clearer for the reader if we show only one timepoint, i.e., the most active in control cells (week 6). However, as suggested by both reviewers, we decided to aggregate all the data and build a new Figure 3, showing how the activity changes over time, and between donors, and even between different biological replicates for each donor. Now, we think this is a better way to appreciate the variability of MEA, and consider potential difference in maturation rate of various lines. Line 19-2 is generally more active than most other familial lines, and this may have delayed emergence of the hyperactive phenotype in isogenic cells until week 10, as now discussed in the new version. Moreover, we have previously detected hypoactive phenotypes in line 19-2 at week 8 and before , the results at 11 weeks appear to be the reverse of that seen at 7 weeks, yet this outcome may have little to do with synaptic functions. To this reviewer, since time plays an important role in genetic variant effects , and the authors seem to tentatively conclude that changes in MEA measured activity depend on changes at the synapse, it would strengthen their case if single cell electrophysiology of these cells at this or an earlier time were correlated with the MEA outcomes, to demonstrate that CNTN5 effects are mediated by changes in synaptic currents, and not in cell survival, distribution, or adhesion. Cellular analysis at MEA culture termination would address this unexplored mechanism.There is also concern that cell survival plays an important role. In CNTN5CNTN5+/- neurons should help to support our MEA findings in the future. However, we have used weighted MFR (wMFR) , in addition to MFR , to take into account electrodes that might be inactive due to cell survival, distribution or adhesion.We agree that patch-clamp recordings of The definition of weighted mean firing rate is not clear; the authors state, "We monitored the weighted mean firing rate (MFR), which represents the MFR per active electrode,.…". If understood correctly, this measure only averages active electrodes in any MEA well. Thus if there is significant (and currently un-described) failure of electrode activation in a well, that data is excluded. A major feature of a cell line may be that in one case only 20-30% of electrodes have signal due to differences in cell survival or dispersion, whereas in another line, it may routinely be 80-100% coverage. If this is the case, this type of information may impact the kinds of cellular mechanisms that one considers to explain the outcomes. This kind of information should be assessed, if not already done, and should be reported transparently in a Table that includes all families, perhaps in the supplement. One might find a correlation of decreasing/increasing cell-electrode contact (appearing as active/inactive electrodes/well) to the outcomes reported.EHMT2-/+ lines in could be due to a better capacity to survive, disperse or adhere than EHMT2+/+ cells, without affecting synaptic activity. However, excluding all inactive electrodes would then result in a comparable wMFR, which is not the case . Indeed, both MFR and wMFR are significantly higher in EHMT2-/+. That does not exclude the possibility of a better survival, dispersion or adhesion, but it is likely not the only reason for the observed increase in spiking activity, suggesting greater synaptic activity, as supported by patch-clamp recordings in Figure 6B. We now discuss this in the fifth paragraph of the Discussion.We apologize if the definition of wMFR was not made clear in the previous manuscript. It is defined as the MFR divided by the number of active electrodes per well. An electrode was considered active if it detected at least five spikes per minute, as explained in the section “Multi-electrode array (MEA)” of the Materials and methods. If there is significant failure of electrode activation in a well, for example, due to differences in cell survival, dispersion or adhesion, that data is indeed excluded. However, a detailed analysis of cell survival, dispersion and adhesion, will be required for each different cell line involved in this study to clarify this issue in the future. This is one of the reasons why we decided in the new version of the manuscript to present the MFR  in order to support the wMFR to take into account potential issue with cell survival, dispersion or adhesion. For example, the increased MFR observed in +/-, demonstrates abnormal MFR and burst frequency only from weeks 4 to 7 when compared to family controls , a result that is lost by week 8. In contrast, the 19-2 isogenic line only shows differences at week 9 or later. Similar differences are observed for CNTN5+/- in Figure 3. Second, the MFR in line 19-2 is 4 Hz or more, whereas analysis of all the other 10 family control lines (Sendai based) examined at 6 weeks (sometimes 4-8 weeks) are mostly less than 1 Hz, and always below 2 Hz. These consistent differences raise concerns that the 19-2 line brings a special test system in which perhaps most of the analyzed variants could/should be assessed. But this may also bias results because only this single line is used for isogenic studies. Would this same effect occur in a Sendai non-integrating virus-induced iPSC model for isogenic line production?There is concern about the sole use of retrovirally-induced line 19-2 for all isogenic experiments. In contrast to control lines from the 10 families , whose study led to selecting 6 weeks for MEA analysis, line 19-2 with introduced CRISPR gene variant only reveal differences at 8-12 weeks. There raises two issues. Firstly, MEA analysis of, for example, EHMT2SHANK2 , and PTCHD1-AS . Furthermore, a pattern of outward/inward currents comparable to that found in 18C/18CW lines  was monitored in a different and unrelated isogenic pair, in which our StopTag fragment was previously inserted within the ANOS1 coding sequence in line 19-2 . This result was found on the right panel of Figure 5D in the original manuscript. However, we prefer now to exclude this panel from the revised manuscript and put this profile only in the response to reviewers . Since line 19-2 could be used to validate both hypoactive and hyperactive electrophysiological phenotypes, we don’t