Stearoyl CoA Desaturase-1 (SCD) is considered as playing an important role in the explanation of obesity. The aim of this study was to evaluate whether the DNA methylation SCD gene promoter is associated with the metabolic improvement in morbidly obese patients after bariatric surgery. The study included 120 subjects with morbid obesity who underwent a laparoscopic Roux-en Y gastric by-pass (RYGB) and a control group of 30 obese subjects with a similar body mass index (BMI) to that found in morbidly obese subjects six months after RYGB. Fasting blood samples were obtained before and at six months after RYGB. DNA methylation was measured by pyrosequencing technology. DNA methylation levels of the SCD gene promoter were lower in morbidly obese subjects before bariatric surgery but increased after RYGB to levels similar to those found in the control group. Changes of DNA methylation SCD gene were associated with the changes of free fatty acids levels  and HOMA-IR  after surgery. RYGB produces an increase in the low SCD methylation promoter levels found in morbidly obese subjects. This change of SCD methylation levels is associated with changes in FFA and HOMA-IR. Stearoyl CoA desaturase-1 (SCD) has been proposed as playing a vital role in the explanation of obesity in Mediterranean countries3Bariatric surgery has proved to be the most effective treatment for clinically severe obesity and its associated co-morbidities810et al. showed that promoter methylation of peroxisome proliferator-activated receptor gamma coactivator-1αlpha (PPARGC1A) and pyruvate dehydrogenase kinase isozyme-4 (PDK4) were altered with obesity and were restored to non-obese levels after RYGB induced weight loss18Recently, epigenetics has been acquiring great importance and has been recognized as playing a significant role in complex diseases, providing mechanisms where by environmental factors can influence complex diseases such as obesity and type 2 diabetes (T2DM)121516Since SCD seems to be related with obesity and insulin resistance, we aimed to study a) whether the methylation of the SCD gene promoter is associated with obesity and insulin resistance in morbidly obese subjects, b) its evolution after bariatric surgery and c) whether its change after bariatric surgery is associated with the changes found in different metabolic variables.The characteristics of the control group and the morbidly obese subjects are shown in DNA methylation levels of the SCD gene promoter in peripheral blood were lower in morbidly obese subjects before bariatric surgery (1.42 ± 0.91%) when compared with the control subjects (2.13 ± 0.68%) , but incSCD mRNA expression was measured in peripheral blood mononuclear cells (PBMC) in a subset of morbidly obese subjects (n = 35) before and after RYGB. SCD mRNA expression was lower after RYGB compared to baseline levels, although these differences were not statistically significant . Before SCD activity indices were measured in the serum phospholipids fraction in a subset of morbidly obese subjects (n = 35) before and after RYGB, and in the control group (n = 30) . SCD actNo significant correlation was observed between SCD methylation levels and mRNA expression, neither with activity indices . NeitherThere were no significant correlations between the SCD gene promoter methylation levels and anthropometric and biochemical variables in morbidly obese subjects, neither were there before or after RYGB .vs 0.53 ± 0.16 mmol/L). While, those that decreased the SCD gene promoter methylation did no change the free fatty acids levels (0.54 ± 0.19 mmol/L vs 0.51 ± 0.17 mmol/L). On the contrary, a positive association was found between changes in SCD gene promoter methylation levels and changes in adiponectin levels .To evaluate whether the changes observed in the anthropometric and biochemical variables in morbidly obese subjects after RYGB were related with changes in the DNA methylation levels of the SCD gene promoter, correlation tests were performed. A negative association was observed between changes in SCD gene promoter methylation and changes in free fatty acids (FFA)   and HOMAIn those morbidly obese subjects who lost a greater percentage of weight (above percentile 75th) (n = 30), a significant association was found between the change of the SCD gene promoter methylation level and the change of weight  . In thosTo test which variables could explain the variation of the SCD gene promoter methylation levels after RYGB, a linear regression model was performed. We showed that FFA levels at baseline were significantly associated with SCD methylation level changes in a model adjusted for age, sex, weight, HOMA-IR and adiponectin levels at baseline. The 36% of the variability of SCD is explained by FFA or other covariables .The main finding of our study was that SCD gene promoter methylation levels were decreased in morbidly obese subjects before bariatric surgery, raising six months after RYGB to similar levels to those found in a group of subjects with similar BMI. This increase was associated with changes of insulin resistance related metabolic parameters after RYGB. Of special relevance are the inverse relationships between the DNA methylation of the SCD promoter and FFA and HOMA-IR, and the direct association with adiponectin levels.et al. observed that promoter-specific DNA methylation of PGC-1α and PDK4 genes is modified after RYGB in skeletal muscleet al. showed an increase of methylation levels in whole blood in PDK4, interleukin 1B, interleukin 6 and tumor necrosis factor (TNF) promoters one year after RYGBet al., also demonstrated small changes in the DNA methylation of 51 gene promoters from whole blood after RYGB (at six months), being similar to a control groupet al. found differential methylation within genes associated with obesity, epigenetic regulation and development in human adipose tissue from obese women before and after a gastric bypassSCD is the enzyme that catalyses the conversion of saturated fatty acids to MUFA. Animals and human studies have shown the association between SCD and obesity and related metabolic disorders, such as insulin resistance and inflammation23et al. provided evidence that elevated levels of FFA induce acute hypermethylation of the PGC-1α and mitochondrial transcription factor A (TFAM) promotersEpigenetic modifications are regulated by numerous nutritional and environmental factorsSCD is regulated, among other factors, by fatty acids. SCD mRNA levels in 3T3-L1 adipocytes were repressed by 80% of the original levels when these cells were exposed to 300 μM arachidonic acidFurthermore, FFA might modify DNA methylation in other CpG sites of the SCD gene that have not been analyzed in our study, thus affecting mRNA expression levels of SCD. In this study, we only analyse 8 CpG in the SCD promoter region. This requires more studies about the effect of different fatty acids on the SCD methylation promoter levelsAs in our previous study, serum SCD activity indices were not associated with SCD mRNA expression in PBMC, neither with SCD methylation levels33A limitation of our study is that we have not analyzed the whole SCD promoter. This region contains a high number of CpG sites and is also highly polymorphic, which makes the design more complicated. Anyway, the studied region has also been previously associated with metabolic parameters, reinforcing the resultsFinally, our previous work performed in a prospective cohort intervention study with a control group, showed that the subjects who lost weight after a nutritional intervention had higher levels of SCD gene promoter methylation after the interventionIn conclusion, we found that RYGB produces an increase in the low SCD methylation promoter levels found in morbidly obese subjects. This modification of methylation levels is associated with changes found in HOMA-IR, adiponectin, and above all in FFA levels. These variables are closely linked to insulin resistance state. But in those morbidly obese subjects with a higher weight change, SCD methylation promoter levels are associated with weight changes. More studies are needed in order to understand the association between gene methylation levels and obesity and insulin resistance state.The study included 120 subjects with morbid obesity who underwent laparoscopic Roux-en Y gastric by-pass (RYGB) and 30 obese subjects with a similar body mass index (BMI), HOMA-IR, age and percentages of males/females as the morbidly obese subjects six months after RYGB. Morbidly obese subjects were studied before and 6 months after RYGB. Subjects were excluded if they had cardiovascular disease, arthritis, acute inflammatory disease and infectious disease. Samples were processed and frozen immediately after their reception at the Regional University Hospital Biobank . The research was carried out in accordance with the Declaration of Helsinki (2008) of the Word Medical Association. All the participants gave their written informed consent and the study was reviewed and approved by the Ethics and Research Committee of the Regional University Hospital, Malaga, Spain.Blood samples were collected after a 12-hour fast. The serum was separated and immediately frozen at −80 °C. Serum biochemical variables were measured in duplicate as previously described36Blood samples were obtained after a 12-h fast. PBMCs were only isolated from a subset of morbidly obese subjects before and after RYGB (n = 35). PBMC were isolated by Ficoll standard density gradient centrifugationTotal RNA from PBMC was prepared using Trizol reagent  according to the manufacturer’s instructions. Complementary DNA was obtained and quantified as previously describedSCD activity index in serum were only analyzed from a subset of morbidly obese subjects before and after RYGB (n = 35) and in control subjects (n = 30). Total lipids from serum were extracted with chloroform-methanol 2:1 (v/v). The phospholipids were separated by thin-layer chromatography on silica gel plates  with hexane-ethylic ether-acetic acid  as the developing solvent. The fatty acid composition of phospholipids was analyzed as describedVenous blood samples were collected from all subjects and genomic DNA was isolated from peripheral blood using QIAmp DNA Blood Kit  in a QIACUBE instrument (Qiagen) according to the manufacturer’s recommended protocols. DNA samples were stored at −20 °C.DNA methylation analyses were performed on bisulfite-treated DNA using highly-quantitative analysis based on PCR-pyrosequencing. The bisulfite conversion was performed with 500 ng genomic DNA isolated from peripheral blood using the EpiTect bisulfite kit (QIAGEN) as recommended by the manufacturer. The PyroMark™Q96 ID Pyrosequencing System (QIAGEN) was used to determine the methylation status of the CpG island region of the SCD gene promoter.For the methylation analysis of the SCD gene promoter (NM_005063.4) we used the primers and PCR conditions previously publishedThe degree of methylation was expressed for each DNA locus as the percentage of methylated cytosine over the sum of methylated and unmethylated cytosines. Non-CpG cytosine residues were used as built-in controls to verify bisulfite conversion. Correlation between methylation at all eight CpG sites was high (p < 0.001), therefore the mean for all the sites was expressed as %5 mC. The values are expressed as the mean for all the sites. We also included unmethylated and methylated DNA as controls in each run (New England Biolabs).baseline − variableat 6 months) × 100/variablebaseline. All analyses were performed using R statistical software, version 2.8.1 The continuous variables are shown as the mean and standard deviation and the classification variables as proportions. Normal distribution was tested by the Kolmogorov-Smirnov test. The Wilcoxon paired-sample and the Kruskall-Wallis tests were used for intra-group and inter-group comparisons, respectively. Correlation analyses were performed with the Spearman correlation coefficient. Multiple linear regression models were used to evaluate the predictive capacity of different metabolic parameters on SCD gene promoter methylation changes. The models were adjusted for possible confounding variables such as age, gender and body mass index (BMI) at baseline. Changes in the variables due to RYGB were expressed as percentages and were calculated as .