Delays in recognizing stroke during pre-hospital emergency medical system (EMS) care may affect triage and transport time to an appropriate stroke ready hospital and may preclude patients from receiving time dependent treatment. All EMS transports in a large urban area in the stroke belt were evaluated for transport destinations, triage and transport time and stroke recognition following distribution ofan educational training video to local EMS services.Following video training, local paramedics will improve stroke recognition and shorten triage and transport time to appropriate stroke centers of care.A training module (<10 min) containing a stroke triage scenario, instruction on the Cincinnati Prehospital Stroke Score (CPSS) and the Los Angeles Prehospital Stroke Score (LAPSS) and ‘where to transport’ stroke patients was distributed and viewed by 96 paramedics. Data was collected from February to October 2016. Stroke recognition was determined from one primary stroke center (PSC) hospital’s confirmation of EMS delivered patients (Site A). Yearly stroke recognition percentages of 44% from Site A in 2014 were used as baseline.A total of 34,833 emergency 911 response transports were made with a total of 502 (1.4%) suspected strokes identified by paramedics. Median [IQR] triage and transport time for stroke transports was 33 [2741] min. The PSC hospitals received a 5% increase in stroke transports and non-specific care facilities decreased by 7%. From 8,554 transports to site A (PSC) confirmed strokes totalled 107 transports with 139 suspected strokes by paramedics. Of these transports, 60 were correctly identified by paramedics . By the second month following training, recognition percentages increased from baseline to 64%. At five months, percentages of correct stroke identification had dropped to 36%.Video based training improved stroke recognition by an additional 19%, but continual monthly or quarterly training is recommended for maintenance of increased stroke recognition. Stroke is the third leading cause of mortality in the world . It is aPre-hospital notification by emergency medical services (EMS) has been associated with improved clinical assessment and higher rates of tPA delivery with reduced door- to-needle times in the hospital ,12. ThesHere we present the results over the nine-month study period to determine the training module’s efficacy.A review of de-identified data collected by three area Emergency Medical Systems Agencies in Central Arkansas, the Arkansas Stroke Registry (a Get-With-the-Guidelines registered program) from the Arkansas Health Department (ADH) and the University of Arkansas for Medical Sciences stroke program (Site A) was conducted. All data encompasses a review of de-identified transport data and hospital stroke confirmations from February 1, 2016 to October 31, 2016. The study was approved by the local Institutional Review Board as not human subject research.A video utilizing professional stroke actors, paramedics and an ambulance was made to simulate a real event scenario of a 911 call for stroke. During the call and throughout the simulations of examination, a stroke educator registered nurse discussed the need to decrease time to delivery and treatment, precise methods for triage and care provided in the ambulance. Methodology for both the Los Angeles Pre-Hospital Stroke Screen (LAPSS) and the Cincinnati Pre-Hospital Stroke Screen (CPSS) were presented and discussed. Also detailed during the video was the need for early notification to alert the hospital emergency department for arrival. A directional delivery map was presented during the video illustrating the location and names of the test county’s primary stroke centers (PSC) and the one acute stroke ready hospital (ASRH). There were no comments made on the use of lights and sirens (L&S) during transport. The ADH disseminated the video link and a survey. An incentive gift card was offered to encourage video training and was given after completing an opinion survey regarding the video training.To measure the impact of the video training on correct paramedic identification and management of ‘suspected’ strokes all transport data during the study period was collected for nine months following video release and survey return and then compared to pre-video release data that was collected a year prior to determine baseline stroke recognition rates and destination for stroke suspect transports according to hospital qualification in stroke care (non-specified care facilities (NSCF), ASRH or PSC) during the same months.To determine the number of ‘positive’ and ‘missed’ strokes by the paramedics, all 911 ground response EMS transports delivering to one PSC (Site A) were matched against hospital ED records of strokes. Transport data used to match hospital ED cases of stroke included de-identified EMS information such as date, ER ‘seen time’, patient race, gender and age. Paramedic experience was taken into account as Years of Service (YOS) which was collected and categorized into 5 groups: <1 year, 1 to 5 years, 5 to 10 years, 10 to 15 years and over 15 years of experience. The deidentified paramedic variables of years of service and reported suspected cases of stroke were evaluated based on correct and incorrect recognition.The paramedic’s baseline percentage of correct stroke identification was determined in a previous study which indicated that positive stroke recognition percentages were 44%. This percentage was used as a comparison for the subsequent months of positive percentages of stroke recognition.Triage time and transport time from the scene to hospital destination were obtained for all paramedic suspected stroke transports during the study period. Both time epochs were calculated as one ‘transport time’ in min [IQR] and compared by distance travelled and travel mode (L&S or none) during the study period.We calculated diagnostic sensitivity, specificity, accuracy and related measures across the different groups of paramedics YOS and overall sensitivity across time before and after video training.A total of 96 paramedics underwent the training with a 100% rate of reporting compliance. A total of 70 paramedics were from the test county with another 26 from surrounding areas. The survey results indicated that 9% of the paramedics had not previously performed the CPSS and 4% had not performed the LAPSS. Ninety-five percent of the paramedics thought the video would be helpful in demonstrating the use of both tests.There were 34,833 EMS transports in the Test County during the study period (excluding 373 interfacility transports) out of which 502 (1.4%) were suspected strokes. These ‘stroke suspects’ were either transported to the ASRH, NSCF, or to area PSCs at n=5 (1%), =142 (28%) and =355 (71%), respectively. The PSC hospitals received a 5% increase in stroke transports and the NSCFs decreased by 7%. Site A received a representative sample of 139 of suspected cases out of 8,554 total EMS transports (1.6%) and took care of approximately 107 stroke patients.Sixty paramedics transported suspected stroke patients to Site A during the study period. Their YOS are presented in The paramedics correctly identified stroke transports  with a sOverall combined triage and transport time for all stroke suspects was 33 min [IQR 27–41]. Data on median triage and transport time by vehicle territory is shown in The data indicates that transports inside the county area and metro areas had similar median times to destination hospitals. Some stroke transports used lights and sirens (L&S) during commute and influenced transport time as shown in vs. no L&S (None) transports (56.6 [IQR 36.7–58] vs. 62.4 [IQR 47–69] min).Vehicles originating from outside county areas (OCA) that used L&S had shorter median delivery times vs. None at 35.7 [IQR27.7–44] vs. 32.9 [IQR 29–43], respectively.Transports within the county area (ICA) did not decrease delivery time with L&S vs. None at 25.3 [IQR 20.2– 32] vs. 30 [IQR 25–35] min, respectively.Those transports using L&S in the metro county area (MCA) had an improvement Pre-hospital stroke assessment and notifications have been known to decrease the door to needle time, increase the rate of tPA delivery, and improve stroke outcomes –14. PrevFollowing stroke recognition training in this study, the dramatic 19% increase from baseline in sensitivity  was seenThe training may have also helped the paramedics in directing potential stroke patients to PSCs, which are better equipped at handling stroke cases. Although rates of endovascular interventions on these patients were not studied, this might be important to analyze in future studies. Rates of endovascular intervention could be compared to large vessel occlusion detection scores learned from video-based training in prehospital screening and triage. The use of L&S added an additional variable on triage and transport time . The medFalling stroke recognition rates , suggestSince the overall percentage of stroke patients assessed by paramedics in our study was very low (1.4%), the paramedics may be less adept at recognizing stroke cases as compared to areas yielding a higher stroke volume . The vid


Deafness is a highly heterogenous disorder with over 100 genes known to underlie human non-syndromic hearing impairment. However, many more remain undiscovered, particularly those involved in the most common form of deafness: adult-onset progressive hearing loss. Despite several genome-wide association studies of adult hearing status, it remains unclear whether the genetic architecture of this common sensory loss consists of multiple rare variants each with large effect size or many common susceptibility variants each with small to medium effects. As next generation sequencing is now being utilised in clinical diagnosis, our aim was to explore the viability of diagnosing the genetic cause of hearing loss using whole exome sequencing in individual subjects as in a clinical setting.We performed exome sequencing of thirty patients selected for distinct phenotypic sub-types from well-characterised cohorts of 1479 people with adult-onset hearing loss.NEDD4 and NEFH that were mutated in multiple individuals.Every individual carried predicted pathogenic variants in at least ten deafness-associated genes; similar findings were obtained from an analysis of the 1000 Genomes Project data unselected for hearing status. We have identified putative causal variants in known deafness genes and several novel candidate genes, including The high frequency of predicted-pathogenic variants detected in known deafness-associated genes was unexpected and has significant implications for current diagnostic sequencing in deafness. Our findings suggest that in a clinic setting, efforts should be made to a) confirm key sequence results by Sanger sequencing, b) assess segregations of variants and phenotypes within the family if at all possible, and c) use caution in applying current pathogenicity prediction algorithms for diagnostic purposes. We conclude that there may be a high number of pathogenic variants affecting hearing in the ageing population, including many in known deafness-associated genes. Our findings of frequent predicted-pathogenic variants in both our hearing-impaired sample and in the larger 1000 Genomes Project sample unselected for auditory function suggests that the reference population for interpreting variants for this very common disorder should be a population of people with good hearing for their age rather than an unselected population.The online version of this article (10.1186/s12920-018-0395-1) contains supplementary material, which is available to authorized users. Hearing loss is one of the most common sensory deficits in the human population, and it has a strong genetic component . HoweverSIK3 and ESRRG [GRM7 [PCDH20 and SLC28A3 [ISG20 or ACAN and TRIOBP [Although several ARHL genome-wide association studies (GWAS) have been carried out –11 and pnd ESRRG , 11, onlRG [GRM7 , PCDH20  SLC28A3 , and ISGd TRIOBP . Furtherd TRIOBP , 16. ThuHere, we have taken a more in-depth approach than GWAS, using whole-exome sequencing (WES) to study thirty patients carefully selected from a total sample of 1479 patients with a variety of adult-onset hearing loss phenotypes to represent the mixed phenotypes and varied genetic aetiology that might be present in a clinical scenario, targeting specific sub-phenotypes to maximise power to detect shared variants. Our aim was to establish to what extent exome sequencing is an effective and appropriate tool for genetic diagnosis of hearing loss in a clinic setting, where there is usually only a single adult patient involved and family members are not available for segregation analysis. Whole exome and genome sequencing are beginning to be