think that this line create any significant bias on its own.We agree that line 19-2 is generally more active than other tested lines in our hands, and we cannot exclude at this stage the possibility that it could be due to the use of retrovirus for reprogramming. However, we don’t think that these differences in intensity and time window should preclude the use of this line for validation/isogenic experiments. Indeed, we have previously shown that a completely independent line, i.e., 50B (Sendai-reprogrammed), was able to reproduce significant electrophysiological and RNAseq results obtained in isogenic KO studies performed in line 19-2 . Line 19-2 has also been extensively used in other projects to validate different results obtained with familial lines defective in wers see  since th-/+ CRISPR-Isogenic Pair Confirms Neuronal Hyperactivity”: The authors should reserve interpretations of differences in expression of MEA in the isogenic line compared to the family lines, 38B/E. They state: "This increased activity in mutant neurons occurred later than that observed in the familial lines 38B/E, possibly due to different genetic backgrounds." Alternatively, there are changes in cell survival, aggregation, electrode contact, or distribution that have not been assessed. It is premature to raise the issue of genetic background as explanation. Furthermore, the authors seem to be mistaken, reporting there is "lower resting membrane potential," but that is not what is shown in Figure S5 where the mean values appear identical; please correct this results error.Subsection “EHMT2CNTN5 Isogenic Pair to Control for Genetic Background Contribution” section of the Results: “However, the wMFR of line 19-2 increased up to nearly 3 Hz at week 8  while the CNTN5 family controls stayed around 0.5 Hz . In this context of a more active cell line, we extended the recordings until week 11, and the hyperactive wMFR of 19-2-CNTN5StopTag/+ was only evident from week 10 .”We agree that it is probably premature to raise the issue of genetic background as explanation. Accordingly, we have modified “possibly due to different genetic backgrounds" for “possibly due to the more active 19-2 line”. As now explained earlier in the second paragraph of the “Moreover, based on our response to one of the other points raised above by this reviewer, we think that the issue of cell survival and electrode contact is not a major contributor to the different time window observed in the activity in lines 38B/E and 19-2. We apologize for the confusion regarding the lower “resting membrane potential”, we have now changed it for lower “input resistance” in the main text.The discussion fails to mention any limitations to the approach of MEA at 6 weeks, the absence of cell characterization, and possible alternative mechanisms to the currently pre-conceived notions of the gene's function. The authors might in fact suggest that a full time course analysis would be beneficial. They fail to discuss the very different behavior of retrovirally created 19-2 line used at 9-12 weeks that show outcomes not observed in the Sendai virus created lines assessed at 6 weeks. They fail to address the possibility of explanatory mechanisms in their studies such as changes in cell survival, composition, distribution, or electrode contact. In all, these studies seem preliminary in nature and set a stage for further analysis before we recommend this specific set of procedures to be adopted by the field.In the new version of the manuscript, we present a time course of the wMFR and MFR from week 4 to 8 PNI, which addresses the reviewer’s concern about the use of only week 6 as a timepoint. We now discuss the different behavior of line 19-2 at the end of the second paragraph in the Discussion. Moreover, we discuss some limitations of using MEA as a primary read-out, e.g., “It is possible that some phenotypes were missed since we cannot exclude the possibility that differences in cell number or composition across individuals in a family actually masked potential phenotypes.” We also discuss that since the familial controls are often not very active, this screen might be biased towards the identification of hyperactive phenotypes rather than hypoactive. Better MEA controls will need to be developed in the future.Additional data files and statistical comments:A major feature of a cell line may be that in one case only 20-30% of electrodes have signal due to differences in cell survival or dispersion, whereas in another line, it may routinely be 80-100% coverage. If this is the case, this type of information may impact the kinds of cellular mechanisms that one considers to explain the outcomes. This kind of information should be assessed, if not already done, and should be reported transparently in a Table that includes all families, perhaps in the supplement. One might find a correlation of decreasing/increasing cell-electrode contact (appearing as active/inactive electrodes/well) to the outcomes reported.This point was addressed above.[Editors’ note: further revisions were suggested before publication.]· We added a few new words to the Abstract (it now slightly exceeds 150 words). This additional text was added to address the reviewer's suggestion about the 75% of lines without detectable phenotypes. If you need to have the Abstract at 150 words please just remove the additions (the previous presentation was fine with us too);· We made a few new modifications to the 4th paragraph of the Discussion to respond to the reviewer's first two requests;· We clarified the nomenclature of Figure 2–figure supplement 1, and modified the corresponding legend;· We modified the last box of the transparent reporting file to include the new Source data files that were recently uploaded;· We brought more precision on the type of multiple t-test comparisons that were used in the legend of Figures 3-6;· Regarding the last request of the reviewer, we acknowledge that we did not completely resolve whether deficits (or phenotypes) in CNTN5 and EHMT2 result from impaired maturation rather than impaired synaptic function (we will never be able to do everything), however, we feel that we have already explained this extensively in the 5th paragraph of the Discussion.