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Cannabinoids exert dynamic control over many physiological processes including memory formation, cognition and pain perception. In the central nervous system endocannabinoids mediate negative feedback of quantal transmitter release following postsynaptic depolarization. The influence of cannabinoids in the peripheral nervous system is less clear and might have broad implications for the therapeutic application of cannabinoids. We report a novel cannabinoid effect upon the mouse neuromuscular synapse: acutely increasing synaptic vesicle volume and raising the quantal amplitudes. In a mouse model of myasthenia gravis the cannabinoid receptor agonist WIN 55,212 reversed fatiguing failure of neuromuscular transmission, suggesting future therapeutic potential. Our data suggest an endogenous pathway by which cannabinoids might help to regulate transmitter release at the neuromuscular junction. While cannabinoids can act directly upon some ion channels, in most cases they modulate synaptic transmission via G-protein-coupled cannabinoid receptors4. The endogenous cannabinoid (endocannabinoid) system plays a well-established role as a retrograde regulator of synaptic plasticity in the brain5. Endocannabinoids are produced on demand following postsynaptic depolarization and generally act via presynaptic cannabinoid-1 (CB1) receptors to acutely increase K+ currents and inhibit Ca2+ currents8, and consequently reduce the number of quanta released during neurotransmission1.Cannabinoids are ubiquitous regulators of synaptic transmission in the brain: acutely modulating neurotransmitter release, mediating numerous forms of short- and long-term plasticity, and strongly influencing synapse formation and neurogenesis10. Sanchez-Pastor et al. reported reductions in both frequency and amplitude of spontaneous miniature endplate potentials (mEPPs) at the frog NMJ11. In contrast, a 1970’s study on the rat NMJ reported that delta9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana (Cannabis sativa)12, elevated the amplitude of the spontaneous miniature endplate potential (mEPP) without altering the evoked EPP13. Little else is known of the actions of cannabinoids at the mammalian NMJ.While cannabinoids have been intensely studied in the brain for many years, their effects on the mammalian neuromuscular junction (NMJ) have been curiously overlooked. The NMJ provides the essential link between motor nerves and skeletal muscle and has been central to our understanding of fast communication throughout the nervous system. Cannabinoids such as 2-Arachidonoylglycerol (2-AG) and WIN 55,212 (WIN) act on central synapses by reducing the amount of transmitter released per nerve impulse . Studies of the NMJs of lower vertebrates similarly revealed a reduction of the quantal content (QC) upon application of cannabinoids such as 2-Arachidonoylglycerol and WIN14, being expressed at high levels by neurons in the hippocampus, cerebellar cortex, amygdala, striatum, hypothalamus, cerebellum and the dorsal horn of the spinal cord and at lower levels in the peripheral nervous system15. CB1 receptors are found to control the release of GABA and glutamate as well as neuromodulators such as serotonin, acetylcholine, dopamine, opioids, norepinephrine, and cholecystokinin16. CB2 receptors were initially identified on immune cells17. CB2 receptors are also detectable on neurons and glia in the CNS (particularly in the cerebellum and brainstem) but at much lower levels compared to CB1 receptors19. Given the well-established role of cannabinoids in negative-feedback regulation of transmitter release at central synapses we set out to investigate their function at the mouse NMJ.CB1 receptors are amongst the most abundant G-protein coupled receptors in the CNSHere we show that cannabinoids have a different effect at the mammalian NMJ compared to synapses in the brain. Cannabinoids increased the miniature end plate potential, which represents the quantal amplitude, and the evoked end plate potential, thereby enhancing neuromuscular transmission. We show that the increase in quantal amplitudes could be explained by an increase in synaptic vesicle volume. In a mouse model of myasthenia gravis, acute cannabinoid treatment restored disease-impaired neuromuscular transmission. We therefore conclude that cannabinoids positively modulate synaptic transmission at the mammalian NMJ through a hitherto undescribed mechanism. Our results suggest that cannabinoids might play a role in sustaining neuromuscular transmission.ex vivo preparation. Unlike for many central synapses, WIN produced no change in quantal content  produced a significant rise in mEPP amplitude. The pharmacology of the cannabinoid receptors is complex and at this widely used concentration AM630 might act as a CB1 agonist22. In any event, WIN produced no significant additional increase in the mEPP amplitude in the presence of either AM251 (5 µM) or AM630 (10 µM)  led to a 1.6-fold rise in mEPP amplitude, mimicking the effects of exogenous AEA  produced 1.6 fold increases in the amplitudes of both the evoked EPPs  Fig. . NeitherµM) Fig. . Next weAEA Fig. . Togethe+-ATPase that is needed to drive the acetylcholine transporter25. Neither of these drugs on their own affected mEPP amplitudes in our recordings , but both inhibitors blocked the WIN-induced increase in mEPP and EPP amplitudes  and AM630 (10 µM) caused no change in the area or density of postsynaptic nicotinic acetylcholine receptors (AChR) that might explain the increase in mEPP amplitude Fig. . WIN alsdes Fig. . Thus, t0 µM and 27. We tested the potential of WIN in a mouse model of myasthenia gravis. Mice received daily injections of IgG from an anti-MuSK-positive MG patient. The autoantibody-induced failure of neuromuscular transmission was demonstrated by a decrement in the compound muscle action potential (CMAP) during repetitive stimulation28  autoantibodies deplete postsynaptic nicotinic acetylcholine receptors (AChRs), thereby reducing quantal amplituden28 Fig. . In thesn28 Fig. . These r1.Here we report a hitherto unknown synaptic effect whereby cannabinoids acutely elevated quantal amplitudes via a mechanism that changed the filling of synaptic vesicles and lead to an expansion of vesicle volume. These effects of WIN and AEA at the mammalian NMJ differ from the classical modulatory effect of cannabinoids at central synapses, where cannabinoids generally act via presynaptic CB1 receptors to acutely reduce the number of quanta releasedex vivo phrenic nerve-diaphragm muscle preparation. This delay was intended to ensure complete diffusion of the bath-applied WIN into the muscle such that a steady state would exist during the extended timespan of recordings from 12–16 muscle fibers. We attempted to determine the kinetics of the WIN effect by recording mEPPs from single impaled muscle fibers for 15–60 mins immediately after application of WIN. The few fibers that yielded a steady resting potential over this time frame revealed no immediate effect of WIN on mEPP amplitude or frequency. These results suggest that some time is required for the increase in vesicle filling following exposure to WIN.The effects of WIN on synaptic potentials were recorded approximately 1–2 hours after the addition of WIN to the 29. Interestingly, blood concentrations of AEA increase after athletes exercise31. It is conceivable that during sustained exercise, muscles produce AEA and that this serves to raise quantal size and thereby compensate for the natural decline in quantal content, as a physiological response. Our study reports a cannabinoid-induced increase in the content of ACh within each vesicle rather than change in the number of vesicles released per nerve impulse. The distinct mechanism through which vesicle filling is regulated, independently of the control of quantal release, will require further detailed investigation.Inhibition of the AEA-degrading enzyme FAAH likewise elevated mEPP amplitude, suggesting that endogenous cannabinoids can regulate quantal size at the NMJ. During tetanic activation of muscle there is a decline in quantal content/number, which can be a challenge to effective neuromuscular transmission32. Moreover, in the pineal gland, nucleus accumbens and globus pallidus CB1 and CB2 receptors can form heteromers with bidirectional cross-antagonism of downstream signalling pathways and activation of either CB receptor can lead to modulation of the partner receptor33. The pathways typically engaged by CB receptors include regulation of cAMP levels, stimulation of MAPK activity, elevation of intracellular calcium and opening of K+ channels6. A recent study reported that the neuropeptide CGRP can increase mEPP amplitudes at the mouse NMJ via a PKA-inhibitor (H-89)-mediated process. This CGRP effect was blocked by vesamicol34 suggesting that it too was dependent upon synaptic vesicle refilling. Given the emerging complexity of cannabinoid signal transduction, further detailed pharmacological studies will be needed to: (i) characterise the types of cannabinoids that elevate quantal size, (ii) identify the receptor(s) involved, and (iii) characterize the second messenger pathways responsible for cannabinoid-induced increase in quantal size at the mammalian NMJ.The pharmacological profile of the cannabinoid-induced effects suggests that WIN activates different pathways at the mammalian NMJ, compared to its known effects in the CNS. In the presence of the CB1 inverse agonist AM251, WIN produced no significant increase in quantal amplitude. This was also the case