used in this scenario for diagnosis of adult-onset hearing loss with the clinician faced with challenges in evaluating the candidate variants identified. Our results demonstrate the value of targeting well-characterised phenotypic subtypes and cross-species data comparison in exome sequencing analysis, and highlight issues which need to be considered in interpreting genetic variants of unknown pathogenicity in current genetic diagnosis and gene discovery studies, in particular the finding that many individuals have multiple predicted-pathogenic variants in different genes known to underlie deafness.Twenty patients  with non-syndromic sensorineural adult-onset hearing loss (self-reported age of onset between 20 and 50) were selected from a larger group of 700 patients recruited from the adult hearing aid clinic at the Royal National Throat Nose and Ear Hospital, London, U.K. (described in ). The twA second group of 10 older individuals were selected from the 779 people in the database of the longitudinal study of ARHL being conducted at the Medical University of South Carolina (MUSC) since 1987 (described in [All patients were recruited by written informed consent.DNA was submitted for WES using either the Agilent SureSelect Human All Exon V3 kit or a custom library designed by Agilent for human whole exome sequence capture (which predates the SureSelect kit). Sequencing was carried out on either the Illumina Genome Analyzer IIx or Illumina HiSeq 2000 platform as paired-end 54 bp, 75 bp or 76 bp reads according to the manufacturer’s protocol.The data were filtered on quality, Minor Allele Frequency (MAF) in the ExAC non-Finnish European population , most seCandidate variants were re-sequenced using Sanger sequencing (Source Bioscience). Primers were designed using Primer3   were analysed to find genes common within each sub-group.n = 33, Additional file We first examined known deafness genes in all thirty people together and found that every person had at least ten known deafness genes with one or more predicted pathogenic variants Fig. . As thisall the predictors used instead of the majority of predictors. The distributions were again similar for all groups of people and sets of disease-associated genes with both MAF filters , and found the same pattern  bore variants in all ten individuals with recessive hearing loss, MON1B was mutated in all five individuals with metabolic deafness (but see later), and the five people with sensory hearing loss all had variants in NEDD4 and ZAN  present in all members of each sub-group. No known deafness genes were common to any of the sub-groups. We found four other such genes: Our common variant analysis has highlighted several potential problems with detecting common variants of small effect size in a common disorder. Although each individual had many deafness genes with predicted pathogenic variants, the overall spread didn’t look any different to that observed in the 1000 Genomes dataset  Fig. 2)2). HowevLPP (chr3:g.188327555 T > C).Another factor to consider is whether variants which are rare in the most relevant ethnic populations and passed the MAF filter but are more prevalent in other populations should be retained; 1234 of the 10,482 unique variants in the thirty patients which passed all our filtering steps were present in other ExAC populations at a MAF of more than 10%. To give one extreme example, it is worth considering whether a variant which is present in 60% of the African ExAC population should be included even if it is very rare (0.3%) in the non-Finnish European population, as for a T > C missense variant in SIRPA (a candidate for exclusion) and ZAN  . No indiGPR98. One individual from the dominant group had five predicted variants in WFS1  linked to adult hearing ability or ARHL in GWAS [GRM7, a missense variant in SIK3, and three missense variants in ACAN, each present in one individual except for one of the variants in ACAN, which is present in two people  encoding a G protein coupled receptor, and frameshift mutations in this gene cause one form of Usher’s syndrome, USH2C, an autosomal recessive disorder [Details of the 9 predicted variants in disorder  causing WFS1 in individual 2590 were confirmed by resequencing. Mutations in WFS1 can cause either a dominantly-inherited non-syndromic hearing loss typically affecting the low frequencies (below 2 kHz) or Wolfram Syndrome (OMIM #222300), a recessive neurodegenerative disease which can include mental retardation, childhood diabetes, optic atrophy and deafness which typically is progressive and affects the high frequencies [https://lovd.euro-wabb.org/home.php?select_db=WFS1).All five predicted variants in quencies . The famOMIM #22200, a recquencies , 38. IndGRM7 and one with a SIK3 SNV , which has only one isoform. The 18 bp duplication is predicted to duplicate six amino acids in a low complexity region. The missense variant results in a substitution of lysine, which is positively charged, for glutamate, which is negatively charged, which may affect protein function, but without functional studies it is hard to predict what difference either variant would make to the function of the protein.NEFH, the neurofilament heavy chain, is expressed in the rat cochlear nucleus  and the on Table . Both vaZAN were found in patients from each subgroup, all in the heterozygote state except for three dominant patients who were homozygous for a 35 bp deletion (g.7:100385563_100385597del).ZAN is a multiple-domain transmembrane protein found on the apical region of the sperm head , which fDNAH2 is an axonemal dynein heavy chain, about which very little is known. Six missense variants, one intronic splice site SNV and one single base pair insertion were predicted in seven patients (MAF < 0.5%), and six further missense variants with a MAF < 10% were predicted in 5 patients, of which we have confirmed one missense variant present in the heterozygous state in two patients from the dominant and recessive subgroups.Nedd4 and Zan were detected during this period. Nedd4 is expressed at reasonably high levels in sensory cells, and only slightly lower levels in non-sensory cells, in accordance with previous publications [Zan has very little expression in the hair cells but is strongly expressed in the supporting cells at P0 [Nefh is expressed in the neurons under the inner hair cells [Dnah2 is expressed in the cochlear duct at P0, but not as strongly as Zan or Nedd4.We examined the expression of our novel candidate deafness genes using the gEAR portal , which dications . Zan hasls at P0 . Nefh isir cells , and is ir cells . Dnah2 iNedd4, Zan, and Nefh, but the only mouse line with a variant in Dnah2 has a large inversion on chromosome 11 covering 1898 genes. No hearing phenotype has been reported for any of the mutant alleles of these genes , but thNEFH and NEDD4. It is perhaps surprising that our relatively small sample size of thirty individuals was able to identify good candidate variants and genes. By targeting our approach to specific phenotypic subtypes within large, well-characterised patient cohorts based on audiogram shape, family history and similar age of onset, we have sought to increase power to detect causal genetic variants. In addition, by cross-referencing our data in the filtering pipeline with data from the mouse we were able to prioritise the strongest candidate variants.Our analysis has identified several candidate variants and genes for involvement in adult-onset progressive hearing loss, in particular variants in NEDD4 and NEFH, should be followed up in larger cohorts and by functional studies to confirm whether they are causal mutations.The two strongest candidates from our analysis, the variants in Our study also highlights the potential pitfalls of using targeted sequencing to diagnose the cause of adult-onset hearing loss in a typical clinical scenario, where relatives are not available for segregation analysis especially on a gene by gene basis. Of the 30 individuals WES was only able to identify the likely causal mutation in one individual with five WFS1 variants. For the remaining patients the variants identified are of uncertain pathogenicity without further validation. Interpretation of these variants in single individuals is extremely challenging given that even when we limited our analysis to dominantly-inherited human deafness genes, 2334 of the 2504 individuals in the 1000 Genomes Project data carried at least one predicted pathogenic variant in a known dominant human deafness gene. These findings reveal the need for allele frequency databases from carefully-selected controls with good hearing for their age rather than the existing unselected general population controls in studies of highly-prevalent disorders such as hearing loss. Here, we have explored the use of WES in undiagnosed individuals, based on our results it would be interesting to pursue a similar study in individuals who have received a genetic diagnosis to ascertain the number of other predicted pathogenic variants that are present in deafness genes. Furthermore, our study demonstrates that confirmation of candidate variants by Sanger sequencing is always a necessary step. Our findings further suggest that for diagnostics in a clinical situation, novel candidate variants identified by sequencing should be investigated by family analysis if possible, to look for segregation. Without these additional steps, our data suggest that it is not possible to determine with confidence the causative mutation responsible for a patient’s hearing impairment from exome sequence alone. Even with these secondary steps great caution should be exercised in interpreting predicted disease-causing variants, given our findings of the incidence of such variants in every genome.Additional file 1:Figure S1. showing the audiograms of each participant in the recessive patient group. (PDF 486 kb)Additional file 2:Figure S2. showing the audiograms of each participant in the dominant patient group. (PDF 485 kb)Additional file 3:Figure S3. showing the audiograms of each participant in the metabolic and sensory patient groups. (PDF 428 kb)Additional file 4:Table S1. which lists the software prediction tools used to call and annotate variants, with relevant references. (XLS 30 kb)Additional file 5:Figure S4. and legend detailing counts at each filtering step for common variant analysis. (PDF 259 kb)Additional file 6:Table S2. detailing primers used for confirming selected variants. (DOCX 20 kb)Additional file 7:Table S3. listing genes reported to underlie deafness in humans and/or mice. (XLS 45 kb)Additional file 8:Table S4. and lists candidates for exclusion - genes with predicted pathogenic variants in many exome projects or across multiple families exhibiting a variety of phenotypes. This list consists of the genes in Supplementary Tables S1 and S2 from [ S2 from . Additional file 11:Table S7. listing individuals with more than one very rare mutation in the same gene. (DOCX 20 kb)Additional file 12:Table S8. giving details of the single very rare variants in known deafness genes found in multiple samples. (DOCX 16 kb)Additional file 13:Table S9. giving details of genes with identical variants found in more than one person. (DOCX 16 kb)Additional file 14:Table S10. detailing very rare mutations in GWAS candidates. (DOCX 20 kb)Additional file 15:Table S11. which contains further details of the validated variants listed in Table Additional file 16:Table S12. giving details of very rare mutations identified in GPR98. (DOCX 19 kb)


