Recently developed small molecule PKD inhibitors, CID755673 and CRT0066101, provide potentially important pharmacological approaches to further investigate the effect of PKD in pancreatitis therapy. The aim of this study was to explore whether CID755673 and CRT0066101 block NF-κB activation with in vitro and in vivo models of experimental pancreatitis and whether the small molecule PKD inhibitors have therapeutic effects when given before or after the initiation of experimental pancreatitis. Freshly prepared pancreatic acini were incubated with CID755673 or CRT006101, followed by hyperstimulation with CCK or CCh. For in vivo experimental pancreatitis, rats were treated with intraperitoneal injection of CID755673 or CRT0066101 prior to or after administering cerulein or saline. PKD activation and NF-κB-DNA binding activity in nuclear extracts from pancreatic acini and tissue were measured. The effects of PKD inhibitors on pancreatitis responses were evaluated. Our results showed that both CID755673 or CRT0066101 selectively and specifically inhibited PKD without effects on related protein kinase Cs. Inhibition of PKD resulted in significantly attenuation of NF-κB activation in both in vitro and in vivo models of experimental pancreatitis. NF-κB inhibition by CID755673 was associated with decreased inflammatory responses and attenuated severity of the disease, which were indicated by less inflammatory cell infiltration, reduced pancreatic interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased intrapancreatic trypsin activation, and alleviation in pancreatic necrosis, edema and vacuolization. Furthermore, PKD inhibitor CID755673, given after the initiation of pancreatitis in experimental rat model, significantly attenuated the severity of acute pancreatitis. Therapies for acute pancreatitis are limited. Our results indicate that small chemical PKD inhibitors have significant potential as therapeutic interventions by suppressing NF-κB activation.Nuclear factor-kappa B (NF-κB) activation is a key early signal regulating inflammatory and cell death responses in acute pancreatitis. Our previous  Acute pancreatitis is a disorder with pathologic features of inflammation and necrosis in pancreatic tissue. Increasing evidence has demonstrated that inflammatory response plays a pivotal role in the development of the disease  family, composed of PKD/PKD1, PKD2, and PKD3, has recently characterized as an important target in the signaling cascades initiated and transduced through G protein coupled receptors, phospholipase C, second messengers, and PKC-dependent or -independent mechanisms in various cell types including pancreatic acinar cells ; Medium 199 was from GIBCO . ATP and [γ-32P] ATP were from Perkin Elmer . CRT0066101 and CID755673 were obtained from TOCRIS . Nitrocellulose membranes were from Schleicher and Schuell BioSience. Carbachol and GF1  were from Calbiochem . Antibodies against PKD C-20, IκB-α, or LDH were from Santa Cruz Biotechnology . Phosphoserine 744/748 PKD antibody that detects primarily the phosphorylated state of Ser 744  and humidity- (55 ± 5%) controlled room with a 12-h light/dark cycle (lights on at 07:00 a.m.). The animals were provided Animal care and all procedures were approved by the Institutional Animal Care and Use Committees of the Veterans Affairs Greater Los Angeles Health Care System and Cedars-Sinai Medical Center, Los Angeles in accordance with the National Institutes of Health guidelines USA. Rats were kept under specific pathogen-free conditions and received humane care according to the guidelines of our institution. All experiments were performed according to the guidelines of the National Institutes of Health guidelines.2-humidified atmosphere. For experimental purposes, the acinar cells were pre-incubated in the medium 199 for 2 h at 37°C with or without inhibitors as described previously (36) and then treated further with or without agonists. Acinar cells were collected and washed with PBS. Aliquots of the acinar cells were lysed and sonicated in lysis buffer as described previously  using a collagenase digestion method as described previously  were pre-treated with intraperitoneal (IP) injection of PKD inhibitor CID755673  or CRT0066101  60 min prior to initiation of pancreatic stress with