for the CB2 inverse agonist AM630 but AM630, on its own, significantly raised the mEPP amplitude. Our preliminary pharmacological investigations, testing the effects of AM630 and AM251 on mEPP amplitudes, will require further careful pharmacological assessment. CB1 ligands are known to behave either as agonists or antagonists depending upon the interaction of the receptor with specific Gi subunits. For example, (R)-methanandamide behaves as an inverse agonist for CB1-Gi1 and -Gi2, but as an agonist for CB1-Gi3 complexes37. The increasing usage of cannabinoids in a variety of medical conditions39 highlights the crucial need to better understand the physiological roles of cannabinoids in the periphery. Here we reveal evidence of their involvement in regulating neuromuscular transmission, and a possible therapeutic potential for cannabinoid signaling in myasthenia gravis.Cannabinoid drugs have already been approved for the treatment of nausea in chemotherapy patients and clinical trials are underway to test for treatment of spasticity and other muscle-debilitating symptoms of multiple sclerosisFemale C57BL/6 J mice (6–10 weeks) obtained from  were used in this study. Mice were killed with an overdose of pentobarbitone . All mouse experiments described in this paper were conducted with the approval of The University of Sydney Animal Ethics Committee  in compliance with the NSW Animal Research Act 1985 and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes 7th Edition NH&MRC 2004. In relation to the collection of plasma, informed, written consent was obtained from patients in accordance with the Declaration of Helsinki . The project was approved by the Human Research Ethics Committee of the Sydney South West Area Health Service.2PO4-1, NaHCO3-12.8, MgCl2-1, CaCl2 and glucose-10, pH adjusted to 7.3). Spontaneous miniature endplate potential (mEPP) and nerve-evoked endplate potential (EPP) recordings were made at room temperature as previously described40. Contraction was blocked using the muscle sodium channel blocker, µ-conotoxin GIIIb . EPP and mEPP recordings were started 1–2 hrs after the diaphragm was placed in the bath solution. Spontaneous mEPP amplitudes were recorded for 1–3 min and were normalized to a resting potential of −80 mV. A train of 40 stimuli (1 Hz) was used for EPP analyses . EPP amplitudes were normalized to −80 mV and then corrected for non-linear summation41. Quantal content was calculated by dividing the normalized and corrected EPP amplitudes by the normalized mEPP amplitude for each muscle fiber. Recordings were made from 12–18 muscle fibers from each muscle to derive mean mEPP and EPP values for each sample. Only fibers with a stable resting membrane potential (RMP) ≤ −60 mV were analyzed. To calculate the 90–10% decay times before and after pyridostigmine treatment the average decay time for all sample traces (irrespective of peak amplitude) for n = 3–4 mice was compared.The diaphragm with the phrenic nerve attached was quickly dissected, pinned on a Sylgard-coated dish and bathed in oxygenated Ringer’s solution containing physiological calcium levels . Stock solutions for R(+)-WIN 55,212 (WIN), AM251, AM630, L-+/-vesamicol, bafilomycin, anandamide and URB597 were first dissolved in DMSO, stored at −20 °C, and added to the bath 1–2 hrs before the start of recordings. This drug exposure time allowed for dissection of the tissue and set up of the in vivo experiments, compound muscle action potentials (CMAP) were recorded from myasthenic mice (see below). The mice then received an intraperitoneal injection of R(+)-WIN 55,212  and a second set of CMAP recordings were made 3 hrs later. At the dose employed mice showed some signs of hyperactivity but did not display behavior suggestive of intoxication . After the phrenic nerve-diaphragm preparation was dissected it was thoroughly washed for 1.5 hrs in Ringer’s solution to remove traces of the injected WIN prior to baseline EPP recordings. The acute effects of WIN were then tested by adding WIN to the bath solution for 1.5 hrs.For the 42. For comparison of AChR staining intensities, sections were processed together and examined during the same imaging acquisition using identical imaging settings. Confocal Fluorescence Resonance Energy Transfer (FRET) was used to compare the extent to which AChRs were tightly packed within endplate AChR clusters (<10 nm AChR-AChR spacing) as described previously43. FRET efficiency was calculated from the increase of the fluorescence intensity of the donor after the acceptor fluorophor was selectively photobleached.Immunostaining, imaging  and endplate morphometry was performed as described previously44. Diaphragms were then sliced into approx. 1–2 mm strips (perpendicular to the main nerve) and, fixed for a further 45 min followed by heavy metal staining45. Finally, each strip of diaphragm was flat embedded in EPON resin and polymerized overnight at 60 °C. An ultramicrotome  was used to produce sections for LM and EM observations. Semi-thin sections (500 nm) across the entire length of the diaphragm were produced, stained with Toluidine blue and observed under a light microscope: this step aided in trimming the diaphragm down to only the areas located within 2–3 mm of the main nerve (where most NMJs are located). Ultra-thin sections (70 nm) were then produced from the trimmed area, collected on 200 mesh copper grids, stained with Uranyl acetate and Lead citrate for 10 min each before being observed with a TEM, operating at 120 kV .Diaphragms prepared as described above were divided into two hemidiaphragms. The right hemidiaphragm was bathed in WIN (10 µM) for ~2 hrs while the left hemidiaphragm was incubated in control solution (without WIN). Samples were then immersion fixed in 4% paraformaldehyde +2.5% glutaraldehyde in 0.05 M cacodylate buffer  for 10 min (modified from)et al.26. Briefly, the outer diameters of synaptic vesicles were measured for at least 200 vesicle profiles, which was sufficient to obtain a characteristic distribution46. Only clearly distinguished vesicles that were circular in shape, had a grey core and a uniform membrane were used for the analysis. At least 15 NMJs were sampled from each of four hemidiaphragms/mice. A greater number of micrograph images were analysed from WIN-treated muscles than from control muscles . Vesicle diameters were corrected using the method of Froesch so as to take into consideration EM section sampling47. Volumes were calculated assuming spherical geometry (Volume = 4/3*π*radius3) based upon the inner diameter of the vesicles .Measurements of vesicle diameter were performed on digital micrographs by an operator who was blind to the treatment group and following the protocol outlined in Karunanithi 49. Briefly, mice received daily injections of IgG from anti-MuSK-positive patient 4 . Passive transfer of IgG from this patient was previously reported to cause overt myasthenic weakness after 12 daily injections50.The passive IgG transfer model of anti-MuSK myasthenia gravis was described previously40. Briefly, the mouse was first anesthetized with 2–3% isoflurane/oxygen. Two ~3 mm custom-made single monopolar recording electrodes were glued to the surface of the skin: one over the dorsal aspect of the gastrocnemius muscle and the second electrode at the ankle of the same hind limb. Electrolyte gel  was applied directly at the electrode sites. Stimulation of the sciatic nerve was accomplished via a 4 mm incision in the sciatic notch and by placing the nerve on custom-made silver hook electrodes (0.6 mm diameter). Ten stimuli were delivered at 3 impulses/sec and three such trains recorded from each muscle were averaged.Compound muscle action potentials (CMAP) were recorded from the gastrocnemius muscle during repetitive stimulation of the sciatic nerve, as previously describedGraphpad Prism  was used for statistics and to visualize the differences between the treatment groups. EPP recordings before and after drug treatment were made from the same muscle and differences between the means were evaluated using a paired Student’s t-test. For all statistical tests significance was taken as P < 0.05. Unless otherwise indicated the data was symmetrically distributed with equivalent variances and values are presented as the mean ± standard error of the mean (SEM).Supplementary file


GLRB) has recently been associated with increased neural fear network activation and enhanced acoustic startle reflexes. On the basis of two independent healthy control samples, we here aimed to further explore the functional significance of the GLRB genotype (rs7688285) by employing an intermediate phenotype approach. We focused on the phenotype of defensive system reactivity across the levels of brain function, structure, and physiology. Converging evidence across both samples was found for increased neurofunctional activation in the (anterior) insular cortex in GLRB risk allele carriers and altered fear conditioning as a function of genotype. The robustness of GLRB effects is demonstrated by consistent findings across different experimental fear conditioning paradigms and recording sites. Altogether, findings provide translational evidence for glycine neurotransmission as a modulator of the brain’s evolutionary old dynamic defensive system and provide further support for a strong, biologically plausible candidate intermediate phenotype of defensive reactivity. As such, glycine-dependent neurotransmission may open up new avenues for mechanistic research on the etiopathogenesis of fear and anxiety disorders.Representing a phylogenetically old and very basic mechanism of inhibitory neurotransmission, glycine receptors have been implicated in the modulation of behavioral components underlying defensive responding toward threat. As one of the first findings being confirmed by genome-wide association studies for the phenotype of panic disorder and agoraphobia, allelic variation in a gene coding for the glycine receptor beta subunit ( GLRB), play a major role for inhibitory neurotransmission. Recently, a genome-wide association of GLRB with categorical (panic disorder (PD)) and dimensional (agoraphobia (AG)) forms of fear and anxiety has been reported,1 in particular for rs7688285 which was associated with GLRB expression changes in post mortem tissue and reporter gene assays. Increased neural fear network activation during fear conditioning and increased acoustic startle reflexes were observed in risk allele carriers, thus qualifying as a potential intermediate phenotype of defensive system reactivity across different levels of analyses, corresponding to the Research Domain Criteria (RDoC) approach.2Glycine receptors, including its beta receptor subunit  findings in an independent sample as performed in the initial report) provide information about the reproducibility of a specific phenomenon, but not necessarily about the theoretical construct.3 Hence, direct replications need to form a synergy with conceptual