Dysfunction or death of retinal pigment epithelial (RPE) cells is involved in some forms of Retinitis Pigmentosa and in age-related macular degeneration (AMD). Since there is no cure for most patients affected by these diseases, the transplantation of RPE cells derived from human pluripotent stem cells (hPSCs) represents an attractive therapeutic alternative. First attempts to transplant hPSC-RPE cells in AMD and Stargardt patients demonstrated the safety and suggested the potential efficacy of this strategy. However, it also highlighted the need to upscale the production of the cells to be grafted in order to treat the millions of potential patients. Automated cell culture systems are necessary to change the scale of cell production. In the present study, we developed a protocol amenable for automation that combines in a sequential manner Nicotinamide, Activin A and CHIR99021 to direct the differentiation of hPSCs into RPE cells. This novel differentiation protocol associated with the use of cell culture robots open new possibilities for the production of large batches of hPSC-RPE cells while maintaining a high cell purity and functionality. Such methodology of cell culture automation could therefore be applied to various differentiation processes in order to generate the material suitable for cell therapy. RPE cells are altered in 5–6% of Retinitis Pigmentosa cases  and in Age-related Macular Degeneration (AMD)3. AMD is the leading cause of blindness in developed countries with more than 150 million people affected worldwide, a figure that will increase in the coming years4. It can be classified into two groups, dry (atrophic) or wet (exudative), which is based on the presence of a choroidal neovascularization. While the understanding of the molecular mechanisms underlying wet AMD, which accounts for approximately 10–15% of AMD patients, led to the development of effective anti-VEGF drugs, there is still no treatment for dry AMD and for most of RPs6. As such, the transplantation of RPE cells derived from human pluripotent stem cells (hPSC-RPE) represents an attractive strategy for treating retinal degenerative diseases8.Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are characterized by unlimited self-renewal and their ability to differentiate into any cell type. Due to these properties, extensive efforts have been done to use them as a source material for cell therapy to repair damaged tissues. At the forefront of cell therapy, the replacement of retinal pigment epithelium (RPE), the monolayer of pigmented cells localized between the neural retina and the choroid, acts as a proof of concept. RPE cells play crucial roles in sight and their dysfunction or their loss may engender the secondary loss of photoreceptors11. The distinctive cobblestone morphology of RPE cells as well as their pigmentation allow to manually collect pigmented areas that appear upon differentiation of hPSCs to obtain a pure population of hPSC-RPE cells. Such approach of RPE cell production is used as cell replacement material in on going and planned clinical trials16. However, this spontaneous method remains fastidious, inefficient and time consuming (8 to 12 weeks of hPSCs differentiation) making it incompatible with the industrial large-scale production which is required to treat the potential millions of patients.hPSCs spontaneously differentiate into RPE cells after removal of basic fibroblast growth factor (bFGF), used to maintain the pluripotency state, from the culture medium20. One of the quickest and most efficient protocol was published by Leach et al. in 201522. Following data demonstrating that RPE and neural retina progenitors (NRPs) have the same embryonic origin, they combined a protocol allowing the efficient differentiation of NRPs23 with previously described RPE inducing factors such as Nicotinamide (NIC) and Activin A24, a member of the TGF-β super family. Using this method, they obtained a large majority of cells expressing the pigmentation marker PMEL17 after 14 days of differentiation allowing bypassing manual enrichment of pigmented cells. It is, however, important to note that even with this protocol the banking of mature RPE cells is only performed after several additional weeks of maturation (Day 84)22.During the last ten years, several teams have developed improved differentiation protocols by combining the use of an increasing number of cytokines and small molecules selected on the basis of results obtained from developmental studies26 and the quality of hPSCs and the efficiency of their differentiation into RPE cells are currently highly dependent on technical skills. In this regard, automation should not only allow scaling up the production of hPSC-RPE cells but should also increase its robustness. It could enable larger and more reliable cell production for clinical and disease modeling applications. Robots, such as Cellhost system or CompacT SelecT system, as well as appropriate programing were already developed for the amplification of hPSCs. Differentiation into mesenchymal and neural stem cells has been also implemented in such systems32. Until recently, the requirement of a manual enrichment to obtain a pure population of hPSC-RPE cells prevented the use of these automated systems for the differentiation of this cell type. However, the recent development of protocols allowing efficient differentiation of hPSCs into RPE cells offers now the possibility to automate the production of these cells.Therefore, although the differentiation of hPSCs into RPE cells became more efficient during the last years, it still remains a long and laborious process requiring meticulous manipulations from hPSCs thawing to hPSC- RPE cell banking. Many cell culture parameters, such as seeding homogeneity, the time spent by the cells out of the incubator or the method used to isolate pigmented clumps, could impact on the proliferation and the differentiation of hPSCs. Thus, manual processing implies operator to operator variabilityThus, our aim was to develop a fully automated process allowing the large-scale production of hPSC-RPE cells. Considering that, in addition to considerably complicate the process, the use of numerous growth factors and small molecules on a large scale should be very expensive, especially for an automated process which requires significant dead volumes, we implemented a simplified RPE differentiation protocol amenable for automation that only requires the treatment of hPSCs with NIC, Activin A and CHIR99021, an activator of the Wnt canonical pathway, in a sequential manner. Our protocol recapitulates the main steps of retinal development and is sufficient to obtain a pure population of RPE cells without manual enrichment. We then programmed a culture robot to automate this protocol in order to upscale the production process. 16 billion of mature and functional RPE cells could now be produced within 12 weeks with only one round of production. Such efficient and reproducible automated protocol should be useful for the treatment of the millions of patients affected by RPE associated retinal degeneration.In an effort to simplify previous directed differentiation protocols for automation, we evaluated whether the simple use of NIC, Activin A and Chir99021 in a sequential manner (referred as “directed protocol”) improves RPE cell differentiation of adherent hESCs enough to bypass manual enrichment. We compared the efficiency of our “directed protocol” with the one of the classical spontaneous differentiation.SIX3) and Retinal homeobox (RAX) concomitantly to a higher decrease of the expression of the pluripotency marker NANOG at mRNA level when compared to the spontaneous protocol  and the melanocyte inducing transcription factor (MITF), when compared to the spontaneous differentiation . Immunostaining assays confirmed the absence of VSX2 positive cells at day 21 and the increased number of MITF+ cells (87.5% ± 12.5%)  Fig. . At this5%) Fig. .11  was covered by pigmented cells on day 42 Fig. . By cont11 Fig. . Importa11 Fig. .Figure 2Taken together our results indicate that the sequential use of Nicotinamide, Activin A and Chir99021 recapitulates the main steps of retinal development and efficiently directs the differentiation of hPSCs into a highly-enriched RPE population within 42 days compared to the spontaneous differentiation. Thus, cell differentiated through the directed protocol could be amplified directly while a prior manual selection of RPE clusters is required for the spontaneous protocol.Using the “directed differentiation” protocol, we set up a fully automated process by performing media changes and enzymatic passaging using the CompacT SelecT automation platform. This automated cell culture platform is composed of an incubator, bar-coded flasks for cell process tracking, multiple connected pumps to dispense culture media, a six-axis anthropomorphic robotic arm and a live-cell imaging system (Incucyte) Figs  and S2.F2 flasks. Then, cell proliferation and differentiation initiation by medium switching were performed in the robot until day 42. At this stage, hESC-RPE cells form a cohesive epithelium in culture that requires long incubation times with dissociation reagents to trigger cell detachment for further replating and amplification. In order to eliminate a maximum of cell contaminants we took advantage of this characteristic by performing a differential dissociation treatment with TrypLE Express  of cells co-expressed the two transcription factors PAX6 and MITF Fig.  indicaticol Fig. . We furtcol Fig. .Figure 4bona fide hESC-RPE cells in an automated system with a quality similar to the cells obtained through the widely used spontaneous differentiation method.All together these data demonstrate that we were able to obtain pure Important issues concerning cells differentiated from hPSCs are their maturity and functionality. As an indicator of epithelial maturity, we evaluated the apico-basal polarization of specific RPE markers. As expected, hESC-RPE cells homogeneously expressed the microvilli protein EZRIN , the tight junction marker Zonula Occludens-1  and the MER proto-oncogene tyrosine kinase receptor  at their apical membrane while the calcium activated chloride channel, BESTROPHIN  was localized at the baso-lateral compartment Figs  and S4.FOne of the most important functions of RPE cells is the phagocytosis of the outer segments shed by the photoreceptors. To determine whether the cells differentiated according to our directed protocol on the automated cell culture platform were functional, we assessed their ability to phagocyte pig fluorescein isothiocyanate (FITC)–labeled photoreceptor outer segments and quantified the fluorescence signal of pH-sensitive particles that become fluorescent after cell entry and phagosome formation. hESC-RPE cells were able to phagocyte FITC-labeled photoreceptor outer segments as shown by the cytoplasmic localization of the FITC signal under the apical limit Ezrin positive Fig. . hESC-RPin vitro.All these results indicate that RPE cells differentiated from hPSCs using a fully automated protocol are functional 36. In line with these studies, hESC-RPE cells obtained with an automated process adopted a mesenchymal phenotype starting from passage 4  and FIBRONECTIN 1 (FN1), two extracellular matrix proteins, starting at passage 5 when compared to passage 3 and 4 e 4 Fig.  despite EST Fig. . Indeed,EST Fig. . This mi24. Previous works described that the sequential use of NIC and Activin A allowed increasing embryonic body commitment into retinal lineage. In another hand, Leach et al. showed that the same factors, in addition to many others including bFGF, Noggin, DKK1 , Insulin Growth Factor (IGF)-1, Chir99021, B27 and N2 supplements, increased RPE cell differentiation of hPSCs21. We demonstrated in this study that most of these cytokines and supplements were not essential to trigger an efficient and pure RPE cell differentiation. Indeed, the use of only 3 compounds  in a sequential manner allowed obtaining a pure population of RPE cells without 3-dimensional culture and manual dissection of pigmented foci during the differentiation process. This optimized differentiation is thus amenable to automation.Since the publication of the first protocol showing that hESCs spontaneously differentiate into RPE cells, extensive efforts have been made to reduce the duration and increase the efficiency of differentiation protocols24, the use of Nicotinamide quickly reduced the expression of pluripotent markers, suggesting the rapid exit from pluripotent state, while increasing eye field genes expression in treated cells. In this study, Activin A increased both expressions of the neural retina marker VSX2 and the RPE marker MITF. These two markers are initially co-expressed by the same cells at day 10 suggesting that at this stage the cells could be bipotent37 and able to give rise to both RPE and neural retina progenitors. But quickly, two subpopulations of cells expressing either one of these two genes appeared. This result is consistent with the known role of VSX2 on MITF repression during optic vesicle patterning33. The increased VSX2 expression following Activin A treatment is however surprising as it is known to induce RPE specification rather than neural retina38. This result could be due to the endogenous expression of bFGF that positively regulates the expression of VSX2. The simultaneous use of a FGF inhibitor, such as SU5402, already used in a previously described protocol, may be necessary at this stage to repress neural retina markers39. The presence of cells expressing only VSX2, meaning that they are potentially engaged into the neural retinal lineage, could be a concern at this stage. However, the later use of the small molecule CHIR99021 rapidly leads to the acute decrease of VSX2 expression concomitantly with an increased expression of MITF, indicating that the activation of the Wnt/β-catenin pathway at this stage might induce the death of VSX2 positive cells or