TP53 mutations and its downstream pathway is usually intact. This study assessed the efficacy of the p53 activator, PRIMA-1MET, in inducing neuroblastoma cell death.Neuroblastoma is the most common extracranial solid tumor in children. This cancer has a low frequency of MET. Real-time PCR and western blot were used to assess the most common p53 transactivation targets. Induction of p53 and Noxa, and inhibition of Cas3/7, were used to assess impact on cell death after PRIMA-1MET treatment. Flow cytometry was used to analyze cell cycle phase and induction of apoptosis, reactive oxygen species, and the collapse of mitochondrial membrane potential.CellTiter 2.0 was used to study susceptibility and specificity of NB cell lines to PRIMA-1MET than were primary fibroblasts and keratinocyte cell lines. PRIMA-1MET induced cell death rapidly and in all cell cycle phases. Although PRIMA-1MET activated p53 transactivation activity, p53’s role is likely limited because its main targets remained unaffected, whereas pan-caspase inhibitor demonstrated no ability to prevent cell death. PRIMA-1MET induced oxidative stress and modulated the methionine/cysteine/glutathione axis. Variations of MYCN and p53 modulated intracellular levels of GSH and resulted in increased/decreased sensitivity of PRIMA-1MET. PRIMA-1MET inhibited thioredoxin reductase, but the effect of PRIMA-1MET was not altered by thioredoxin inhibition.Neuroblastoma cell lines were at least four times more susceptible to PRIMA-1MET could be a promising new agent to treat neuroblastoma because it demonstrated good anti-tumor action. Although p53 is involved in PRIMA-1MET-mediated cell death, our results suggest that direct interaction with p53 has a limited role in neuroblastoma but rather acts through modulation of GSH levels.PRIMA-1The online version of this article (10.1186/s13046-019-1066-6) contains supplementary material, which is available to authorized users. MYCN amplification (MNA) [TP53, and some estimations contend that almost all cancers evolve a way to circumvent the p53 pathway [TP53 [TP53 mutation, which dramatically reduces the efficacy of Nutlin-3 [Neuroblastoma (NB) is the most common extracranial solid tumor in children. Current event-free survival rates range from 75% to more than 85% for low- and very low-risk groups, to less than 50% for high-risk patients despite dose-intensive therapy , 2. NB pon (MNA)  and 11q on (MNA) . NB show pathway . NB has ay [TP53 , 11. Theay [TP53 . Nutlin-ay [TP53 . In vivoay [TP53 . Clinicaay [TP53 . In addiNutlin-3 .MET, is currently being investigated in several early-stage adult clinical trials . In vivo, PRIMA-1MET is converted into the active compound methylene quinuclidinone (MQ), which reacts with the thiol group of cysteine in proteins. Studies by Lambert et al demonstrated that PRIMA-1MET binds to p53, thus restoring p53 function by refolding the protein in its native structure [MET on adenocarcinoma and non-small cell lung cancer [MET induced death was not always p53 dependent. Different off-target effects involving ROS toxicity or autophagy were reported .MET in NB cell lines and to explore the roles of p53, MYCN, glutathione (GSH) and thioredoxin (TXN) systems in PRIMA-1MET efficacy and cellular response to PRIMA-1MET.This study aimed to evaluate the efficacy of PRIMA-1MET , pan-caspase inhibitor Z-VAD-FMK , etoposide , glutathione (100 mM in H2O), JQ-1 (1 mM in H2O), L-cysteine (50 mM in H2O), L-homocysteine (100 mM in H2O), L-methionine (12 g/L in H2O), N-acetyl-cysteine (100 mM in H2O), orlistat (20 mM in DMSO) , buthionine sulfoximine . A stably expressing p53 LA1–55 N cell line was obtained by transfection of pcDNA3, containing the p53 gene, using X-tremeGENE HP DNA Transfection Reagent  and subsequent selection with G418  according to the manufacturer’s recommendations. Concentrations of PRIMA-1MET were adjusted for each cell line to obtain precise 50% inhibitory concentration (IC50) values. Vehicle control was used in all experiments.The NB cell lines CHP212, LAN6, NBL-S, NGP, SK-N-DZ and SK-N-SH were provided by Dr. E. Attiyeh and Prof. J. Maris . The CLB-GA NB cell line was provided by Dr. V. Combaret . BE-(2)C, LA1–55 N, and SK-N-DZ were purchased from ATCC (USA). All NB cell lines were maintained in a standard NB medium composed of DMEM supplemented with 10% FBS, 1% antibiotic/antimycotic solution, and 1% L-glutamine. All NB cell lines passed identity and mycoplasma testing performed independently by Microsynth AG (Switzerland). Human normal primary keratinocytes and fibroblasts  were maintained in a dermal cell basal medium supplemented with keratinocyte growth kit and low serum fibroblast basal medium, respectively, prepared according to the manufacturer’s recommendations . LCL  were maintained in RPMI 1640 supplemented with 10% FBS and 1% antibiotic/antimycotic solution according to manufacturer’s recommendations. The following compounds were used: PRIMA-1MET for 24 h in 100 μL of medium. Plates were incubated for 2 min on a horizontal shaker at medium speed and at room temperature. Chemiluminescence was measured using Victor3  for 1 s, after shaking at medium speed and without the use of filters. Real-time cell viability after treatment with PRIMA-1MET or etoposide and inhibition of caspase by pan-caspase inhibitor was followed using RealTime-Glo MT Cell Viability Assay  according to the manufacturer’s recommendations. Chemiluminescence was measured using SpectraMax iD3  every 10 min for 22 h at 37 °C.CellTiter 2.0  was used according to the manufacturer’s recommendations to measure the number of live cells. 