replications targeting the same construct with divergent methodological approaches and paradigms.3 Here we focus on ‘defensive reactivity’ on a neurobiological level of analysis aiming to conceptually replicate the previous report1 on fear conditioning and startle responding.In light of the recent ‘replication crisis’ in psychology and neuroscience,6 The defensive system encompasses phylogenetically older adaptive behaviors  to higher-level integrated cognitive-affective coping systems.7 The underlying neural circuit is represented by the interplay of diverse cortical structures such as the medial prefrontal cortex, anterior cingulate cortex (ACC), insular cortex, hippocampus, and amygdala that dynamically interact with midbrain structures  mediating hard-wired defensive reflexes under proximal threat.10Defensive behaviors are part of an evolutionary conserved dynamic defense cascade that depends upon the proximity of threat, as described in the predator imminence model.12Phylogenetically old defensive responses are modulated by individual learning experiences. Fear conditioning enables the organism to avoid future threats in that important information (conditioned stimuli (CS)) signaling a potential threat (unconditioned stimulus (US)) elicits defensive reactions (conditioned fear response). Fear conditioning is considered to be involved in the pathogenesis of pathological anxiety and serves as experimental model for the development of anxiety disorders. On a neural level, fear conditioning involves multiple areas associated with defensive responding such as the (pre-) motor cortex, medial prefrontal cortex/ACC, anterior insula, amygdala, hippocampus and thalamus.14 and animal studies15 evidence that genetic factors represent a significant source of individual variation in (aversive) emotional-associative learning. At least one third of the variance in human fear conditioning14 and the risk to develop anxiety disorders16 has been attributed to genetic factors. The overall startle magnitude in humans has been shown to be heritable  appears to be a temporally stable and trait-like measure.21 This evidence presents fear conditioning and affective startle paradigms as prime laboratory models for investigating genetic influences on neurobiological mechanisms contributing to defensive responding, fear and anxiety.Human1 and focusing on rs7688285 which phenotypically showed the most robust impact, we aimed to conduct a conceptual replication by studying defensive reactivity on the level of brain function, structure, and physiology. We expected GLRB risk allele carriers to (a) exhibit increased fear network activation during two different fear conditioning tasks in regions related to the defensive system’s neuro-architecture such as the brainstem, thalamus, amygdala, hippocampus, insula, medial prefrontal cortex/ACC, and (pre-) motor cortex with different effects for the early vs late acquisition  and morphology (n=416). In addition, we present data of a further sample on GLRB dependent variation of startle habituation . Subjects were screened for psychiatric conditions via the mini-international neuropsychiatric interview,22 and excluded if screening for any lifetime mental disorder was positive. They provided written informed consent and protocols were approved by the General Medical Council of the State of Hamburg.Sample 2: This healthy control group sample was recruited within the national research network PANIC-NET23 .25 After complete description of the study, subjects provided informed written consent. The study had been approved by the ethics committees of all participating centers.IC-NET23  and not 1 Focusing on rs7688285 as main SNP of interest, the Risk group was defined as carrying at least one risk (A) allele (G/A). Complying with the previous report,1 rs7688285 and three further GLRB variants  were used for the Combined GLRB Risk group analyses , as described previously.yses see . Combine28). Subjects viewed a sequence of three different rooms (45 s each) as context CSs, which were assigned counterbalanced to a cue conditioning (predictable: p), a context conditioning (unpredictable: u) or a safe condition (s). During fixed time windows, three different symbols that served as cue CSs (5 s) were superimposed on the assigned room. A black screen with a white fixation cross served as inter-trial interval for 6–8 s (mean 7 s). An individually adjusted electro-tactile stimulus served as US. To mirror experimental designs reported in28 Data of seven subjects were excluded because their proportion of zero or missing responses exceeded a threshold of 66% during the full fear acquisition phase. An α level of P<0.05 was considered significant and Greenhouse–Geisser correction was applied if necessary.The fMRI assessment employed a combined cue and context fear conditioning paradigm (for details see29) with SPM8 (http://www.fil.ion.ucl.ac.uk/spm/) running on MATLAB2013a  smoothed with a 6 mm full-width at half maximum (FWHM) isotropic Gaussian kernel. For first-level analyses, data were analyzed within the framework of the general lineal model for differences in general and differential cued fear responses . Linear models for each of the three sessions included block regressors for three contexts  and one block regressor for the rating phase. Event-regressors were included for three cue-  and US-onsets and convolved with the canonical hemodynamic response function. Parameter estimates (β−) images were calculated and linearly combined for contrasts for main effect responses (CS+ & CS−), as well as differential responses (CS+ vs CS−). As the previous study1 reported findings particularly during early acquisition, we computed first-level contrasts for early acquisition (session one only), late acquisition (session two and three), as well as full acquisition . Second-level group analyses were performed using general lineal model analyses for early, late and full acquisition by two-sample t-test designs. Contrasts of interest included the main effect of group  and the interaction effect of group * CS (CS+ vs CS−). Because of significant group differences in mean age , thalamus, amygdala, hippocampus, insula, ACC, medial prefrontal cortex , supplementary motor areal and precentral gyrus. Small volume corrections were based on a cluster forming threshold of P<0.001 and a family-wise error threshold of P<0.05. Beta values from significant peak voxels were extracted for bar graph visualization.Functional and structural magnetic resonance imaging data were acquired on a 3 T MRI scanner . For fMRI, 38 continuous axial slices  were acquired using a T2*-sensitive gradient echo-planar imaging (EPI) sequence (repetition time (TR): 2.34 s; echo time (TE): 26 ms; flip angle: 80° field of view (FOV): 220 × 220 mm, 2 × 2 mm in-plane resolution) in three sessions (229 volumes/~9 min each) using a 32-channel head coil . To account for T1 equilibrium effects, the first four volumes of each time series were discarded. fMRI preprocessing included realignment, unwarping, co-registration and normalization  were acquired using a magnetization prepared rapid gradient echo sequence (MPRAGE) using a 12- or a 32-channel head coil (coil number was included as covariate). Grey matter differences were analyzed using the voxel-based morphometry (VBM) toolbox  as provided for SPM8. Default settings included a ‘non-linear only’ modulation of the grey matter (GM). Pre-processed images were smoothed with a Gaussian kernel of 8 mm FWHM. Analysis of the covariance between the images identified no images as outliers (mean−2 × s.d.). Correction of multiple comparisons followed the regions of interest approach of the fMRI analyses above. An additional exploratory whole-brain analysis was computed using an uncorrected threshold of P<0.001 and a minimum cluster size of k>=15. Where appropriate, covariates were added if groups differed in demographic or personality properties .Details of structural (s) MRI for brain morphometry have been reported previously.1 startle habituation was assessed for 12 blink responses during inter-trial intervals interspersed during passive viewing of emotional pictures in an affective startle modulation paradigm. Although the task was conceptually similar to the paradigm employed by the previous report, subtle differences with respect to timings, experimental breaks, or selected stimulus material exist. An ANOVA testing for a direct interaction effect of the within- (time block) and the between-subject (genotype) factors was computed. All behavioral and psychophysiological data of the sample were analyzed using SPSS 22  for Windows.Startle reactions were measured by recording electromyographic activity over the orbicularis oculi muscle beneath the left eye using miniature Ag/AgCl electrodes. A 50 ms burst of 95 dB(A) white noise with an instantaneous rise-time was presented binaurally via headphones  as startle-eliciting stimulus. In correspondence with,32 It consisted of three phases (familiarization (F) with 16 trials; acquisition (A) with 32 trials and extinction (E) with 16 trials of each CS ; presentation time: 2000 ms with a variable inter-trial interval of 4.785 to 7.250 s) and an aversive tone  as US between 70 and 105 dB. In the acquisition, the US was pseudorandomly paired with one of the CSs , resulting in equal proportions of CS+paired and CS+unpaired trials of which only CS+unpaired trials were used to avoid confounding effects between CS+ and US processing. Structural data with sufficient high-resolution were not available for sample 2. After each phase, valence and arousal ratings using the Self-Assessment Manikin33 for CSs were obtained using a five-point Likert Scale . Two subjects that rated the US aversiveness below 4  were excluded from analyses. Because of technical problems ratings of one subject were missing. Task duration was 16:49 min. Stimuli were presented by MR-compatible LCD goggles or back projection systems and standard headphones using Presentation 11 . Ratings were analyzed using a three-factorial analysis of covariance with the between-subjects factor ‘risk group’, the within-subjects factors ‘phase’ and ‘CS’ and Beck Depression inventory II (BDI II)34 scores as covariate, with P<0.05 serving as the statistical threshold. If sphericity assumptions were violated, Greenhouse-Geisser correction was applied.A differential fear conditioning and extinction task was employed that has been described elsewhere.35 was performed using a target smoothness of 12 mm FWHM Gaussian isotropic kernel . A detailed description of measures for quality control is given in.32 At first level, realignment parameters were included as regressors of no interest. The BOLD response for each event type  and phase  was modeled by the canonical hemodynamic response function within the framework of the general linear model. Parameter estimates (ß-) and t-statistic images were calculated. At the second level, a group analysis was performed