their conversion into RPE precursors. CHIR99021 treatment could also induce the differentiation of VSX2 positive cells into other mature lineages that are further eliminated by the differential dissociation.As previously describedFoltz et al.22 where cells are banked at day 84, we bank hESC-RPE cells with the same timing (D84). Thus, the removal of factors other than NIC, Activin A and CHIR99021, used in Foltz and collaborators study did not delay the overall production of hPSC-RPE cells nor affected the quality of the cells obtained.The “directed protocol” developed in this study enabled us to obtain 87.5% of RPE precursors that expressed MITF by day 21. However, cells were maintained further in culture in order to allow more maturation. Once a cohesive epithelium is formed at day 42, cells were passaged. This cohesive epithelium is suitable to a differential dissociation that eliminates the last cell contaminants without manual enrichment. Compared with the protocol published by 40 that range from 0.05 to 0.8 × 109 cells, and could be produced by a single operator supervising the robot. Moreover, the use of HYPERflask (Corning) with a growth area of 1720 cm2 (compared to 75 cm2 flask used in this study) could even dramatically increase the number of cells produced per batch. Another way to increase the size of the bank would be to delay the EMT. Indeed, the number of passages without EMT might be extended as previously described by the addition of a ROCK inhibitor in the culture medium35.Using the automated differentiation process described in this study, it is theoretically possible to produce about 16 billion of hESC-RPE cells at passage 2 per batch Fig. . A bank 42 or a polarized epithelium resting on a synthetic basement membrane43. The use of cell suspension formulation considerably simplifies the logistical and surgical procedures but several studies made in animal models, including our own, suggest that the survival of the RPE cells and the visual benefits for the animal are improved when the cells are grafted as an epithelial tissue rather than a cell suspension44. In human, these two approaches have shown both satisfactory safety results and promising efficacy results, even if the extent and the causes of visual improvement in transplant recipients remain ambiguous16. Nevertheless, considering that 1 × 105 hESC-RPE cells are currently used to graft a human eye with the both methods43, the automated process presented here should allow to produce enough cells to treat several thousands of patients with retinal degeneration even if some steps of the production, such as the simultaneous banking of a huge numbers of cryovials, remains challenging. The automated culture system could be qualified for clinical cell productions, as actually already done by others45. However, before reaching a clinical application, the raw materials used with this automated protocol might be adjusted to comply with Good Manufacturing Practice (GMP) guidelines.hPSC-RPE cells have been already grafted in AMD patients either as a cell suspension46, we described a fully automated RPE cell differentiation process from the hPSCs thawing to the banking of differentiated cells. Such automated process is a step towards the scale up and the industrialization of RPE differentiation that will be necessary to treat large numbers of patients. Finally, any differentiation protocol that doesn’t require 3D culture or manual selection could theoretically be adapted to this automated culture system opening new perspectives concerning the scale up and the industrialization of the production of many cell types differentiated from hPSCs.In conclusion, following the previously published amplification of hPSCs using CompacT SelecT automate47 was used and cultured in feeder free conditions using mTeSR™1 Medium (StemCell technologies) and hESC qualified Matrigel (Corning)  supplemented with 50 μM β-mercaptoethanol, 1× minimum essential medium–nonessential amino acids (Thermo Fisher Scientific) and 20% (D0-D42) or 4% (after passage 1) of knockout serum replacement . During all the differentiation process the medium was changed every 2/3 days.Clinical-grade hESC line RC-09ng) Fig. . Cells w25. Briefly, hESCs were grown to confluence and switched to a bFGF deprived culture medium. Pigmented patches were then dissected under a stereomicroscope with a fine 15° ophthalmic knife and plated onto culture dishes coated with hESC qualified Matrigel (corning).hESC-RPE cells were obtained by spontaneous differentiation of hESCs as previously describedFor the referred “directed differentiation” protocol, 10 mM Nicotinamide (Sigma), 100 ng/ml Activin A (Peprotech) and 3 µM CHIR99021 (Tocris) were added sequentially to the basal differentiation medium at specific time points Fig. . On day Mature hESC-RPE cells were dissociated and cryopreserved in liquid nitrogen vapors with CryoStor CS10 medium (StemCell technologies) at passage 1 or 2.The CompacT SelecT (Sartorius) is a fully automated cell culture platform which allows the expansion and differentiation of large batches of adherent cells in a controlled environment Fig. . The sysAutomated medium changes consisted in (i) retrieving T-flask from the incubator, removing flask cap and pouring media into a waste funnel, (ii) adding the appropriate culture medium using the peristaltic pump system (“dispensing” step), and (iii) replacing T-flask in the flask carousel incubator. Automated passaging consisted in step (i) described above and (ii) add PBS, (iii) swirl flask with the robot arm to ensure an even coverage of PBS across the cell sheet to wash cells, (iv) empty flask, (v) add TrypLE Express, swirl flask and incubate at 37 °C for 45 min, (vi) add fresh medium to dilute the dissociation reagent, (vii) shake flask from side-to-side at a given speed a number of times to dissociate the cell sheet into single cells or small clumps (“shaking” step), (vii) seed the cell suspension into new T-flasks with a split ratio of 1:3 or 1:5, swirl and replace in the incubator (“seeding” step). Step (v) was modified for the first cell passaging at the end of the differentiation (P0) to perform a two-step enzymatic dissociation procedure to enrich the culture for RPE cells by: (i) incubating the cells for 10 min with TrypLE Express, (ii) gently shaking the flask and pouring the dissociation agent into a waste funnel to remove non-RPE cells, (iii) incubating the cells for 35 min with TrypLE Express .−ΔΔCt.Total RNAs were extracted using RNAeasy Plus Mini kit (Qiagen) and cDNA synthesized using SuperScript III (Invitrogen). Quantitative real-time RT-PCR was performed using a Quant Studio 12 K flex (Applied Biosystems) with HiGreen qPCR Master Mix (Thermo Fisher Scientific). Primer sequences are listed in Supplementary Table hESC-RPE cells were grown on Matrigel-coated 96 or 24-well plates. Adherent cells were fixed in 4% PFA for 10 min at room temperature (RT) and rinsed 3 times with PBS. After 30 min in blocking solution (10% FBS in 0.1% Triton PBS) at RT, cells were incubated with primary antibodies overnight at 4 °C (Antibodies are listed in Supplementary Table Images were acquired with an Axio observer Z1 microscope (Zeiss) with a Hamamatsu ORCA-flash 4.0 camera and a spinning disk unit  with Metamorph software or with a LSM-800 confocal microscope (Zeiss) with Zen software. Images were exported, analyzed and processed with Fiji software. For zx images, xy stacks (0.33 μm z step size) covering cell width were resliced in zx. The quantification of pigmented areas was performed after manual delimitation of culture dish areas using Fiji software. Pictures were then binarized to 8-bit images using a fixed intensity threshold and the black area fraction was measured.Cells were detached from culture plates, fixed in 4% PFA for 10 min at RT and permeabilized with PBS containing 0.1% Triton for 30 min before labeling with TYRP1 antibody for 1 hr at RT. Labeling of the cell surface markers TRA-1-81 and SSEA4 was performed on freshly dissociated cells for 15 min at 4 °C. Cells were then incubated with fluorochrome-conjugated primary antibody for 30 min at RT and rinsed twice with PBS. The antibodies used and their working dilutions are listed in Supplementary Table hESC-RPE cells were exposed for 24 hours to purified FITC-labeled photoreceptor outer segments of pig (gift from Dr. E. Nandrot). After washing with PBS, cells were fixed in cold methanol and labelled with DAPI. Images were taken with LSM-800 confocal microscope (Zeiss). hESCs derived RPE cells were also exposed to pHrodo Green Zymosan Bioparticles (Thermo Fisher Scientific) overnight at 37 °C. These particles are pH-sensitive and become fluorescent after cell entry and phagosome formation. As a negative control, phagocytosis assays were performed at 4 °C to block the phagocytic process. Plates were then read using a microplate reader (Clariostar-BMG LABTECH) and values were normalized to DAPI intensities.VEGF measurements were done in triplicate using the human VEGF Quantikine ELISA kit (R&D System) according to manufacturer instruction.All experiments were performed in triplicate. Summary statistical analyses were performed in XLSTAT software. Comparisons between experiments were performed using the unpaired t-test and statistical significance was established as *p < 0.05, **p < 0.01.Supplementary informations


Background: Irritable bowel syndrome (IBS) is characterized by visceral pain and abnormal bowel habits that are worsened during stress. Evidence also suggests altered intestinal barrier function in IBS. Previously, we demonstrated that stereotaxic application of the stress hormone corticosterone (CORT) onto the central nucleus of the amygdala (CeA) induces colonic hyperalgesia and anxiety-like behavior in a rat model, however the effect on intestinal permeability and mucosal function remain to be evaluated.Methods: Male Fischer 344 rats underwent bilateral stereotaxic implantation of CORT or inert cholesterol (CHOL)-containing micropellets (30 μg) onto the dorsal margin of the CeA. Seven days later, colonic tissue was isolated to assess tissue permeability in modified Ussing chambers via transepithelial electrical resistance (TEER) and macromolecular flux of horseradish peroxidase (HRP). Secretory responses to electrical field stimulation (EFS) of submucosal enteric nerves as well as activation with forskolin were used to assess movements of ions across the isolated colonic tissues. In a separate cohort, colonic histology, and mast cell infiltration was assessed.Key Results: Compared to CHOL-implanted controls, we determined that exposing the CeA to elevated levels of CORT significantly increased macromolecular flux across the colonic epithelial layer without changing TEER. Nerve-mediated but not cAMP-mediated active transport was inhibited in response to elevated amygdala CORT. There were no histological changes or increases in mast cell infiltration within colonic tissue from CORT treated animals.Conclusion and Inferences: These observations support a novel role for the CeA as a modulator of nerve-mediated colonic epithelial function. Irritable bowel syndrome (IBS) is a common gastrointestinal (GI) disorder associated with chronic abdominal pain accompanied by a change in stool frequency or consistency, and a 2:1 female predominance Chang, . AlthougVisceral hypersensitivity characterized by hyperalgesia and allodynia has been measured in patients with IBS  or tight (colon), depending on its ability to transport fluid and electrolytes. Sustaining gut barrier function and mucosal permeability are tight junction (TJ) proteins, which connect adjacent epithelial cells and regulate the paracellular movement of substances across the small and large intestinal mucosa. This paracellular movement of gut contents between the epithelial cells is therefore dependent on the composition of the TJ proteins found between cells, which include various combinations of zonula occludens (ZOs), occludins, claudins, and junctional adhesion molecules. Additionally, a layer of mucus covering the mucosal epithelium represents an “unstirred” layer and barrier to movement of contents between intestinal epithelial cells . The animal protocol was approved by the Institutional Animal Care and Use Committee at the Oklahoma City VA Medical Center (1302-001) and the University of Oklahoma Health Sciences Center (13-028). After facility acclimation, all rats were randomized to treatment groups and underwent stereotaxic bilateral implantation of CORT or cholesterol (CHOL) control micropellets on the central nucleus of the amygdala (CeA) (Day 0). During recovery from the surgery all rats were single-housed. On day 7 after the surgery, rats were euthanized and colonic tissue was isolated for the