up to 4 hourly IP injections of cerulein (20 μg/kg), a CCK analog used for experimental pancreatitis models. Control animals received similar injections of physiologic saline. We choose the dosages of the PKD inhibitors in the animal experiments based on our previous dose response  or vehicle was administered by IP injection to rats at early and late stages after the initiation of pancreatitis . The animals were sacrificed in 1 h after the 6 hourly cerulein injections.Nuclear protein extracts were prepared using ActiveMotif Nuclear Extract Kit  following the manufactory instructions. NF-κB DNA binding activities were measured with electrophoretic mobility shift assay (EMSA) as described in our laboratory previously  and 2.5 mg/ml syntide-2 (PLARTLSVAGLPGKK) in kinase buffer. After 10 min of incubation at 30°C, the reaction was stopped by adding 100 μl of 75 mM H3PO4, and 75 μl of the supernatant was spotted on P-81 phosphocellulose paper. Free [γ-32P]ATP was separated from the labeled substrate by washing the P-81 paper four times for 5 min in 75 mM H3PO4. The papers were dried, and the radioactivity incorporated into syntide-2 was determined by Cerenkov counting.PKD in pancreatic tissue lysates was immunoprecipitated at 4°C for 3 h with the PKD C-20 antibody (1:100) and protein-A-agarose as previously described  Custom Service. Active trypsin in pancreatic tissue was measured by using Boc-Gln-Ala-Arg-AMC as a substrate by a fluorescent assay as described previously  sections stained with H&E as described previously  sections from at least 4 mice per group and expressed as the number of inflammatory cells or vacuoles per 100 acinar cells. Pancreatic edema grading was made on the H&E stained tissue sections from 0 to 3 according to the Schoenberg grading system  followed by Bonferroni multiple comparison post-hoc tests with the GraphPad Prism software . T-tests were used to analyze differences between two groups. P < 0.05 was considered statistically significant.Results were expressed as the mean ± in vitro  proteins. Upon activation, the inhibition of NF-κB by IκBs is removed as soon as IκBs are phosphorylated by IKK kinases and the phosphorylated IκBs rapidly degrade via proteasome-involving pathways. Active NF-κB will redistribute into the nucleus and promote the expression of a number of genes that contain κB-binding sites in their promoters/enhancers  or CRT0066101 (10 mg/kg) and the control rats received the same volume of vehicle (DMSO). Sixty minutes later, cerulein  or saline only were administered to the inhibitor- or vehicle-pretreated animals. Thirty minutes after the cerulein injection, we harvested the pancreas and measured pancreatic PKD/PKD1 catalytic activity with in vitro kinase assays. As showed in Figure in vitro experimental results shown above, the two inhibitors also suppressed PKD Ser916 autophosphorylation . The combined results in Figures We then investigated whether the inhibition of PKD activation by the two small molecule chemicals has effect on NF-κB activation in cerulein pancreatitis animal model. The nuclear extracts prepared from the rat pancreatic tissue were used in EMSA measurement for NF-κB DNA binding activity. Figures To test whether the inhibition of NF-κB by the PKD inhibitor altered expression of inflammatory molecules in pancreatitis, we examined pancreatic level of cytokine IL-6 and chemokine MCP-1. As shown in Figure Subsequently, we evaluated the effect of NF-κB inhibition by the PKD inhibitor CID755673 on the pathologic changes in pancreatitis. Following 1 h injection of CID755673 (15 mg/kg), we gave the rats up to 4 hourly IP injections of cerulein (20 μg/kg) and the control rats received saline IP injections. The rats were then sacrificed at 30 min and 4 h after the first cerulein injection.in vivo animal model of pancreatitis indicated that NF-κB inhibition by the PKD inhibitor CID755673 was associated with significantly decreased severity of acute pancreatitis.The results in Figure P = 0.054) between CID755673-treated and the control groups  or vehicle by intraperitoneal (IP) injection to rats in early and late stages after the initiation of pancreatitis . The animals were sacrificed in 1 h after the 6th hourly cerulein injections and the pancreas were collected. We found that