100 μL of CellTiter 2.0 was added to each well containing 7500 cells treated with PRIMA-1CyAn ADP flow cytometer  was used with the following kits according to manufacturers’ recommendations: FITC Annexin V Apoptosis Detection Kit I , CellROX Oxidative Stress Reagent , Autophagy detection kit  and mitochondrial JC-1 dye . Cell cycle, induction of caspase 3 and 7 (Cas3/7), and cellular membrane stability were investigated simultaneously using the FORTESSA flow cytometer system . Results were analyzed using Kaluza software .MET. The following primary mouse antibodies were used for protein labelling. Abcam (UK): Anti-ATM (ab-78), Anti-TXNRD1 (ab16847), Anti-Bax (ab77566) and Anti-Actinβ (ab6276). Cell Signaling Technology (USA): Phospho-p53 (Ser-15) (16G8) (9286), Phospho-ATM (Ser1981) (4526). Santa Cruz (USA): Anti-p53 (sc-126), Anti-p21 . Antibody specificity was assessed using WB as described in our previous publication [The intracellular concentration of proteins was measured using CellTag 700 Stain and In-Cell Western (ICW) Assay Kit 1  according to the manufacturer’s recommendations, 6 h after exposure to 60 μM PRIMA-1lication . Etoposilication , was usep21, 14–3-3 (YWHAQ), GADD45, PUMA (BBC3), Bax, MDM2, Noxa (PMAIP1), CXCR4, and NMYC. GAPDH, EEF1A1, and TBP were used as a normalization control . The pRL-SV40 Renilla luciferase control reporter vector  was used as a normalization control at a 1:10 weight ratio. Plasmids were transfected into SK-N-SH cells using X-tremeGENE HP DNA Transfection Reagent  according to manufacturer’s recommendations. The Dual-Luciferase Reporter Assay System  and Victor3  were used to measure luciferase activity (5 s shaking at medium speed prior to 1 s measurement time without the use of filters).pGL10.4  was used to study the impact of PRIMA-1The concentration of total intracellular glutathione , cysteine (Cysteine Assay Kit (Fluorometric), Abcam, UK), S-adenozyl-homocysteine (S-Adenosylhomocysteine (SAH) ELISA Combo Kit, Cell Biolabs, USA), glutathione transferase activity (Glutathione S-Transferase (GST) Assay Kit, Sigma Aldrich, USA) and the activity of thioredoxin reductase (Thioredoxin Reductase (TrxR) Assay Kit, Abcam, UK) was measured according to the manufacturer’s recommendations. The Victor3 reader and Versamax microplate reader  were used to measure luminescence and absorbance (412 nm), respectively.ALK and TP53 variants. Affymetrix expression data from GSE3960 were normalized using Robust Multichip Average (RMA) and was used to investigate the association between MNA and expression of genes in the TXN and GSH pathways.The Illumina HiSeq 2500 sequencer  was used for whole exome sequencing according to manufacturer’s recommendations. Illumina paired-end reads were aligned to the human UCSC hg19 reference sequence using BWA software (version 0.7.10). Single-nucleotide variants were called with GATK (version 3.4.46) and annotated with Annovar (version 2016 Feb 01) and SnpEff (version 4.3). Ingenuity variant analysis  was used to predict the number of cancer driver mutations and to search for p-values were used to determine significant differences in expression levels between MNA and non-MNA samples. A t-test was used to determine association of MNA and difference in expression of GCLM versus GCLC genes. A two-way t-test was used to calculate statistical significance for cell-based experiments. Robust nonlinear fit of dose vs. response method  was used to calculate the IC50 from relative cell survival, as determined by using CellTiter2.0. The coefficient of drug interaction (CDI) was calculated according to the following formula: CDI = AB/(AxB), where AB represents the relative cell viability for the combination of drugs and A and B represent the relative viability for each compound alone. CDI less than 0.8 was considered indicative of synergy. CDI between 0.8 and 1.2 as additive, while CDI more than 1.2 indicated antagonism [ANOVA tagonism . ActivitMYCN amplification and 11q-deletion were detected in four cell lines each. These two features overlap in the cell lines NGP and SK-N-DZ, which were found to have both MNA and 11q-deletion. MNA and 11q-deletion were assessed using real-time PCR . Mutation R110L, found in SK-N-DZ, has been reported in patients with Li-Fraumeni syndrome and results in high expression and abnormal localization of p53, as well as impairment of proper p53 oligomerization [TP53 mutations found in the NGP cell line, A159D (COSM11496) and C141W (COSM44204), are both deleterious mutations leading to amino acid substitution. No loss of heterozygosity at the TP53 locus was detected in NGP. This last result suggests that the NGP cell line has no gross deletions at this locus, but it is unknown whether both alleles are inactivated. BE-2C carries the previously described homozygous C135F mutation and has a loss of chromosome 17 at the site of TP53 [TP53 variants, MYCN was found to be amplified ; BE-2C was resistant to high PRIMA-1MET concentrations (58.8 + − 10.72 μM). Next, the cell lines were compared for IC50 and presence of 11q-deletion and MNA, the two most prominent molecular events leading to highly aggressive clinical behavior. No significant difference in IC50 was observed in the subgroupings by these features. However, the two most resistant cell lines (BE-2C and CHP212) have MNA, while the two most sensitive (CLB-GA and SK-N-SH) have normal MYCN expression . A 20 μM concentration of PRIMA-1MET was used to induce partial cell death, allowing us to analyze the cell cycle after a 24-h exposure. The results show that PRIMA-1MET induces the accumulation of cells in the sub-G1 phase  without apparent side effects [MET does not induce phosphorylation of either ATM or p53 . Investigating the induction of reactive oxidative species (ROS) after PRIMA-1MET treatment in the NGP cell line revealed an increased presence of ROS  was demonstrated between IC50 and PRIMA-1MET-induced Noxa expression, suggesting that together with mitochondrial dysfunction  or 1.3-fold (p < 0.026) decrease in activity when the p53 or CREB site is mutated, respectively  and CREB-mutated  plasmids, but no significant difference in cells with plasmids containing a p53 binding site mutation  and no change was observed in SK-N-SH cells bearing plasmid with p53 binding site mutation   of GSH