measurement of transepithelial electrical resistance (TEER), macromolecular transport, active ion transport induced following nerve stimulation and/or the addition of forskolin. In a separate cohort of rats on day 7 after the stereotaxic bilateral implantation of CORT or CHOL micropellets on the CeA colonic tissue was isolated mast cell quantification and histology. Post mortem, the rat brain was isolated to confirm the accurate placement of the bilateral micropellet implant on the dorsal margin of the CeA  rats, 250–300g . F-344 rats were chosen because they have been demonstrated to possess low basal levels of anxiety  micropellets onto the dorsal surface of the CeA. Rats were anesthetized with a ketamine-xylazine cocktail administered intraperitoneally  and body temperature was monitored during the surgical procedure and maintained at 37°C using a homeothermic heating blanket . Animals were mounted in a stereotaxic frame and secured by both the ear and bite bars . A midline incision was made from the frontal region to the apex of the skull, and the epidermis and fascia were retracted. The stereotaxic frame was equipped with a digital coordinate system, which allowed for precise localization of the targeted brain region using skull landmarks for bregma and lamda. Following a midline incision, small holes were made in the skull at the coordinates 2.5 mm posterior to bregma and 4.2 mm to the right and the left of midline using the Paxinos-Watson stereotaxic atlas  were decapitated and colonic tissue was isolated and immediately immersed in ice cold oxygenated Krebs buffer containing (mM): 121 NaCl, 5.95 KCl, 1.19 MgCl2, 2.50 CaCl2, 1.34 NaH2PO4, 14.3 NaHCO3, 11.4 C6H12O6. Colonic tissue was opened along the mesenteric border and 1.5 cm segments of mucosa stripped of myenteric plexus and longitudinal muscular layers were mounted in four modified Ussing chambers  with an aperture of 0.6 cm2. Exposed surfaces of the epithelial segments were bathed in identical formulations of Krebs buffer, continuously perfused with 95% O2 and 5% CO2, while bath temperature was maintained at 37°C by a water heating circulator . All Krebs buffer chemicals were purchased from MilliporeSigma .Within the main laboratory, starting at 10:00 a.m., after preparation of Krebs buffer and equipment calibration, following anesthetization with isoflurane , rats  and short-circuit current (Isc) across the epithelial segment were monitored. If the chamber did not demonstrate a stable baseline Isc after the 30 min equilibration period, the tissue was replaced with another segment from the same animal  and the new chamber received an additional 30 min equilibration period. No electrophysiological measurements were conducted if a stable Isc could not be recorded. Two voltage sensing, agar-salt bridge electrodes on either side of the tissue monitored the PD across the tissue, while two Ag-AgCl current passing electrodes injected current into the system to maintain a PD of zero. PD and Isc were measured using an EVC 4000 Precision V/I clamp . Stable baseline values for PD and Isc confirmed viability of the tissue preparation. Transepithelial electrical resistance (TEER) across the epithelial layer was calculated from Ohm's law using measures of PD and Isc and corrected for the area of the aperture normalized to 1 cm2SO4 (25 μl) to stop the HRP-TMB reaction. Samples were read at 450 nm in a Dynex MRX colorimetric plate reader , using Revelation plate reading software . The concentration of HRP present in each sample was determined using a standard curve. During the 2-h sampling period six samples were collected for HRP analysis and the average HRP flux was expressed as ngHRP/hr/cm2.Macromolecular transport was assessed by measuring horseradish peroxidase (HRP) type II (~44 kDa),  flux from the mucosal to the serosal sides of the colonic epithelial layer. HRP in Krebs solution (0.8 mg/mL) was added to the mucosal bath of the Ussing chamber, and samples (0.25 mL) were taken from the submucosal bath at 20 min intervals for 2-h following equilibration. An equivalent volume of fresh Krebs buffer was added to the serosal bath to maintain equal hydrostatic pressure on either side of the mounted tissue. A colorimetric endpoint assay was used to quantify the amount of HRP contained in each sample. 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate (25 μl)  was incubated at room temperature with the HRP containing sample (1 μl) for 10 min followed by the addition of 0.1M HTo assess whether elevated amygdala CORT affected net active ion transport, electrical field stimulation (EFS) was used to induce nerve mediated increased in short-circuit current (Isc). With an electromotor force of 100 mV, electrical current  was passed through two foil electrodes situated on the serosal side of the mounted colon segment, connected to a Grass S88 Stimulator using a Grass SIU5 stimulus isolation unit . The maximum increase in Isc was compared with pre-stimulation baseline Isc to quantify the post-stimulation response at each protocol frequency. EFS data acquired using PowerLab 8 data acquisition system and LabChart7 Software . In a sub-set of tissues, forskolin (10 μM) was added bilaterally to the mucosal and serosal sides of the Ussing chamber. Forskolin is known to increase intracellular cAMP production and induce an increase in Isc across the epithelial cell layer  were decapitated and the colon tissue was isolated, opened along the mesenteric border, and immediately immersed in ice cold oxygenated Krebs buffer. 1.5 cm colonic segments were placed in 10% neutral buffered formalin at room temperature for histological evaluation.Within the main laboratory, starting at 10:00 a.m., following anesthetization with isoflurane , rats . Tissue segments were paraffin embedded and sliced at 5 μm using a rotary microtome and slides were cured at 37°C. Slides were deparaffinized in xylene (2 × 10 min) and successive treatments of EtOH  and then rehydrated in diH2O. Slides were stained with commercially available Hematoxylin solution  followed by Eosin counterstaining  followed by dehydration in EtOH . Tissues were visualized using a light microscope and scored for histological damage by an expert pathologist using the Chui score for mucosal damage . Slides were immersed in the working solution for 1 min then rinsed in diH2O 3 times. Slides were dehydrated in 95 and 100% EtOH (3 rapid immersions each) and cleared by 2 × 3 min in xylene. Tissue was visualized by light microscopy. Mast cell counts were quantified by a blinded observer by counting the number of mast cells visible in 10 high powered fields at 100 × magnification from the distal colon of 4 rats/treatment, with the final result expressed as the average number of mast cells per μm2.Colonic epithelial segments were evaluated for mast cell counts across all treatment and control groups. Slides were deparaffinized in successive treatments of xylene and EtOH as described above (see Hematoxylin/Eosin Staining) and stained using the standard toluidine blue mast cell staining procedure. Toluidine blue stock solution was prepared using 1 g toluidine blue-O  in 100 ml 70% EtOH. NaCl stock solution (1%) was prepared with 0.5 g NaCl:50 ml diH*p < 0.05, **p < 0.01) using an unpaired Student's t-test for parametric data or a Mann-Whitney test for nonparametric data. Results for TEER, Isc, macromolecular flux of HRP, and mast cell counts were evaluated in this manner. Data from electrical field stimulation were evaluated using a repeated measure 2-factor analysis of variance (ANOVA), with Bonferroni post-hoc testing (**p < 0.01). Graphpad Prism 7  was used for all analysis. Although the experimenter was not blinded to the experimental treatments, the histologist was blinded. While a power analysis was not conducted, n-values for this study were similar to our previously published reports using the same techniques  in baseline Isc across the distal colonic epithelium of CORT (31.78 ± 3.0 μA/cm2) and CHOL (33.31 ± 3.1 μA/cm2) implanted rats . In the same experimental preparation, we measured TEER and found that there was no significant change in TEER  across the colonic epithelium from CORT implanted rats compared to tissue from CHOL implanted controls  across the colonic epithelium of rats with amygdala CORT implants  compared to amygdala CHOL control animals . In all animals, we verified the correct localization CORT or CHOL micropellet on the dorsal margin of the CeA  = 37.75, p < 0.0001], a significant effect of micropellet implant , and a significant interaction between the terms  as demonstrated in colonic tissue isolated from CORT implanted rats  where there was a blunted Isc response to EFS at frequencies of 32 and 64 Hz compared to the Isc responses in colonic tissue from CHOL controls . In a separate cohort of experiments, we investigated whether forskolin (10 μM)-induced Isc was effected by elevated amygdala CORT. Addition of forskolin caused a rapid and significant increase in Isc, however there was no difference in the magnitude of the forskolin-induced increase in Isc  between tissue isolated from rats with CORT or CHOL micropellets implanted on the dorsal margin of the CeA    is the most predominant neurotransmitter in the ENS and in response to EFS the release of ACh stimulates chloride secretion from the colonic crypts measured electrophysiologically as an increase in Isc. In the current study, we found that in control CHOL implanted rats, EFS induced a frequency dependent increase in Isc; however, in rats with CORT-containing micropellets on the CeA there was a significant attenuation in the EFS-induced increase in Isc, which was in contrast to the lack of change in the EFS response following a chronic psychological stressor (Hattay et al., Important to the interpretation of our latest results is that changes in colonic epithelial transport occurred in the absence of changes in colonic histology. These data mirror IBS patient pathology where there is no obvious tissue damage to explain patient symptomatology including visceral pain and abnormal bowel habits (Kirsch and Riddell, Our study was not designed to mimic physiological concentrations of CORT in response to stress, rather an advantage of our experimental approach is that we are able to investigate the effect of amygdala-specific CORT activation on the integrity of colonic barrier function. The concentration of CORT was chosen based on our previous observations showing increases in colonic hypersensitivity in rats without spread of the CORT to other structures in the region of the amygdala (Myers and Greenwood-Van Meerveld, Taken together our findings support a role for the CeA as a key modulator of colonic epithelial function that may play a role in producing the previously observed increase in visceral hypersensitivity seen in response to elevated amygdala CORT (Greenwood-Van Meerveld et al., In summary, many studies have examined various aspects of stress-induced symptom exacerbation as it pertains to colonic barrier dysfunction in animal models of IBS. To our knowledge, we are the first focus on the role of the amygdala, specifically the CeA, in altering colonic barrier function and nerve-mediated active ion transport. Our findings support a role for the amygdala as a key central modulator of colonic epithelial function and integrity as it relates to gut health and the pathophysiology of functional bowel disorders such as IBS.PH and BG-VM conceptualized the experiments. PH and DP performed the experiments. PH, DP, and AJ analyzed the results. PH and BG-VM wrote the first draft of the manuscript. PH, DP, AJ, and BG-VM revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We tested whether a single nucleotide polymorphism (SNP) that affects splicing of CD33 predicted response to treatment in adults with acute myeloid leukemia (AML) who received the novel CD33 antibody-drug conjugate SGN-CD33A. This genotype, for the CD33 splice site SNP rs12459419, was not associated with clinical response (30% CR/CRi in both groups), event-free survival, or overall survival. CD33 [CD33 gene and affects the expression of the extracellular epitope recognized by GO. Variation from the common C allele to the rare T allele abrogates the splice site for inclusion of exon 2, which codes for the IgV domain of CD33; without the C allele, the exon is skipped during transcription. Thus, there is a plausible biological mechanism for altered response to GO. However, when Gale and colleagues performed a similar analysis in adult patients with AML treated with GO plus chemotherapy, they did not find an association between the SNP and outcomes [Antibody-drug conjugates (ADCs) are among the most promising immunotherapies developed in the last few decades for patients with acute myeloid leukemia (AML) . The CD3CD33 . This SNoutcomes , 10. Furn = 2) and the previously reported decreased response to anti-CD33 ADCs in patients carrying even one T risk allele [n = 10), and those carrying CT or TT genotypes (n = 10): both groups had 30% complete response (CR) or complete response with incomplete hematologic recovery (CRi). The genotype for the CD33 splice site SNP rs12459419 was also not associated with event-free survival or overall survival  or in combination with hypomethylating agents (10 mcg/kg SGN-CD33A and standard doses of hypomethylating agent) were tested for the CD33 SNP genotype rs12459419) using TaqMan SNP genotyping . Clinical characteristics of disease, prior treatments, and outcome data were collected and analyzed for association of the SNP genotype with response rate, the primary objective. Event-free and overall survivals were secondary objectives assessed by the Kaplan-Meier estimator. We included adults with de novo and secondary AML who had either experienced disease relapse or declined intensive chemotherapy. Much as would be expected at the population level, we saw a 50%/40%/10% distribution of genotypes CC, CT, and TT, respectively. The CT and TT genotypes were combined in our analysis because of the low numbers of TT genotype (419 usingCD33 SNPs are germline mutations, so these could result in different expression of CD33 in off-target tissue. An alternative hypothesis, therefore, is that increased toxicity in the CC genotype would offset the drugs’ benefit; however, neither our study nor the other adult study showed any difference in response rates between genotypes. This lack of any benefit for the CC genotype in adult AML suggests that age-related or other biological differences between adult and pediatric AML may explain disparate results between these groups.While limited by the small sample size of this study, our data show that genotype of the CD33 splice site SNP was not associated with outcomes of patients treated with an anti-CD33 drug conjugate, aligning with previously reported data for adult patients  and exteCD33 genotype is a poor biomarker for broad use in adults with AML to predict response to CD33-targeted ADCs. While these results are disappointing—because genotype is much more reliably measured, reported, and interpreted than CD33 expression by flow cytometry, another potential biomarker for anti-CD33 ADC response—larger studies may nonetheless show a benefit for genotype testing in specific patient subsets identified by age, disease risk, or mutational subtype.This study suggests that


There are no reliable biomarkers to predict thyroid eye disease (TED) in patients with autoimmune thyroid disease (AITD) currently. Several evidences support the involvement of the lacrimal gland in TED. The aim of our study was to quantitatively correlate the changes in tear protein profile with increasing severity of TED. Tear samples were collected from four groups of patients; AITD without TED (AITD), AITD with mild TED (mild TED), AITD with severe TED (severe TED) and normal controls. A total of 72 patients were recruited for the study. In discovery phase, isobaric tags for relative and absolute quantification (iTRAQ) 4-plex was used for quantitative proteomics analysis. For verification of results from discovery phase, sequential window acquisition of all theoretical fragment ion spectra (SWATH) was used to analyze an independent cohort from normal controls, AITD, mild TED and severe TED. Two proteins, S100A4 and PIP showed consistent dysregulation trends in the discovery and validation phase experiments. Our study demonstrated the differences in tear proteome across the spectrum of different severity and activity of TED in patients with AITD. Two tear proteins, S100A4 and PIP may serve as potential biomarkers to predict progression to severe TED in patients with AITD. Majority of the presentation is mild, with moderate to severe form affecting 5% of cases, which can progress to sight-threatening disease. Current literature suggests several risk factors for development of TED in patients with AITD, such as smoking and thyroid dysfunction2. However, there are no reliable biomarkers currently which could predict development of severe TED in patients with AITD. Consequently, if one could predict the patients in whom significant ocular complications would develop; closer monitoring and appropriate early intervention could be instituted in these susceptible individuals to avoid potential serious consequences.Thyroid Eye Disease (TED) is the most common extra-thyroidal manifestation of autoimmune thyroid disease (AITD)4. Potential tear biomarkers have been investigated in several ophthalmic diseases5, such as dry eye6, keratoconus7 and glaucoma8. Several evidences support the involvement of the lacrimal gland in the pathogenesis of ocular surface damage in TED. Eckstein et al. demonstrated that the acinar cells from lacrimal glands were positive for thyrotropin receptor (TSH receptor) immunoreactivity and thus susceptible to activation by TSH receptor autoantibodies9. Enlargement of lacrimal glands have also been reported in clinical and radiological studies in TED patients11. Recently, this increase in size has been found to be accompanied by changes in tear inflammatory cytokines in TED and correlated with disease inflammatory activity12.Tears are complex fluid comprising of secretions from several sources including the lacrimal gland, goblet cells, cornea and vascular sources. Tear composition reflects the physiological condition of the underlying tissues. Thus, tear fluid is useful in the evaluation of health and disease states and represents a valuable source of biomarkers for objective ocular and systemic diseasesHence, we postulated that tear protein profile can vary with different stages of TED. The aim of our study was to quantitatively correlate the changes in tear protein profile with severity and activity of TED. In this study, quantitative proteomics methods, which were iTRAQ (isobaric tags for relative and absolute quantification) technology coupled with two-dimensional nanoLC-nano-ESI-MS/MS and SWATH-MS , were used to discover and verify candidate biomarkers respectively.Human tear samples were collected from the following groups of patients: (1) AITD without TED (“group AITD”) (2) AITD with mild inactive TED  (3) AITD with severe active TED  and normal subjects without AITD (group “Ctrl”). Subjects were excluded from the study if they had pterygia, previous ocular surgeries or radiation, concomitant chronic ocular surface inflammation, already been treated with systemic immunosuppressants or had underlying systemic inflammatory conditions or malignancies. In addition, patients receiving medications that could affect tear film constitution  were also excluded.13, i.e. TED is present if eyelid retraction occurs in association with thyroid dysfunction, exophthalmos, optic nerve dysfunction or extraocular muscle involvement and other confounding causes such as idiopathic orbital inflammation are excluded. The subjects’ disease severity was assessed based on ITEDS VISA classification14. Briefly, this classification system is based on four disease end points: Vision, Inflammation, Strabismus, and Appearance/Exposure. Subjective input and reproducible objective measurements are recorded for each section, and a global severity grade can be assigned for each function. The subjective and objective progress and tempo of disease can be documented to reflect disease activity. Based on this classification, TED is considered active when the inflammatory score is more than 4/10; mild when only lid signs, proptosis and/or mild extraocular muscle restrictions are present, and severe if vision is affected because of compressive neuropathy, exposure keratopathy or disabling diplopia.A total of 54 subjects with AITD (46 with Graves’ disease and 8 with Hashimoto’s thyroiditis) and 18 control subjects without AITD were recruited from the outpatient clinics for the whole study. All the patients with TED were examined by an Ophthalmologist. The diagnosis of TED was based on diagnostic criteria defined by Bartley and GormanNone of the patients were treated with steroids, immunosuppressant (e.g. Methotrexate) or topical eye drops (e.g. steroid or lubricant eye drops) prior to recruitment for the study. Further information on subjects’ autoimmune thyroid disease duration, thyroid receptor antibodies levels (TRAb) and thyroid hormone levels measured at the time of recruitment (for patients with AITD only) or at the time disease presentation (for patients with TED) were collected from electronic medical records.For the discovery phase, eight patients with underlying AITD without TED , eight patients with mild inactive TED  and eight patients with severe active TED  were studied. Another eight healthy subjects  served as the normal control group (group “Ctrl”). There were no statistically significant differences in the age, duration of AITD, thyroid hormone levels or TSH receptor antibody titer between the groups in the discovery phase. A separate group of patients were recruited for the verification phase. This group consisted of ten patients with underlying AITD without TED , ten patients with mild inactive TED  and ten patients with severe active TED . Another ten healthy subjects  were recruited as normal controls. There were also no statistically significant differences in the age, thyroid hormone levels or TSH receptor antibody titer between the groups in the verification phase. The patients with severe TED had significantly shorter duration of AITD compared to patients with AITD only (p = 0.010) and mild TED (p = 0.029).Informed consent was obtained from all participating subjects and the study was approved by the SingHealth Centralized Institutional Review Board. The current study was performed in accordance with the Declaration of Helsinki. The clinical characteristics of subjects recruited in the discovery and validation stages are summarized in Table 15. No stimulation, anesthetic or eye drops were used before sample collection. External factors known to affect the content of tear samples, such as harsh lighting, background noise and extremes of room temperature were strictly avoided. After collection, Schirmer’s strips were immediately frozen at −80 °C until further analysis. The Schirmer’s strips were cut into small pieces and soaked in 100 µL of 50-mM ammonium bicarbonate  solution containing protease inhibitor  for three hours to elute tear proteins. Total tear protein concentration of each sample was measured using a Micro BCA Protein Assay Kit .Tear fluid was collected with gloved hands from each eye using Schirmer’s paper strips. These strips were inserted in the inferior conjunctival cul-de-sac (fornix) 1 cm from the lateral canthus for five minutes with eyes open and in a dim room. Special care was taken not to touch the eyelids or ocular surface to avoid eye irritation and contaminationThe overall experimental design is given in Fig. For each sample, 100 µg total protein amount was used. Briefly, protein samples were reduced using final concentration of 5 mM TCEP  and incubated for one hour at 60 °C. This was followed by alkylating the reduced protein sample with 15 mM IAA  for 20 minutes in the dark. The sample was then digested with trypsin  at a trypsin-to-substrate ratio of 1:50, at 37 °C overnight. Finally, the digested samples were reconstituted in dissolution buffer (0.5 µM TEAB) and labeled with iTRAQ reagents according to the experimental design in Fig. NanoLC-MS/MS [Ultimate 3000 nanoLC system  coupled with AB Sciex 5600 TripleTOF ] was used for the proteomic analysis. The LC separation is based on the reversed phase LC with an Acclaim PepMap RSLC C18 column . A step linear gradient of mobile phase B (2/98 v/v of water/ACN with 0.1% formic acid) from 5% to 12% over 30 min, 12–40% for 60 min, and lastly, 40–90% over 20 min at flow rate of 300 nL/min was used for the peptide separation. Two microliters of sample were injected for the analysis. Proteomics data was acquired using information-dependent acquisition (IDA) mode using Analyst TF 1.7 software . Data acquisition parameters were listed as follows, (1) TOF-MS survey scan: 250 ms; (2) mass range for TOF-MS: 400–1250; (3) product ion scan: 50 ms; (4) mass range for MS/MS: of 100–1500; (5) switching criteria: ions greater than m/z 400 and smaller than m/z 1250 with charge state of 2–5, maximum number of candidate ions to monitor per cycle was 40 spectra and an abundance threshold of >120 counts; former target ions were excluded for 12 s; (6) collision energy (CE): IDA Advanced “rolling collision energy”.The iTRAQ data was processed with ProteinPilot 5.0  using protein database version “uniprot_all_Oct2014”.To