by entering contrast images into a flexible factorial analysis. FMRI center variables and the BDI II were introduced as covariates of no interest. Contrasts of interest included the main effect of group  during acquisition and - in order to test for the specificity of effects for fear learning - extinction, as well as the interaction effect of group × CS (CS+ unpaired vs CS−) for each experimental phase. Corresponding to,1 we further split acquisition as well as extinction into early vs late parts. Two-sample t-tests were used in order to localize the direction of effects. Identical ROI analyses were performed as in sample 1. For exploratory whole-brain analyses and in line with previous analyses of this dataset,36 a Monte-Carlo simulation was conducted to establish an appropriate voxel contiguity threshold.37 Assuming an individual voxel type I error of P<0.005, a cluster extent of 142 contiguous resampled voxels was indicated as sufficient to correct for multiple voxel comparisons at P<0.05. Beta values from significant peak voxels were extracted for bar graph visualization.Images were acquired using 3 T Philips Achieva (Aachen and Münster), 3 T Siemens Trio (Dresden), and 3 T General Electric Healthcare (Berlin) scanners. Five-hundred and five axial functional images , covering the whole brain were recorded. Images were analyzed using SPM5 implemented in MATLAB 6.5. The first five volumes were discarded to minimize T1 saturation effects. Data were filtered to 1/128 Hz to remove low-frequency noise. Functional images were slice-time corrected, temporally and spatially aligned and normalized into standard stereotactic space (2 × 2 × 2 mm). To account for differences in intrinsic smoothness between scanners, an iterative smoothness equalization procedureSample 1. While cue-related acquisition of fear during fMRI was indicated by a significant main effect of CS-type for subjective ratings and skin conductance reactions, no main or interaction effect with GLRB risk genotype was observed (observed .GLRB Risk group during early fear acquisition in the left (anterior) insula / frontal operculum area . During the full course of acquisition, increased activation was observed for GLRB risk carrier in right insula  and left amygdala . Similar findings were obtained when using the Combined Risk group definition as in the previous report1 . During late acquisition, this pattern reversed and showed neural indicators of sustained differential conditioning in the Risk group towards the CS+ while the No-Risk group showed decreased differential CS responding =0.526, P=0.470, see 1 reported strong translational evidence for glycine neurotransmission as a modulator of the brain’s evolutionary old defensive system and provided a strong, biologically plausible candidate intermediate phenotype of defensive reactivity within the domain of negative valence and arousal systems as laid out in the RDoC matrix.Recent findingsGLRB risk allele carriers (rs7688285) in both samples despite two procedurally different fear conditioning tasks, thus supporting the initial report.1 Also in line, we showed increased differential conditioning (CS+>CS−) during late acquisition in the GLRB risk groups on a neurofunctional level. Extending previous evidence, GLRB risk allele carriers showed an inverse pattern of responding by increased processing of the CS− compared with the CS+ during the early acquisition. These findings were also observed when using an alternative Combined Risk group definition previously employed1 .The insular cortex serves as a cortical representation of upstream visceral information.GLRB), have been associated with human hyperekplexia, or startle disease.41 This condition is characterized by exaggerated startle reflexes in response to sudden unexpected auditory, visual or tactile stimuli. On a translational level, this phenotype is confirmed by the spastic mouse , showing exaggerated startle reactions43 as a basic indicator of defensive system reactivity. Evidence on attenuated startle habituation in GLRB risk allele carriers in the present analysis was however negative. Paralleling previous findings1 on a descriptive level, we observed different slopes of startle habituation according to GLRB allelic variation. Single-trial data -allele increased the mean expression of GLRB significantly by 1.34 fold. Neither in the forebrain nor in the amygdalae, rs7688285 affected mRNA expression. Transcript and protein determination of Glrb in spastic mice pointed to low transcript expression of Glrb particularly in the cortex and thalamus.1 We assume that our findings reflect a bottom-up modulatory effect on higher-order defensive system components by more hard-wired reflexes in the brainstem (as reflected by pons and midbrain activation in sample 2), which is corroborated by high-resolution diffusion imaging47 and resting-state48 fMRI studies showing that the insula is functionally connected to the brainstem and that coma-causing brain stem lesions are associated with a dissociation of this network.48Being more ancient than the GABA system, glycine receptors are mainly distributed in evolutionary older brain regions encompassing the spinal cord, brainstem including the midbrain, and olfactory bulb,GLRB gene for enhanced activation of the anterior insula as a core component of higher-order threat-processing systems. However, the GLRB Risk group did not report higher symptoms of anxiety or showed different subjective or autonomic conditionability. This observation is in line with present findings representing an intermediate phenotype, where variance in defensive system responding is related to allelic variation of the GLRB, yet does not lead to symptoms of pathological anxiety. We thus provide further support for the role of GLRB, which is listed under the arousal gene list in the RDoC matrix, for a neurofunctional intermediate phenotype associated with enhanced defensive system reactivity that may increase the vulnerability to develop pathological forms of anxiety.Altogether with the previous report, we provide converging evidence from a total of three different samples on the effect of allelic variation in the 49 Depending on the stimulus presented, subjects will either activate the defensive system towards the CS+, or inhibit it towards the CS-. Despite procedural differences across the fear conditioning tasks, we observed surprisingly strong evidence for enhanced CS discrimination (CS+>CS-) in GLRB risk allele carriers from both samples particularly during the late phase of fear conditioning in fear processing regions such as the thalamus (sample 1) or amygdala, hippocampus, ACC, and insula (sample 2), showing an overlap particularly in the insula and thalamus with previously reported findings.1 Extending these, we additionally observed an inverse effect during early fear conditioning: here the GLRB risk group showed impaired CS- discrimination  or even enhanced responding towards the CS−  that resembles impaired fear inhibitory learning as reported for pathological states of anxiety. While the main group effect of GLRB was driven by enhanced responding in the insula during both acquisition and extinction (sample 2), GLRB effects on differential conditioning were observed during fear learning only, thus pointing towards a specificity in modulating the acquisition of conditioned fear, but possibly not its recall or fear inhibitory learning (as induced during extinction training). Evidence from fear conditioning studies50 suggests that anxiety disorders (and particularly PD51) are not characterized by enhanced reactivity towards the CS+, but rather by attenuated fear inhibition in the face of a safety signal leading to reduced discrimination between both CSs. Using fMRI, enhanced activation of defensive circuit networks in response to a safety signal has been reported as a pathophysiological feature of PD52 and as a marker of non-response towards exposure-based treatment36 that is based on fear-inhibitory learning, reflecting the relevance of safety signal processing for the etiopathogenesis of anxiety disorders. Given further evidence on the relevance of GLRB allelic variation regarding the underlying etiopathogenesis of anxiety disorders, it could represent an additional source in explaining altered patterns of fear conditioning, thus offering potential to stratify patient subgroups to individually tailored, novel therapeutic strategies .Fear conditioning is a basic learning mechanism that allows the organism to flexibly adapt to environmental dangers. Importantly, during differential fear conditioning, the organism learns to process the CS+ as a threat signal, while associating the unreinforced CS- with safety.a priori studies upon. As not enough AA homozygote subjects were available in the fMRI samples, no dose–response relationship on the number of risk alleles could be investigated. fMRI and startle data were not available on the same sample, thus precluding multimodal comparisons across different units of analyses. Recent technical advances for combined assessments will allow for more fine-grained investigations of defensive system functioning in future studies.53 Data on brain morphology with sufficient resolution were only available for sample 1, but not sample 2 (due to restrictions in this multicenter study). Findings may serve as a starting point for further replication approaches on brain morphometric effects of GLRB. Although preliminary findings in the pons and midbrain were observed, it should be noted that the scanning parameters of both studies were not optimized for brain stem imaging, e.g. regarding pulsatory artifacts. Animal studies may furthermore inform about the interplay of these hierarchically organized systems and the modulatory function of the glycine receptors.Owing to the post-hoc nature of this analysis, particularly sample 2 was possibly underpowered for subgroup analyses. However, in yielding a significant difference, these findings speak in favor of a large effect size of rs7688285 on brain activation which could be informative to base power calculations of future 56 and AG, converging evidence for GLRB allelic variation in defensive reactivity was derived from a large-scale translational approach, cross-cutting different levels of analyses. Of note, the robustness of this finding is demonstrated by consistent associations across different experimental paradigms and recording sites. As such, glycine-dependent neurotransmission may open up new avenues for mechanistic research on the etiopathogenesis of fear and anxiety disorders.Representing one of the first GWAS-confirmed findings related to the phenotype of PD