concentration was observed in all eight of the NB cell lines  but increased cysteine concentrations (1.27-fold at 11 h), whereas the cysteine/SAH ratio had increased by 1.56-fold at 1 h to 1.92-fold at 11 h  more GSH . To investigate how GSH levels differ between MNA and non-MNA NB, correlation between MYCN status and gene expression of the main enzymes involved in GSH synthesis was analyzed using the Affymetrix expression array dataset consisting of 101 NB primary tumors (GEO accession: GSE3960). The results demonstrate that expression of CBS and GSS, genes involved in cystathionine and glutathione synthesis, are 2.97 times (p < 0.001) and 1.25 times (p < 0.001) higher, respectively, in MNA than in non-MNA tumors  is 1.3 times higher (p < 0.05) than GCLM expression, but there was no difference in non-MNA NB tumors  were also investigated in order to understand the potential influence of MNA on GSH, but no significant differences were detected between MNA and non-MNA NB tumors (data not shown). Finally, to explore the interaction between PRIMA-1MET and the glutathione-S-transferase (GST) system, we measured overall GST activity in the eight NB cell lines. No significant change was observed after PRIMA-1MET treatment  and sensitized p53-null LA1–55 N cells to higher doses of PRIMA-1MET  increase in IC50 , and increased susceptibility to higher doses of PRIMA-1MET . Recently, Peng et al demonstrated that, in lung adenocarcinoma, osteosarcoma, and Burkitt lymphoma cells, PRIMA-1MET can also target thioredoxin reductase 1 (TXNRD1) by binding to its active site, causing a decrease in reductase activity. Interestingly, it was demonstrated that even when inhibited by PRIMA-1MET, TXNRD1 is still functional as a pro-oxidant NADPH oxidase. This kind of modulation of TXNRD1 can cause an increase in oxidative stress and cell death, but implications for NB cell survival are questionable [MET might influence the TXN system in NB cells, we first used ICW to measure intracellular TXN levels. The results demonstrate that PRIMA-1MET treatment induces no change in total TXN levels in NB cell lines  and 1.22-fold (p = 0.02) upregulation in respective expression of TXN and TXN2 in NB tumoral samples. However, no difference in TXNRD1 expression was observed between MNA and non-MNA NB tumors . Finally, to address the possible role of the TXN system in resistance to PRIMA-1MET, cells were treated concurrently with PX-12 (a known TXN inhibitor) and PRIMA-1MET, and survival was analyzed. The results showed an additive effect of PX-12 and PRIMA-1MET, but no synergy was observed in any of the eight NB cell lines tested (data not shown). PX-12 alone at 5 μM exhibited the ability to inhibit growth in all NB cell lines, particularly those with MNA   at 60 μM and in two additional cell lines  at 120 μM Additional file 2:Supplementary information for figures. (PPTX 6460 kb)


Culicoides vector multiple veterinary pathogens and are difficult to control. Endosymbionts particularly Wolbachia pipientis may offer an alternative to control populations of Culicoides and/or impact disease transmission in the form of population suppression or replacement strategies.Biting midges of the genus Culicoides sonorensis cell lines were transfected with a Wolbachia infection using a modified shell vial technique. Infections were confirmed using PCR and cell localization using fluorescent in situ hybridization (FISH). The stability of Wolbachia infections and density was determined by qPCR. qPCR was also used to examine immune genes in the IMD, Toll and JACK/STAT pathways to determine if Wolbachia were associated with an immune response in infected cells.Culicoides sonorensis cell lines (W3 and W8) with a Wolbachia infection  from donor Aedes albopictus Aa23 cells. PCR and FISH showed the presence of Wolbachia infections in both C. sonorensis cell lines. Infection densities were higher in the W8 cell lines when compared to W3. In stably infected cells, genes in the immune Toll, IMD and JAK/STAT pathways were upregulated, along with Attacin and an Attacin-like anti-microbial peptides.Here we have transfected two Wolbachia infections in C. sonorensis cell lines and the upregulation of immune genes, suggest the utility of using Wolbachia for a population replacement and/or population suppression approach to limit the transmission of C. sonorensis vectored diseases. Results support the further investigation of Wolbachia induced pathogen inhibitory effects in Wolbachia-infected C. sonorensis cell lines and the introduction of Wolbachia into C. sonorensis adults via embryonic microinjection to examine for reproductive phenotypes and host fitness effects of a novel Wolbachia infection.The successful introduction of  Culicoides species are small hematophagous insects that have been shown to harbor more than 50 different viruses of veterinary and medical importance [portance . These vportance , 2. Multportance , 4. One portance –10 and tportance . Worldwiportance .Culicoides-vectored arborvirus and zoonotic diseases have limited methods of treatment and prevention and rely on inadequate forms of vector control to combat the spread of the disease [Culicoides are focused on treating livestock with topical insecticides at livestock production facilities and farms, but are typically met with limited success, depending on the Culicoides species targeted [Culicoides species, specifically immature habitat selection, making the effective application of insecticides to control immatures difficult [Culicoides near livestock, but is limited to use in areas near livestock production. The combination of larvicide and adulticidal treatments have also demonstrated some success, but the true efficiency of this type of control has not been assessed, and this type of treatment typically does not reduce the numbers of Culicoides adults, if only treating around a farm property [Culicoides-transmitted viruses. Active virus vaccines are available for BTV serotypes, but