establish the human tear spectral library Fig. , a globaThe experimental conditions for nanoLC-MS/MS analysis were the same as the above.The LC set-up and experimental parameters are the same as the above. For SWATH experiment Fig. , the insR .For between group comparisons, Mann-Whitney U test and Kruskal-Wallis tests were used to compare medians. For iTRAQ data, geometric means of technical replicates were used for further analysis. The average ratios of AITD/Ctrl, TED (mild)/Ctrl and TED (severe)/Ctrl from eight iTRAQ sets were calculated using geometric mean. The p-values were generated using t-test of log transformed data. For SWATH data Fig. , alignmeIn the discovery phase using iTRAQ quantitative proteomics, we identified 1167 unique tear proteins with false discovery rate (FDR) < 1% (95% peptide confidence level). Using a specific cut-off of significant fold change, defined as fold change >1.5 for up-regulation and <0.67 for down-regulation (p < 0.05), we found three up-regulated and 18 down-regulated tear proteins when tear protein profile was compared between AITD and normal controls Fig. . There wTo verify the results from discovery proteomics using SWATH, it is necessary to establish a comprehensive spectral library to process SWATH data. The tear sample used for generation of spectral library was pooled from 1000 healthy subjects  to avoid biasness from individual samples. Thirty 1D LC-MS/MS using IDA mode were combined. The total number of unique proteins identified was 1508  after combining 30 IDA runs  , Mesothelin (MSLN) , and Prolactin Induced Protein were up-regulated  and S100 Calcium binding protein A4 (S100A4) was down-regulated  in the discovery phase. The dysregulation trends of two of these tear proteins, S100A4 and PIP, were confirmed on the verification phase .The use of tears as a source of biomarker discovery is an approach of pivotal importance. Protein profiling of tears reflects the altered states in the eye due to the close spatial relations of the lacrimal gland. Previous evidence supports lacrimal involvement in TED. Recent studies have revealed different tear proteome composition between patients with TED and healthy controlset al. and our experience using this method showed that the recovery of total tear protein was reproducible and acceptable23. Our study is the first to compare the tear proteome across different severity of TED using highly sensitive quantitative proteomics approach. We employed iTRAQ technology coupled with two-dimensional nanoLC-nano-ESI-MS/MS in the discovery phase. The iTRAQ technique is widely used for discovery-based proteomics, which allows for simultaneous protein identification and (relative) quantification. We verified the results from discovery phase (using iTRAQ on data-dependent acquisition mode) by SWATH (data-independent acquisition mode) on an independent cohort. In addition to traditional antibody-based targeted assays (ELISA or western blot), mass spectrometry (MS)-based proteomic technologies, such as multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), and Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) have been used as powerful targeted MS-based methods for verification of the results gained from discovery-phase proteomics studies25. The capability of multiplexing in targeted MS-based is especially suitable for samples with extremely low volume, such as tears. In our study, we demonstrated that a few hundred tear proteins can be quantified in one analysis using SWATH-MS. The two different quantitative approaches (iTRAQ and SWATH) have been shown to complement each other, as for a given instrument time, the iTRAQ experiment will provide more protein identifications; however, the SWATH experiment can identify more differential ratios, being both more sensitive in the detection of small differential ratios and being able to cover a larger dynamic range26.In our study, tears were collected using Schirmer’s strips. Tear proteins were then eluted from the strip by 50-mM ammonium bicarbonate using the same protocol for each sample. Prior work by Grus 27. Expression of S100A4 in different tumor cells has been shown to strongly correlate with aggressive metastatic tumor phenotype. Recently, increased expression of this protein has been implicated in pathologies of non-malignant diseases such as rheumatoid arthritis and disorders in cardiovascular, nervous and pulmonary systems. Interestingly, it is postulated that the common feature of various diseases implicating S100A4 is its participation in the inflammatory component which contribute to the development and progression of the various pathologies28. However, our study results did not support this since S100A4 was down-regulated with worsening severity of TED. Recent studies have linked S100 positivity in the ocular surface tissue in non-inflammatory conditions30. Notably, selective S100 proteins involvement in corneal epithelial cell proliferation and differentiation under normal and pathological conditions was suggested by a study by Jing et al.31. Nubile et al. demonstrated strong cytoplasmic expression of the total S100 family (including S100A4) in all limbal epithelial crypts (LECs) of healthy corneas and marked reduced expression was noted in an inflamed limbus32. In particular, S100A4 protein presented higher limbal expression in healthy samples. The LEC are anatomically well defined structures and are located deeper in the substantia propria of the limbus providing both protection and a microenvironment of extracellular matrix with its multitude of resident cells33. Based on the results of their study, the authors concluded that the drastic reduction of S100 expression, highlighted in limbal inflammation, showed different behavior of this family of proteins in limbal tissues compared to other forms of ocular inflammation in which S100 over-expression was reported. These evidence supports the hypothesis that S100 proteins represent a marker of limbal normality32.The discovery phase experiments demonstrated four tear proteins were differentially expressed between varying severity of TED and normal control group; with three up-regulated  and one down-regulated (S100A4). The dysregulation of two proteins (S100A4 and PIP) showed similar trends in the verification phase experiments and reached statistical significance. S100A4 is a member of the S100 calcium binding protein family. Through binding with different proteins, S100 proteins are involved in the regulation of many important cellular activities such calcium homeostasis, cytoskeleton organization, stress response, cell motility, cell proliferation and differentiation34. The precise cause of SLK in TED is unknown, but has been thought to be due to mechanical factors such as the oculopalpebral asynergy or related to an autoimmune mechanism35. In an earlier study by Kadrmas et al., SLK was noted to be prognostic sign for severe TED36. No studies to date examine in detail the pathological changes of the limbus in patients with TED at different stages of their disease. Although up to a third of SLK cases are linked to TED, the diagnosis of SLK in TED patients is uncommon34. The expression of S100A4 protein is also not exclusive to the limbus and may be found in other ocular surface tissues37. We did not ascertain the incidence of SLK in our subjects in this study. Hence, whether increasing limbal inflammation due to the autoimmune process in TED contributed to the downward trend of S100A4 protein observed in the tear profile in our patients reflects the increasing severity and activity of their orbital condition remains to be elucidated in further studies.Superior limbic keratoconjunctivitis (SLK) is characterized by inflammation of the superior bulbar conjunctiva with predominant involvement of the superior limbus and adjacent epithelial keratitis and has been associated with TED39. Although the function of PIP is not well elucidated, recent evidence suggest that its primary role may be related to host defense and immune modulation40. Several studies have shown that PIP has a strong affinity for the CD4 receptor and is a specific inhibitor of CD4+ T-cell receptor-mediated apoptosis, with potential functions in innate and adaptive immunity40. PIP expression has also been shown to be influenced by several cytokines, namely IL4/13, IL1 and IL639. Since aberrant cytokine expression and breakdown of immune tolerance is central to pathogenesis of TED41, we postulate the changes in PIP in our study could reflect these processes. PIP has recently been reported as a biomarker on keratoconus in human studies42. The fact that PIP was downregulated in keratoconus disease but upregulated in TED in our study further strengthens the specificity of our study results. Interestingly, in the study on the role of PIP as a potential biomarker for keratoconus disease, Priyadarsini et al. noted PIP expression was up-regulated in both healthy and diseased human corneal fibroblasts upon Transforming Growth Factor β (TGF-β) treatment43. Orbital fibroblast is central to the pathogenesis of TED and the cytokine TGF-β plays a key role in this autoimmune inflammatory disorder of the orbit. Two subpopulations of orbital fibroblasts were previously noted; Thy1− (CD90−) orbital fibroblasts which exhibit high capacity to differentiate into adipocytes, and Thy1 +(CD90+) orbital fibroblasts which tend to differentiate into α–smooth muscle actin expressing myofibroblasts. When exposed to TGF-β, the Thy-1- fibroblasts differentiate into myofibroblasts with prominent cytoplasmic actin filaments that can participate in inflammation, repair, and fibrosis44. Within the orbit, relative proportions of Thy-1+ and Thy-1− fibroblasts and their degree of exposure to TGF-β may affect disease expression, including whether muscle or fat expansion predominates and the extent of fibrosis that develops45. The presence of TGF-β, particularly in patients with severe TED, potentially augments PIP levels in the tears of these patients.Prolactin-inducible protein (PIP)/gross cystic disease fluid protein-15 (GCDFP-15) is typically expressed in several exocrine tissues, such as lacrimal, salivary, and sweat glands. Recently, PIP has been investigated as a potential biomarker for metastatic breast cancer and keratoconus6. Dry eye is the most frequent cause of the ocular discomfort in TED and has been found to be present in 85% of patients47. The underlying mechanism is not fully elucidated but was thought to be related to anatomical alterations in TED such as corneal exposure from exophthalmos and lid retraction leading to ocular surface drying, ocular surface inflammation, as well as lacrimal gland dysfunction in TED49. In contrast, the cause of dry eye syndrome is fundamentally due to an insufficiency of tears to maintain the ocular surface. Previous proteomic studies have showed distinctly different proteomics pattern between TED and dry eye syndrome20.It is interesting to note that the dysregulation trend of both S100A4 and PIP is opposite in TED compared to our earlier works which compared dry eyes and healthy controlsThe main limitation of our study is the small sample size. However, all the subjects with TED recruited for the study were not treated  prior to collection of tear samples, since treatment of TED could potentially alter the tear proteome composition. This stringent criterion nonetheless affected the patient numbers that could be recruited for the study. Although the patients with concomitant medical conditions or receiving medications that could affect the tear film constituents were excluded from the study, we cannot be completely sure other medical conditions of the subjects would not influence the levels of the S100A4 and PIP.Our study demonstrated for the first time, differences in tear proteome across the spectrum of different severity and activity of TED in patients with AITD. Of note, two proteins, S100A4 and PIP showed consistent dysregulation trends in the discovery and validation phase experiments. These tear proteins may serve as potential biomarkers to predict progression to severe TED in patients with AITD. Based on previous studies, S100A4 may represent limbal normality and PIP could reflect the breakdown of immune tolerance and ongoing fibrosis in severe TED. Hence, these potential biomarkers appear to complement each other, with S100A4 protein down-regulated in both mild inactive and severe TED, potentially providing a marker for the propensity to develop TED in patients with AITD before clinical manifestation; while PIP up-regulation may signify more ominous progression to severe TED. Further prospective evaluation on larger cohort of patients would be necessary to confirm our findings.Supplementary Tables S1-S4 accompanies this paper.