Cochlear outer hair cells (OHC) receive direct efferent feedback from the caudal auditory brainstem via the medial olivocochlear (MOC) bundle. This circuit provides the neural substrate for the MOC reflex, which inhibits cochlear amplifier gain and is believed to play a role in listening in noise and protection from acoustic overexposure. The human MOC reflex has been studied extensively using otoacoustic emissions (OAE) paradigms; however, these measurements are insensitive to subsequent “downstream” efferent effects on the neural ensembles that mediate hearing. In this experiment, click- and chirp-evoked auditory nerve compound action potential (CAP) amplitudes were measured electrocochleographically from the human eardrum without and with MOC reflex activation elicited by contralateral broadband noise. We hypothesized that the chirp would be a more optimal stimulus for measuring neural MOC effects because it synchronizes excitation along the entire length of the basilar membrane and thus evokes a more robust CAP than a click at low to moderate stimulus levels. Chirps produced larger CAPs than clicks at all stimulus intensities (50–80 dB ppeSPL). MOC reflex inhibition of CAPs was larger for chirps than clicks at low stimulus levels when quantified both in terms of amplitude reduction and effective attenuation. Effective attenuation was larger for chirp- and click-evoked CAPs than for click-evoked OAEs measured from the same subjects. Our results suggest that the chirp is an optimal stimulus for evoking CAPs at low stimulus intensities and for assessing MOC reflex effects on the auditory nerve. Further, our work supports previous findings that MOC reflex effects at the level of the auditory nerve are underestimated by measures of OAE inhibition. Cochlear outer hair cells (OHC) receive direct efferent feedback from the caudal auditory brainstem via the medial olivocochlear (MOC) nerve bundle. The MOC bundle inhibits OHC motility and indirectly modulates basilar membrane motion and inner hair cell (IHC) sensitivity—an effect termed the MOC reflex  likely originate from mechanics associated with OHC motility  from the human auditory nerve  That chirps evoke larger CAP amplitudes than clicks using low to moderate stimulus levels, which engage the cochlear amplifier and are thus more sensitive to MOC effects and (2) That MOC reflex inhibition of chirp-evoked CAPs is larger than for click-evoked CAPs due to the broader basilar membrane area represented in chirp responses. To relate our findings to more commonly used MOC reflex assays, we also compared average chirp- and click-evoked CAP inhibition to click evoked OAE (CEOAE) inhibition measured in the same subjects.The University of Arizona Human Subjects Protection Program approved the following methods which were carried out with written, informed consent from all subjects. Eighteen adult participants without history of neurologic or otologic disease were enrolled in the study; however, due to attrition, 14 subjects  completed all six testing sessions. Otoscopy examinations found that all ear canals were free of excess cerumen and that tympanic membranes (TMs) appeared healthy in all subjects. Participants had normal tympanograms bilaterally, defined as ear canal volume of 0.6–1.5 cc and peak-compensated static admittance between 0.3 and 1.4 mL  and α (−77) are constants. The chirp WAV file was converted into a stimulus file suitable for presentation by the Intelligent Hearing Systems Smart-EP program.A 100-μs click and 10-ms chirp were used to evoke CAPs. The click was created using the Intelligent Hearing Systems Smart-EP stimulus generator . The chirp was created in WAV file format in MATLAB  using a modified “O-Chirp” from Fobel and Dau , as impldB peak-to-peak equivalent sound pressure level (ppeSPL) using a 1,000 Hz tone as a reference  to a 2-cc coupler and calibrated in units of Burkard, . Click aBehavioral thresholds for clicks and chirps were obtained from the right ears of 18 subjects using a modified Hughson-Westlake procedure. Stimuli were presented at a starting presentation level of 50 dB ppeSPL. Presentation level was decreased by 4 dB after every positive response and increased by 2 dB after each failure to respond. Threshold was defined as the lowest presentation level at which three positive responses occurred. These measurements were made without electrodes in the ear canal, as our previous work demonstrated that TM electrode contact with the eardrum can influence audiometric thresholds, particularly to low frequencies , left earlobe (−), and forehead  were interleaved throughout the duration of each 2-h session with the exception that the first 20 min of the sixth session was devoted to CEOAE measurements (described in Section CEOAE Measurements). A 60 dB SPL CAS presentation level is commonly used for MOC reflex experiments, as it is the highest BBN level, on average, that elicits MOC reflex activity without triggering the middle ear muscle reflex Guinan, . CAP levRaw amplitude was calculated as the μV difference between the pre-stimulus baseline average amplitude and the N1 peak, which was automatically selected as the largest waveform minimum within a restricted time epoch at each level based on normative click and chirp latency data from our laboratory. Responses were “not present” if the raw amplitude of a peak was less than one standard deviation of the pre-stimulus baseline amplitude. (2) Normalized amplitude expressed each CAP peak magnitude as a percentage of the maximum raw amplitude (either without or with CAS) in the level-series in which it was acquired:The 48 CAP waveforms for each subject were saved as ASCII files and analyzed offline in MATLAB. CAP waveforms were grouped based on stimulus type (click or chirp), level (50–80 dB ppeSPL), and whether they were obtained without or with CAS. CAP amplitudes for each waveform were expressed in two different ways: (1) Three pairs of CEOAE level-series functions , were calculated at each level and compared for raw and normalized level-series functions for each stimulus type. The amplitude scale producing the smallest coefficients of variation at each stimulus level and across all stimulus levels was used in subsequent analyses of CAP inhibition under the assumption that the less variable scale would be more sensitive to “true” physiologic changes induced by the MOC reflex.The first step in testing the hypothesis that chirp-evoked CAPs were more sensitive to MOC reflex inhibition than clicks was to determine whether level-series functions were less variable when expressed either in units of Amplitude reduction was calculated as the average “vertical” (ordinate) difference in CAP amplitudes without and with CAS at each level of the level-series function. This method of quantifying MOC reflex strength is most commonly used in the OAE inhibition literature; (2) Effective attenuation of chirp and click CAPs was calculated as the “horizontal” (abscissa) difference between linear regression fits to level-series without and with CAS using all subject data. Effective attenuation expresses the amount of dB that the stimulus would need to be increased to overcome the effects of MOC reflex inhibition; it is therefore useful in quantifying inhibition in terms of input level, which allows for gross comparisons of pre-neural and neural responses on the same scale  With few exceptions, chirps produced larger raw peak amplitudes than clicks in individual ears, as evidenced by chirp-to-click CAP amplitude ratios Figure . The sizraw amplitudes of chirp-evoked CAPs without CAS were larger than those for clicks at each level tested  revealed that these differences were significant at 50 , 60 , 70 , and 80 dB ppeSPL .The mean d Figure . Paired Representative chirp- and click-evoked CAP waveforms without and with CAS from a randomly selected participant are plotted in Figure t(14) = 3.46, p = 0.0038]. For clicks, the average coefficient of variation was 47% when expressed in raw amplitude and 29% when expressed in normalized amplitude, which was also a significant mean difference . Thus, we used the less-variable measurements expressed in normalized amplitude for subsequent MOC reflex inhibition of CAP analyses.Figure t(30) = 3.55, p = 0.0006] and 60 dB ppeSPL , respectively, using an alpha level (α = 0.0125) to account for multiple comparisons.Average normalized amplitude inhibitions were largest for stimulus levels below 80 dB ppeSPL for both chirps and clicks Figure . Normaliy = 1.33x–11.96, R2 = 0.47; y = 1.59x–33.22, R2 = 0.57) and clicks , respectively  or clicks . Effective attenuation for each stimulus type was calculated as the difference in the abscissa between without and with CAS linear regression lines for equivalent ordinate values  and with CAS  were also fit with separate linear regression models . Effective attenuation was calculated in the same manner as the CAP data. A comparison of chirp-evoked CAP, click-evoked CAP, and CEOAE effective attenuations at 60 dB ppeSPL revealed that inhibition was largest for chirps (3.42 dB), followed by click CAPs (2.49 dB) and CEOAEs (1.93 dB).Based on our findings that CAP amplitudes were less variable when expressed on a normalized scale (see Section CAP Inhibition), we only report CEOAE inhibition in terms of effective attenuation of normalized responses in the present experiment for comparison. CEOAE normalized level-series data from all subjects obtained without  Chirps evoked larger CAP amplitudes than clicks at low to moderate stimulus levels; (2) Normalized CAP amplitude reductions with CAS were largest at the group level using chirps at 50 and 60 dB ppeSPL . These were the only statistically significant amplitude reductions observed; (3) Effective attenuation measurements were largest at the group level for chirp-evoked CAPs followed by click-evoked CAPs and CEOAEs, respectively, at the lowest stimulus levels where all three could be measured .single stimulus file, did not provide the flexibility to use multiple chirps optimized for different levels in this investigation.The chirp generated larger CAP amplitudes at each stimulus level in most subjects; however, the size of this advantage varied considerably across subjects. This finding is consistent with the observations of Chertoff et al.  who usedThe chirp advantage reported here and by Chertoff et al.  suggestsgroup data, which may have been caused by individual differences in both level-series function contours and magnitude of inhibition. An analysis of individual data using the same method resulted in even poorer linear fits due to the fewer data points in the models. Thus, a limitation of our work is that we were unable to reliably resolve efferent inhibition of CAPs at the single-subject level, which is of interest in studying individual variation in MOC function and in understanding the predictive relationships between pre-neural and neural efferent assays. This issue may have been resolved by focusing recording time on obtaining more response averages to fewer low-intensity input levels  and CEOAEs are generated in a nearly equivalent manner through coherent reflection when the spectral power within a bandwidth on the basilar membrane is equal .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.