are limited in effectiveness due to the large number of serotypes and the potential for genome segment re-assortment of the BTV [Culicoides species. disease , 13, 14.targeted , 13, 14.ifficult . Habitatproperty , 17. Vac the BTV . Inactiv the BTV , 20. TheWolbachia pipientis may offer an alternative environmentally friendly control measure for Culicoides midges and the pathogens they vector. Wolbachia is an obligate intracellular bacterium found in > 55% of insects, as well as filarial nematodes and terrestrial crustaceans [Wolbachia causes alterations in host reproduction, with several phenotypes including feminization, parthenogenesis, male killing and cytoplasmic incompatibility (CI) [Wolbachia has been used as a strategy for mosquito suppression and disease control and has become a topic of global relevance [Wolbachia-based strategies are currently being implemented in the field for mosquito and disease control. The first is a Wolbachia incompatible insect technique (IIT) approach based on mass inundative releases of incompatible male mosquitoes similar to the Sterile Insect Technique (SIT), with the goal of suppressing natural populations through sterile mattings [Wolbachia interfere with viruses and other microbes in the same host [Wolbachia variants (e.g. the wMel strain) can block dengue virus transmission without impacting Aedes aegypti fitness [Wolbachia has also been shown to impact chikungunya virus, Zika virus and the yellow fever virus in their mosquito host [Wolbachia-infected females can mate and produce viable offspring with infected and uninfected males alike, and infected males when mated with uninfected female produce non-viable offspring, the resulting reproductive advantage of Wolbachia infected individuals can drive a given disease refractory phenotype into a natural population limiting disease transmission.staceans , 22. In ity (CI) . Recentlelevance , 25. Twomattings , 27. Theame host –32. Part fitness . In addiito host –32. BecaWolbachia has been limited to disease vector mosquitoes, presumably because of the immediate need for novel tools for hard to control mosquito species such as Ae. aegypti and Aedes albopictus, that are primary and secondary vectors of dengue and Zika viruses, respectively. The success of the recent field applications of Wolbachia control techniques in mosquitoes suggests the potential for the transition of this technology to other insect disease vectors of human and zoological importance. Wolbachia infections have recently been confirmed in multiple populations and species of Culicoides midges in Europe and Australia [Wolbachia infection types into uninfected Culicoides species is possible. Furthermore, the existence of natural infections suggests that reproductive phenotypes such as cytoplasmic incompatibility could exist in natural populations that harbor Wolbachia infections. Here, we demonstrate that Wolbachia infections can be introduced into Culicoides sonorensis cell lines as an initial step towards the investigation of a Wolbachia based control strategy for Culicoides midges. Recent studies have suggested that Wolbachia infections from a donor host can be pre-adapted to a target host cell lines in order to facilitate the adaptation for survival in a novel host [Wolbachia in Culicoides sonorensis cell culture may be a precursor to the successful germ line transfection of C. sonorensis. Furthermore, we use transfected cell lines to investigate a host immune response associated with a Wolbachia infection, which could suggest a reduced ability of Wolbachia infected C. sonorensis to transmit viral pathogens.Biological control using ustralia , 34, sugvel host . The sucAe. albopictus cell lines were cultured in 75 cm2 cell culture flasks  in Schneider’s insect medium (SM)  supplemented with 10% fetal bovine serum (FBS)  in SM (24.5 g/l) supplemented with 0.4 g/l sodium bicarbonate, 0.0585 g/l l-glutatmine, 0.006 g/l reduced glutathione, 0.03 g/l l-Asparagine, 18 µl of 10 mg/l Bovine insulin and 5% FBS  and subjected to sucrose gradient centrifugation (200 mM sucrose solution) at 17,000× g for 10 min at 4 °C. The resulting pellet was re-suspended in SM with 10% FBS and passed through a 2.7-μm syringe filter to collect extracellular Wolbachia. To examine for contamination of extracts with Aa23 cells, environmental bacteria, and/or fungi, a portion of the extract was added to Schneider’s media with 10% FBS in a 25-cm2 flask and incubated at 28 °C with a CO2 concentration of 0.2%.The escribed . The adhWolbachia was applied to W8 or W3 cells. The shell vial was centrifuged at 2500× g for 40 min at 20 °C and the cells transferred to a 25-cm2 flask with 5 ml of fresh media. In an attempt to increase Wolbachia infection rates, a second shell vial experiment was performed on the previously transfected W8-w and W3-w cells (2×) .Infection of W8 and W3 cell lines was carried out using a modified shell vial technique . Briefly2×) Fig. a. One daWolbachia infection was successfully isolated from the Aa23 cells in passage 1 and to confirm establishment in W3-w and W8-w cell lines, DNA was isolated from passage 1, 6 and 14 of 2× Wolbachia infected W3-w, W8-w and uninfected W3 and W8 and Aa23 cells using a Qiagen DNeasy Blood and Tissue Kit . For detection of Wolbachia, a PCR assay that amplified a 438 bp 16S rRNA gene fragment was used with the specific primer set wspecF and wspecR [4)2SO4, and 0.025% Triton X-100, 0.25 mM MgCl2, 0.25 mM dNTPs, 0.5 mM primers, and 1 U of Taq DNA polymerase in a total volume of 25 µl. The PCR amplification protocol was 10 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 54 °C and 1 min at 72 °C, followed by a 10 min extension step at 72 °C using a T100 Thermocycler . A volume of 5–10 µl of each amplicon was separated on 1.5% agarose gel, stained with GelRed  and visualized under ultraviolet illumination.To confirm the in situ hybridization (FISH) was performed on the W8-w and W3-w and W8 and W3 cell