Spatiotemporal gene expression during cardiac development is a highly regulated process. Activation of key signaling pathways involved in electrophysiological programming, such as Notch and Wnt signaling, occurs in early cardiovascular development and these pathways are reactivated during pathologic remodeling. Direct targets of these signaling pathways have also been associated with inherited arrhythmias such as Brugada syndrome and arrhythmogenic cardiomyopathy. In addition, evidence is emerging from animal models that reactivation of Notch and Wnt signaling during cardiac pathology may predispose to acquired arrhythmias, underscoring the importance of elucidating the transcriptional and epigenetic effects on cardiac gene regulation. Here, we highlight specific examples where gene expression dictates electrophysiological properties in both normal and diseased hearts. The human heart starts beating spontaneously about 3 weeks after fertilization . To reacModifications to the nucleosome, the basic unit of chromatin consisting of DNA coiled around histones, controls packaging of DNA and regulates access of transcription factors to the DNA . There aEpigenetic mechanisms have been hypothesized to be a crucial interface between genetic and environmental factors to explain the theory of metabolic memory in diseases such as diabetes. Metabolic memory in diabetes focuses on the interrelationship between stressors , which induce mechanisms that persist after the stressor is removed  . In a stOur group has shown in a clinically relevant murine heart failure model, encompassed by progressive heart failure via transverse aortic constriction (TAC) plus small apical myocardial infarction, that Notch signaling is transiently reactivated in the adult heart, including reactivation in atrial and ventricular cardiomyocytes , 12. Tho+ channels and higher levels of cardiac K+ channels when compared with the LV, which contributes to slower conduction in the RV [+ channels (SCN5A) or the direct Notch signaling pathway target HEY2 (Hairy/enhancer-of-split related with YRPW motif protein 2), among other mutations, can result in ventricular arrhythmias and sudden cardiac death [Embryologically, the left and right ventricles arise from the first and second heart fields, respectively, and are exposed to distinct signaling pathways during cardiac development . This prn the RV . Specifiac death , 17.In addition to regulating baseline physiology, developmental programming may also dictate the differential response to stress in various regions of the heart, a response often referred to as reactivation of the “fetal gene program.” An early study to distinguish cardiac-specific epigenetic regulation of gene expression in normal murine hearts used chromatin immunoprecipitation (ChIP) analysis to compare LV versus RV expression levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). In normal wild-type mice, the group found increased levels of Histone 3 lysine 4 dimethylation (H3K4me2) and Histone 3 lysine 9 acetylation (H3K9ac) with concomitantly higher expression levels of ANP and BNP in the LV compared to RV . In a seHey2 expression levels are upregulated in the LV, whereas Hey2 expression levels are downregulated in the RV. These gene expression changes are associated with increased Histone H3 lysine 4 trimethylation (H3K4me3) at the Hey2 promoter in the LV, while there is loss of the H3K4me3 marks in the RV. Furthermore, the key transcriptional cofactor RBP-J , is dynamically bound to the proximal Hey2 promoter and also to an enhancer specifically within the LV but not in the RV [Hey2 in adulthood following a specific trigger that induces Notch. Collectively, these results demonstrate that chamber-specific histone modifications can result in distinct electrical phenotypes and regulation of gene expression.The Notch signaling pathway regulates multiple aspects of embryogenesis, from processes as diverse as cardiac structure to programming cardiac electrophysiologic properties . We haven the RV . Figure Hey2 is regulated by mechanisms in addition to Notch signaling within the ventricles. We demonstrate for the first time that Hey2 is a direct Wnt target only in the RV and not the LV, which may have relevance to our understanding of RV-specific arrhythmias [Gja1  and Scn5a (encodes Nav1.5 protein), were shown to be differentially regulated. Gene expression changes result in slower conduction velocity in both the Wnt gain and loss-of-function RV, but not LV, though the mechanism of slowed conduction is distinct in these models. Indeed, the chamber-specific gene expression changes predispose the mice to ventricular tachycardia with physiologic stimulation of the RV only. ChIP analysis of wild-type embryonic hearts demonstrates enrichment of the active H3K27ac mark in a Hey2 enhancer region shown to be bound by the Wnt effector β-catenin in the RV only, while a distinct Hey2 enhancer demonstrates H3K27ac enrichment in the embryonic LV. Since Hey2 regulates important electrical properties within the RV, this example highlights how a chamber-specific understanding of embryonic transcriptional and epigenetic programming may help us to better understand arrhythmias which occur later in life. Figure HEY2 in adulthood following a specific trigger where Wnt signaling is reactivated.More recently, we demonstrate how embryonic programming via the Wnt signaling pathway also regulates electrophysiology in a chamber-specific manner , 24. It hythmias . Using b+ currents , alterations to depolarizing Ca2+ currents (ICa-L) and Ca2+ transporters , and downregulation of depolarizing Na+ currents (INa). Connexin 43 is a key cardiac gap junction protein that can be downregulated or improperly distributed which also results in slowed conduction [Two hallmark electrophysiological findings in failing hearts include conduction slowing and action potential prolongation. Electrophysiological heterogeneity within the heart presupposes the dysregulation of multiple key components of the action potential, including downregulation of repolarizing Knduction , 26.+ currents, offering an illustrative example of the interplay between heart failure, electrophysiologic remodeling, and histone modifications. In the heart, KCNIP2  interacts with the subfamily of voltage-gated potassium channel (Kv4) to increase current density, accelerate recovery from inactivation, and slow inactivation kinetics [Ito) and contributes to APD prolongation and conduction slowing in both humans as well as canine models of heart failure [Kcnip2 promoter [Kcnip2 expression levels [Ito and development of premature ventricular beats. Therefore, understanding how post-translational histone modifications are regulated is relevant to understanding cardiac electrophysiology, especially in the setting of heart failure.Several studies have focused on the downregulation of repolarizing Kkinetics . Collect failure , 29. As promoter . Bolstern levels . In addiKCNIP2 has been shown to be a consistent finding across the spectrum of human cardiomyopathies. Exciting work recently demonstrates that KChIP2 protein not only contributes to Ito, but it also regulates a transcriptional network in the heart. In a paper by Nassal et al., KChIP2 was shown to regulate microRNAs (miRNA) important for conduction and maintenance of electrical stability through KChIP2 binding to the miR-34b/c promoter region in the nucleus, supporting the notion that KChIP2 can behave as a transcriptional repressor [KCNIP2 in regulating transcription and electrical remodeling in heart failure, understanding how it is regulated will be of clinical importance.Downregulation of epressor . In bothMore broadly, several exciting studies offer a blueprint for understanding the role of epigenetics in heart failure. Studies by Rosa-Garrido et al. used genome-wide chromatin conformation capture (Hi–C) of isolated adult cardiomyocytes from a TAC heart failure mouse model to reveal alterations in chromatin compartmentalization and looping, demonstrating chromatin remodeling in conjunction with decreased enhancer interactions in heart failure . To suppHEY2 and KCNIP2 in a chamber-specific manner, which may contribute to deadly ventricular arrhythmias.Signaling pathways active during cardiac development program cardiomyocyte identity. In the adult, these developmental pathways can be reactivated in disease states during pathologic remodeling where they can bind to chamber-specific enhancer and promoter regions and are associated with specific histone modifications. These mechanisms of gene regulation coordinate differential transcription of genes such as