Notably, HDAC6, a master regulator of the cytotoxic response, and TP53BP1, which sits at the nexus of the double-strand break repair pathway, both underwent a marked transition towards an intron-retention pattern with a concomitant decline in protein levels. These transitions from coding to noncoding isoforms were recapitulated in a large and independent cohort of 499 colorectal samples taken from The Cancer Genome Atlas (TCGA). The set of altered genes was enriched for multiple components of the Fanconi Anaemia, nucleotide excision and double-strand break repair pathways, and together correlating with tumour status at last contact. Altogether, these data demonstrate a new role for hypoxia-driven alternative splicing in regulating DNA damage response, and highlight the importance of considering alternative splicing as a critical factor in our understanding of human disease.Tumour hypoxia is associated with poor patient outcome and resistance to therapy. It is accompanied by widespread changes in gene expression mediated largely through the transcription factors HIF1/2/3α. Hypoxia impacts on multiple pathways throughout the cell and has widespread effects on phenotype. Here we use sample-specific annotation approaches to determine the changes in transcript architecture that arise as result of alternative splicing in hypoxic cells. Using  Hypoxia has multiple impacts on tumour biology including selection of altered cell signalling, angiogenesis, vasculogenesis, changes in central metabolism, suppression of immune reactivity, enhanced receptor tyrosine kinase signalling and down regulation of DNA repair pathways, promotion of prosurvival phenotypes and increased proclivity for invasion and metastasis;4 extensively reviewed in references 8 Hypoxia mediated transcriptional regulation is also controlled by other factors including HIF2α9 and HIF3α.10 In addition, signalling through both the growth factor receptor pathways  and energy depletion pathways  converge on the tuberous sclerosis complex (TSC1/2) leading to complex patterns of spatial and temporal regulation in response to stress. These signals feed into mechanistic target of rapamycin (mTOR), which, in the context of hypoxia, leads to the rapid suppression of protein synthesis,11 presumably in order to conserve energy.12 Levels of hypoxia vary between and within tumours, correlate with patient outcomes, and can lead to differences in response to therapy.13 A better understanding of heterogeneity in hypoxia-driven changes in gene expression will therefore inform strategies for precision medicine,14 raising the need for reliable biomarkers of tumour hypoxia. To this end, a number of groups have developed multiplex gene expression signatures with the intention of better reflecting the multiplicity of pathways involved in the hypoxic response, e.g., references Hypoxia occurs within the majority of solid tumours and is associated with poor patient outcome and chemo- and radioresistance.In recent years, advances in expression profiling have revealed substantial levels of alternative splicing within the human genome, such that the majority of protein-coding genes are now known to express multiple isoforms (median isoform count per protein coding gene in ENSEMBL 74: 5), of which 44% are annotated as noncoding . Despite their prevalence, the majority of these transcripts have yet to be characterised, raising the question of how much of this ‘dark matter’ is functional, and how much is simply a consequence of aberrant splicing and a passive by-product of gene expression.de novo sample-specific annotation strategies to investigate changes in exon structure alongside multiple events including intron retention and alterations to 5′ and 3′ gene boundaries. We applied these novel annotation approaches both to a time course of colorectal cancer cells in reduced oxygen and to reanalyse a large cohort of colorectal samples from TCGA.Given the widespread alterations in transcript expression that arise in response to changes in oxygen levels, we speculated that similar systematic alterations in splicing might add further levels of transcriptional control. We therefore exploited the increased precision offered by RNA sequencing to investigate how hypoxia affects alternative splicing, since earlier studies using 3′ IVT arrays were not able to characterise the transcriptome at this level of precision. We used 2) and poly(A) RNA sequenced using 100mer paired-end strand-specific Illumina sequencing. FACS analysis revealed no significant effect on cell proliferation or apoptosis at 24 h in response to hypoxia  were unannotated , and included multiple genes associated with hypoxia, glycolysis and MAPK signalling nnotated . In keepgnalling . At the gnalling . MultiplN=869/2,949) of spliced genes were predicted by MATS to exhibit multiple concurrent modifications  protein MutS protein homologue 2 (MSH2).25 Levels of intron expression at the locus increased in hypoxic conditions and were accompanied by a significant decline in protein levels 23 to modulate nonhomologous end joining and genetic stability. These data are therefore particularly interesting in the context of a ‘mutator phenotype’ in which a shift to increased genetic instability is postulated to promote clonal diversity within a tumour.24We hypothesised that this might provide a previously unreported mechanism by which cells could modulate protein levels in response to hypoxia, and therefore sought to confirm that a switch towards a noncoding isoform was indeed associated with altered protein levels. Two notable genes with substantial changes in intron retention were HDAC6 and TP53BP1 . HDAC6 in levels . TP53BP1n levels . It sits29 The samples included AJCC pathologic staging (Stage I–Stage IVB), metastatic staging  and therefore included both patients with early-localised disease as well as metastasis. No information was available about previous treatment with radiotherapy or chemotherapy for the majority of patients. As the TCGA data were processed on two different platforms (an Illumina HiSeq 2000 and an Illumina GAII), we tested whether there were significant differences in mapping rates to introns and exons between the two platforms. No inconsistencies were identified between the two platforms, and major isoforms for each gene were assigned independently for each sample, further mitigating against batch effects. In total, 4,303 genes were found to switch between coding and noncoding major isoforms in at least 5% of samples , suggesting that a significant proportion of these changes are driven by changes in oxygenation status within the tumours. Interestingly, 750 genes changed in major-isoform status from coding to noncoding between these two classes . samples . Unsuper samples . The pro classes ; however classes . These dP value <0.01; P value of overlap: <10−6).Finally, we asked whether these splicing changes in tumour samples were associated with specific pathways, and found significant enrichment for DNA damage and repair pathways, as well as alternative splicing  categories; Benjamini and Hochberg corrected 30–32 there has been no evidence on how widely applicable the phenomenon may be.33 Here we identified a comprehensive remodelling of transcript structures in response to hypoxia, encompassing significant changes in the levels of 12% of all protein-coding transcripts, and a switch to a different major isoform at 7% of all protein-coding loci. We observed similar changes in colorectal carcinomas and a widespread switch to noncoding isoforms that correlated strongly with tumour status at last contact. Many of these changes involved expression of novel transcripts not present in the ENSEMBL reference annotation database.Although hypoxia-dependent splicing changes have been reported for individual loci,34 we have identified a novel role for splicing in which switching between noncoding isoforms modulates overall protein output from a locus. Hypoxia-dependent changes were biased towards loss of the protein-coding isoform under hypoxia and consistent with the rapid decline in protein synthesis that accompanies a shift to hypoxic conditions , the nucleotide excision repair pathway  and the double-strand break repair pathway , together reveal a hitherto unanticipated role for alternative splicing in modulating the DNA damage response through action both at critical regulators  for the given time course 24 h after plating. Cells were obtained from ATCC tested and authenticated using multiplex PCR .HCT116 were cultured in RPMI-1640 media  supplemented with 10% FBS . All cells were maintained in a humidified atmosphere at 37 °C and 5% COCells were fixed with 70% ethanol. Briefly, cells were collected and washed in PBS–2%BSA twice and resuspended to a concentration of 3–6 mill/ml. 500 ml were taken and 5 ml cold 70% ethanol was added dropwise to the cells whilst gently vortexing to prevent cell clumping. Cells were fixed at 4 °C for 1 h and stored at −20 °C until ready for FACS analysis. The cells were stained with FxCycle PI/RNase Staining Solution solution (Life Technologies F10797) according to the manufacturer’s protocol and the cells were analysed using the BD FACS Canto 11 system with the BD FACS Diva software version 8 . Data analysis was performed using the Modfit4 software .Protein was extracted by washing cells in ice-cold phosphate-buffered saline (PBS) and scraping cells in ice-cold cell lysis buffer  supplemented with PMSF  and protease inhibitors . The sample was centrifuged at 4 °C 13,000 r.p.m. for 10 min and supernatent was preserved. Total protein per sample of 50 μg was resolved by SDS-polyacrylamide gel electrophoresis 10% NuPage gels  and transferred electrophoretically to Immobilon-P . The membrane was blocked in 5%milk PBS-T for 30 min and blotted overnight with HDAC6 antibody , TP53BP1 , or tubulin antibody . Detection was performed using a peroxidase-conjugated anti-rabbit or anti-mouse IgG  and chemiluminescence visualisation  was used according to the manufacturer’s instructions. Quantification of western blot signals was performed using the Chemi Genius Bioimaging system  and the Chemi genius gel documentation and analysis system.RNA was extracted using the Qiagen Qiashredder kit (79654) and the Qiagen RNeasy Mini Kit (74104) as per the manufacturer’s instructions . The RNA was DNase treated following the protocol in the RNeasy Mini Kit with Qiagen RNase-free DNase I (79254). Indexed PolyA libraries were prepared using 1 μg of total RNA and 13 cycles of amplification in the NEB Next Ultra Directional RNA Library Prep Kit for Illumina . RNA Integrity Values scores were >9 for all samples. Libraries were quantified by qPCR using a Kapa Library Quantification Kit for Illumina sequencing platforms . Pooled libraries were clustered at 15 pmol/l on the cBot and 2×100 bp sequencing was carried out using the High Throughput mode of a HiSeq 2500 using TruSeq SBS Kit v3 chemistry (Illumina)t-tests and Wilcoxon’s