lines to confirm the presence and absence of Wolbachia, respectively. For the FISH procedures, W8-w and W3-w cells were at passage 4 post-Wolbachia infection. Cells were grown to 90% confluency at 28 °C and 300 µl of the cells were added to an 8-well Nunc® Lab-Tek® Chamber slide system . The cells were incubated in the chambered wells overnight for ~ 15 h at 28 °C. Cells were fixed in 4% formaldehyde (in 1× PBS) for 40 min at room temperature (RT) and followed by two washes with 1× PBS-T. Next, the cells were pre-hybridized for ~ 2 h at RT. The pre-hybridization buffer consisted of 50% deionized formamide, 20% 20× sodium saline citrate (SSC) solution, 1% 50× Denhardt’s Reagent, 10% 1 mol dithriothreitol (DTT), 0.25 mg/ml t-RNA and 0.25 mg/ml poly(A). The pre-hybridization step was followed by an overnight hybridization (~ 18 h) at 37 °C in a moist environment with gentle shaking. The hybridization buffer consisted of pre-hybridization buffer supplemented with 200 mg/ml dextran sulfate, 250 mg/l salmon sperm DNA and Wolbachia specific probes (5′-/56-FAM/ AAT CCG GCC GAR CCG ACC C-3′); 5′-/56-FAM/ CTT CTG TGA GTA CCG TCA TTA TC-3′) [Fluorescence Wolbachia in W8-w and W3-w infected cell lines. DNA was extracted from Wolbachia infected cells using Qiagen DNeasy Kit. Wolbachia density was determined by amplifying a fragment of the Wolbachia wsp gene  following the manufacturerʼs guidelines. To determine the response of the immune pathways to the walbB infection in W8-w cell lines, qPCR was used to determine host gene expression of immune genes involved in the IMD, Toll and Jak/Stat pathways and anti-microbial peptides previously identified in C. sonorensis . Isolated RNA from W8-Wolbachia infected and uninfected Culicoides cells was determined by t-tests, with a significance level of P < 0.05.JMP software  was used for statistical analysis. Statistical significance of immune gene expression levels of Wolbachia transfection procedures, W8 and W3 cell lines were confirmed for absence of a natural Wolbachia infection  and 0.24 ± 0.12 (mean ± SD) for the W8-w and W3-w cell lines, respectively . W8-w infected cells had a significant increase in immune gene expression for STAT , PIAS , Caspar , Relish , Dorsal , Cactus , Attacin  and Attacin-like genes  when compared to W8 uninfected cells , Defensin  and Defensin-like  showed no significant difference in immune gene expression when compared to the uninfected W8 cells  is a notable advancement towards generating novel Wolbachia infections in adult midges.The generation of cell lines with novel sfection , 38. TheWolbachia infection densities in the W8-w and W3-w cell lines are lower than previously reported for Drosophila, mosquito and sand fly host cells [Wolbachia infection densities have been reported to result in pathogen blocking and cytoplasmic incompatibility [Wolbachia infections in native Culicoides spp. populations, and those that have, demonstrated that Culicoides species have low density Wolbachia infections in vivo and a low prevalence in natural populations [Wolbachia infections in Culicoides spp. collected in Australia was accomplished by using qPCR over less sensitive conventional PCR [Wolbachia infections have also been noted in Culicoides spp. collected in Spain, albeit at a low prevalence in populations, which could again be associated with a low Wolbachia density in adult host midges and a low detection level [Wolbachia observed in the W8-w and W3-w cell lines may be common to Culicoides. This observed result could be a function of the ability of Wolbachia to upregulate the immune system in its Culicoides host and inhibiting Wolbachia infection proliferation, or could be the result of environmental parameters such as temperature [Wolbachia strain [Wolbachia density in its insect host. It is currently unclear why the W8-w cell line is able to maintain a higher Wolbachia density than the W3-w cell line, but this observation could be related to predominant cell morphology and type of each culture. The W3 cell line is predominantly, epithelial-like cells that are firmly adherent [Wolbachia can infect multiple cell and tissue types at different rates and densities [The observed st cells , 43, 44.tibility , 45, 46,tibility , 48. Fewulations , 34. Detonal PCR . Wolbachperature –51, hostperature , 52, sexa strain , which hadherent . The W8 adherent . Previouensities .w and W3-w cell lines were infected with a walbB infection from donor Aa23 Ae. albopictus cells. The walbB infection type was an optimal candidate for C. sonorensis cell line transfection because: (i) the walbB infection has been shown to fall within the same B-clade as other Wolbachia infections reported in multiple Culicoides spp. collected in native populations in Spain [wAlbB infection has shown parasite and virus inhibitory effects in multiple insect vectors of disease. In Anopheles stephensi, walbB has been demonstrated to reduce Plasmodium parasite development [walbB has been demonstrated to suppress filarial worm loads and provide resistance to dengue virus transmission in Aedes polynesiensis [The W8-in Spain , suggestelopment , 56. Alsesiensis .Wolbachia insect systems, the mechanism of pathogen inhibition in Wolbachia infected insect hosts is not well understood [Wolbachia induced pathogen inhibition. The first is that Wolbachia primes the host immune system, so when the pathogen enters the insect host, invasion is inhibited. Second, Wolbachia is hypothesized to be competing for metabolic components such as amino acids and cholesterol with the host insect and the pathogen [Wolbachia replication is known to be dependent upon host cell cholesterol production and requires cholesterol-rich host membranes to form the vacuole surrounding each bacterium, which is hypothesized to lead to competition for cholesterol between Wolbachia and pathogens [w cell line. Unfortunately, knowledge of immune pathways in C. sonorensis is limited, but several immune genes have been identified and characterized in a previous study [C. sonorensis annotated genome (GenBank: GCA_900258525) allowed for the identification of genes in Toll, JAK/STAT and IMD pathways [Wolbachia can affect the C. sonorensis immune system pathways in different cascades. Perhaps, this priming of the immune system could have an effect on orbivirus proliferation in its Culicoides host. Previous transcriptome studies have demonstrated 165 genes, including genes in the Toll and IMD pathways and AMPs that were differentially expressed between vector competent or refractory C. sonorensis when challenged with a BTV infection [w and W3-w cell lines with the orbiviruses BTV, AHSV, SBV, and/or EHDV and determining whether a novel Wolbachia infection can induce virus inhibitory effect in C. sonorensis cells. Both the W3 and W8 cell lines have previously been shown to be susceptible to BTV and EHDV infection [While parasite and pathogen inhibitory effects have been reported in multiple derstood –60. Currpathogen , 62. Forathogens , 63. To us study . Furtherpathways . The uprnfection . If pathnfection .Wolbachia infection type on C. sonorensis cells. Future studies could also include the transfection of W8 and W3 cell lines with alternative Wolbachia infections such as wMel, which has also been demonstrated to provide virus inhibitory phenotypes in mosquitoes [Wolbachia and Culicoides host association facilitates a pathogen infection in Culicoides spp. Previous studies have demonstrated an increase in the ability of Culex tarsalis to transmit West Nile virus when infected with Wolbachia and an enhanced flavivirus infection rate in Ae. aegypti [Here we have only examined the effect of one squitoes , 67. It  aegypti , 69.w and W3-w cell lines will continue to be passaged and maintained, with the goal of adapting the walbB infection to C. sonorensis. Subsequently, the walbB Culicoides adapted infection can be extracted from the W8-w or W3-w cell line and used for future microinjection experiments with the goal of generating a walbB germ line infection in C. sonorensis adults. If a stable germ line maternally inherited Wolbachia infection can be attained in C. sonorensis adults, further work would be needed to ascertain whether novel Wolbachia infections have an effect on C. sonorensis fitness, any reproductive phenotypes such as CI associated with Wolbachia infections, or any orbiviruses inhibitory effects. Further investigation is also needed to develop microinjection protocols for introducing Wolbachia infections into C. sonorensis embryos. This future work would open up exciting possibilities to investigate whether Wolbachia-based approaches could be used as an additional tool to control C. sonorensis and other Culicoides species and as an additional tool to limit transmission of veterinary important orbiviruses.The W8-Wolbachia infection in C. sonorensis cells (W8 and W3 cell lines) and that the W8 Wolbachia infected cell line demonstrated an upregulation of the Toll, IMD, JACK/STAT pathways and the production of anti-microbial peptides. The results suggest the potential utility of Wolbachia-based approaches for vector control strategies and to limit disease transmission by C. sonorensis and other Culicoides species of veterinary importance. Further investigation is needed to introduce germline infections into C. sonorensis and examine for Wolbachia induced reproductive phenotypes and pathogen inhibitory effects.Here, we were able to establish a Additional file 1: Table S1. Primer sequences used to confirm Wolbachia infection status, determine Wolbachia density, and host immune pathway gene expression.


First, on p. 4583, line 7, the sentence that starts on this line erroneously referred to the radio-sensitive sensitive cell line, H128; this sentence should have read as follows: “The results demonstrated that peptidyl-prolyl cis-trans isomerase A pseudogene 43 (PPIAP43) transcription was increased 2-fold in cells irradiated with 2 Gy gamma radiation compared with unirradiated cells in pre-reported radio-sensitive sensitive cell lines H69, H146, H209 and H187. Secondly, the following sentence (“These cells shared 259 upregulated and 96 downregulated RNA transcripts following radiation.”) should not have been included in the Abstract. Lastly, further changes should have been made to the subsequent sentence regarding the cell lines described and the general sentence construction.Taken in its entirety, the Abstract should have appeared as follows (the pair of sentences containing changes are highlighted in bold):Abstract. Small cell lung cancer (SCLC) is a highly lethal disease. Although radiation therapy is effective for the majority of patients with SCLC, patient sensitivity to radiation varies. The lack of biomarkers impedes advances in targeting radiation-sensitive patients. In the present study, the changes in transcription patterns of SCLC cell lines were evaluated with or without 2 Gy gamma radiation. The results demonstrated that peptidyl-prolyl cis-trans isomerase A pseudogene 43 (PPIAP43) transcription was increased 2-fold in cells irradiated with 2 Gy gamma radiation compared with unirradiated cells in pre-reported radio-sensitive sensitive cell lines H69, H146, H209 and H187. PPIAP43 was not upregulated 2-fold following irradiation with 2 Gy gamma radiation compared with unirradiated cells in the pre-reported, less sensitive cell lines H526 and D153. The RNA transcript of PPIAP43 was aligned with the mRNA of peptidyl-prolyl cis-trans isomerase A (PPIA) at 2 sections  and the sequences were shown to be 96 and 94% similar, respectively. Therefore, PPIAP43 may act as a sponge for microRNAs which bind with the RNA of PPIA. Therefore, PPIAP43 RNA transcription may serve as a potential biomarker of radio-sensitivity of SCLC.The authors regret that these errors were not identified prior to the publication of this paper, and apologize to the readers for any confusion or inconvenience caused.