tests, were performed in R. Time-course data: 100mer paired reads were aligned to the human genome (hg19) using Mapsplice ,18 which has previously been shown to perform well for de novo splice junction identification.39 An average of 43.8 M (27.5–58.2 M) read-pairs per sample mapped to the genome in the correct orientation and appropriately spaced, corresponding to ~90% of the total reads sequenced. Alternative splicing analysis can be affected by contamination from premature and pre-mRNA transcripts in the poly(A) selected pool. This is manifested by increased numbers of reads mapping to introns. We calculated the read distribution in different regions including exons, introns, untranslated regions, promoters and intergenic regions derived from the genomic annotations in Ensembl (hg19), and found the majority of reads fell within exons and untranslated regions, whereas only a very small proportion of reads mapped to introns. Further, these data were consistent across the samples and there were no significant differences in these overall QC values between replicate groups (https://github.com/cole-trapnell-lab/cufflinks). Resultant models were then merged using Cuffmerge to provide a global model and to classify transcripts as novel, or known, when they mapped to ENSEMBL . In order to minimise false positives, a deliberately stringent filtration was used in order to call novel transcripts: Gene models were first filtered to keep transcripts only when an exon junction was supported by at least 2 reads in at least two samples. This filtration step removed a large proportion of potentially unreliable exon junctions, such that following this step 68.2% of exon junctions completely matched Ensembl annotations, whereas 30% differed in either the start or end site but not both and only 1.8% of which were completely novel <1%) at each time point. Annotation was supplied by the Bioconductor package annmap.42All statistical analysis including e groups . Transcrly novel . The tra43 in R to identify statistically enriched GO terms44 . Non-redundant GO terms were obtained by retaining only one representative term from GO with high semantic similarity, derived using GOSemSim package45 in R.Functional enrichment analysis was performed using the Goseq packageAlternative splicing was detected at both transcript level and exon level. Differential splicing at the exon level was performed for 24 h samples (in hypoxia) versus 0 h using the DEXSeq package in R. DEXSeq uses transcript annotations of each gene to split them non-overlapping bins (exon counting bins) and then uses Generalized Linear Models to identify differentially used exon counting bins. Only exon counting bins that were at least 50-bp long, with mean exonic counts >1 and satisfied a FDR cutoff of 5% were considered as differentially used. Differential splicing at the transcript level was performed for 24-h samples (in hypoxia) versus 0 h using the Cuffdiff program within Cufflinks. In addition to detecting differential splicing, Cuffdiff also reports statistically significant differential promoter usage and differential coding sequence usage. Statistically significant changes were detected at a FDR cutoff of 5%. Following this, the alternative splicing events were annotated into 5 major categories, Exon Skipping, Mutually Exclusive Exons, Intron Retention, Alternative 3′ Splice Sites and Alternative 5′ Splice Sites, using MATS (3.0.8). The splicing events from MATS were detected using both read information and exon junction data and filtered for statistically significant events (FDR<1%).29 comprising of 458 tumour samples and 41 normal samples along with corresponding clinical data was obtained from TCGA. Gene and transcripts quantification and normalisation was performed using the standard Cufflinks pipeline and the annotations in Ensembl (v74). In all cases exon-mapping reads substantially outnumbered intron-mapping reads: median exon/intron ratio per kb=557.5, with minimal difference between HiSeq 2000 and GAII processed samples .Splice-aware aligned RNAseq data (BAM files) of Colorectal cancer cohort (COAD)


The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein that is regarded as one of the markers for tumor initiating cells (TIC) in human hepatocellular carcinoma (HCC). Much work has been directed towards targeting these TICs as a mean of placing these master regulators of cell proliferation and drug resistance under control. Human bone marrow-derived mesenchymal stem cells are known to exhibit an innate property of tumor tropism. However, the possible relationship between MSC and TIC is not well understood. In this study, we show that MSC migration to HCC can be effectively inhibited by TACE and γ-secretase inhibitors that stop the activation of EpCAM signaling event. Silencing of EpCAM expression through siRNA and antibody approaches also resulted in impaired MSC migration. By contrast, increase levels of EpICD proteins in HCC cells and HCC mouse xenografts resulted in enhanced MSC migration. Taken together, these findings show that MSC is drawn to the more oncogenic population of HCC, and could potentially serve as a cell-based carrier of therapeutic genes to target EpICD-enriched hepatic tumor cells. Hepatocellular carcinoma (HCC) is the fifth leading cause of morbidity and mortality in the world . SurgicaHuman mesenchymal stem cells (MSC) have been employed as tumor-tracking missiles for cancer treatment due to their inherent biological property to migrate toward tumors or site of injury and inflammation . The finEpCAM  is a type I transmembrane glycoprotein (40 kDa) consisting of an ectodomain, a transmembrane and an intracellular cytoplasmic domain  and pancIn the present study, we aimed to study the possible relationship between TIC and the tumor tropism of MSC. We demonstrated that MSC migration to HCC tumors can be effectively inhibited by TACE and γ-secretase inhibitors, which prevented the activation of EpCAM signaling. Silencing of EpCAM expression through siRNA and antibody approaches also resulted in impaired MSC migration. By contrast, ectopic expression of EpICD resulted in enhanced tumor cells proliferation in HCC cells via activation of c-Myc, and confers enhanced resistance against sorafenib. Taken together, we have shown that MSC is drawn to the more oncogenic population of HCC and could serve as a cell-based carrier of therapeutic genes whereby specific targeting of TIC among the heterogeneous nature of tumor mass may be made possible, which is otherwise difficult to target by conventional approaches.Bone marrow-derived human MSC were immunopositive for CD44 and double-positive for CD90 and CD105 but negative for CD34 as determined by flow cytometry analysis . MSC alsin vitro. We observed that MSC displayed significant migration towards conditioned medium (CM) derived from various HCC cell lines  and primary HCC cultured from patient-derived xenografts (denoted as HCC 26-1004) when compared to control medium  but not between siEpCAM versus siCtrl parental Huh7 cells (p = 0.06). Together, these findings indicate that the migration of MSC is indeed mediated through the EpCAM associated signaling event.In an attempt to study the possible relationship between EpCAM and MSC recruitment to HCC, surface expression of EpCAM on MSC and tumor cells were first examined by FACS analysis using FITC-conjugated EpCAM antibodies. High levels of EpCAM expression could be detected in representative HCC cell line . The results showed that distinct membrane staining could be detected in EpCAMhigh but not in EpCAMlow tumors  and EpCAMlow (i.e. EpICDhigh) in each animal. The results showed that EpICDhigh tumors attracted more MSC when compared to EpICDlow tumors  expressing either full-length EpCAM or EpICD were demonstrated to confer oncogenicity in a SCID mouse model . To furthigh expressing HCC tumors, which represent a highly oncogenic tumor microenvironment, could recruit more MSC. This provides an additional benefit of using MSC as a cell-based carrier of therapeutic genes to target EpICD-enriched hepatic tumor cells.In the present study, we demonstrated that MSC migration to HCC tumors can be effectively inhibited in the presence of TACE and ɣ-secretase inhibitors, which prevented the activation of EpCAM signaling event. Likewise, silencing the EpCAM expression through siRNA and antibody approaches also resulted in impaired MSC migration. Interestingly, animal studies showed that EpICDIn the field of cancer therapy, the most attractive feature of MSC is its inherent ability to migrate towards tumor cells or “a wound that never heals”. Migration of MSC to tumors has been demonstrated in many types of cancers, indicating that this phenomenon is independent of the type of tumor (reviewed by ). This ahigh –expressing Huh7 formed larger tumor mass in comparison to similar number of inoculated EpICDlow Huh7 cells in the same animal  while Huh7 cells were from Japanese Collection of Research Bioresources . Other HCC cells including Mahlavu and MHCC97H cells were kindly provided by Prof. Antoinette Lemoine  and Prof Zhao-You Tang  respectively. Lung primary fibroblast cell line MRC5 was a kind gift from Dr. Robert Chua . All cells were grown using standard tissue culture conditions. Primary cells derived from tumors were prepared by collagenase-dispase  treatment of tumor samples into a single cell suspension. MSC7F3753 (denoted as MSC) were obtained from Lonza BioSciences  and cultured in MSC growing medium (MSCGM) (Lonza BioSciences) for no more than 8 passages. Cell proliferation was determined at indicated time points by using CCK-8 kit  according to manufacturer's protocol.6 viable cells) at 48 hours post seeding. The harvested media was subsequently centrifuged at 1000rpm for 5 minutes, filtered through 0.2 μm filter to remove cellular debris and added to the bottom well of a modified Boyden chamber as previously described [4 MSC were cultured in 24-well tissue culture insert on the upper well with an 8 μm pore size membrane (BD Biosciences). After 8 h, the number of propidium iodide-stained MSC nuclei was scored on the underside of the membrane under x 200 magnification at five random fields per replicate. For antibody blocking, Huh7 cells were treated with anti-EpCAM clone (EpEx) BerEP4 or normal mouse IgG1 (DAKO) at various concentrations overnight at 37°C prior to migration assay.Conditioned media (CM) was harvested from different cell types . After 72 hours post-transfection, cells were harvested and re-seeded at a density of 1 × 104 cells in 24-well dish in 300 μl of 10% FBS containing DMEM medium. The western blot analysis and migration assays were performed as previously reported [EpCAM-enriched Huh7 cells were treated with 40μM of TAPI-1 (Sigma-Aldrich) or 10μM of DAPT  or a combination of both inhibitors for 24 hours. Migration of MSC towards these inhibitors-treated CM was then analyzed. For EpCAM knockdown experiments, 1 × 10reported . EpCAM-n