blood amylase and lipase dramatically elevated after 6 hourly IP injections of cerulein Figure , but, bos Figure . Histolos Figure . Compares Figure , inflamms Figure , necrosis Figure , and vacs Figure  in CID75Acute pancreatitis is a serious medical disorder and at present, there are no treatments targeting its molecular pathogenesis. The transcription factor NF-κB has been known as an important regulator of the production of the inflammatory molecules and plays a critical role in the inflammatory response and parenchymal injury in pancreatitis (Gukovsky et al., in vitro model of pancreatitis induced by CCK and the cholinergic agonist CCh (Yuan et al., in vivo experimental models of pancreatitis. Recent development of novel small molecule chemical PKD inhibitors brings us a useful pharmacological approach to achieve this goal. We report here that PKD is a critical regulator of NF-κB activation in experimental models of pancreatitis. Either in vitro or in vivo application of the two PKD inhibitors, CID755673 and CRT0066101, significantly prevented NF-κB activation in early stage of pancreatitis.Our previous studies with molecular approach in AR42J cell line showed that PKD was required in NF-κB activation in We first showed that either CID755673 or CRT0066101 was a specific PKD inhibitor that potently blocked agonist-induced PKD autophosphorylation at Ser916 without affecting PKC-dependent Ser744/748 phosphorylation (that was inhibited by PKC inhibitor Go6983, Figure It has been known that NF-κB activation takes place when the inhibitory IκB protein is degraded and dissociated with the NF-κB complex. Activated NF-κB then redistributes to nucleus and promotes the expression of a number of genes that contain κB-binding sites in their promoters (Gukovsky et al., in vivo experimental model of pancreatitis. Consistent with our in vitro experimental results, selective and specific inhibition of PKD catalytic activity and Ser 916 autophosphoryltion by either inhibitor (Figure Next, we employed CID755673 and CRT0066101 in animals to study whether PKD inhibition affected NF-κB activation in r Figure .We then explored whether suppressing NF-κB activation by the PKD inhibitors was associated with attenuation of inflammatory responses and severity of the disease in experimental model of pancreatitis. We found that NF-κB inhibition by CID755673 was associated with significantly decreased inflammatory responses indicated by reduced pancreatic IL-6 and MCP-1 proteins (Figure Cytokines have multiple functions in regulating the immunoinflammatory responses. In particular, IL-6 plays a key role in the convert from neutrophils to macrophages during the transformation from innate to acquired immunity (Maianski et al., The transcription factor NF-κB has been known as a major regulator of the inflammatory molecules expression in pancreatitis. Both IL-6 and MCP-1 expression is under the control of NF-κB because promoters of these genes have DNA binding sites of NF-κB (Ben-Baruch et al., in vitro pancreatitis model in rat pancreatic acinar cells (Thrower et al., in vivo rat cerulein-induced pancreatitis, which supported our previous in vitro experimental results, confirming again PKD regulates zymogen activation in pancreatitis.Previously, we investigated whether the PKD inhibitor CRT0066101 would prevent zymogen activation in pancreatitis in an There was very little information regarding application of the PKD inhibitors in therapy of pancreatitis in experimental animal models. It is exciting that our final results achieved from experiments in which the rats were treated by CID755673 after the pancreatitis has commenced showed that the post-treatment of the PKD inhibitor significantly attenuated pancreatic injury in cerulein-induced pancreatitis (Figure in vivo model of pancreatitis. Small chemical inhibitors of PKD have significant potential as therapeutic intervention for pancreatitis through blocking NF-κB activation, a critical early pathological event in this disease. Combining with our previous reports (Yuan et al., In summary, our studies demonstrate that PKD is a key mediator of NF-κB activation in JY drafted the manuscript and designed the study. JY, TT, MG, GT, and CC acquired and analyzed the data. JY and SP interpreted the data, critically revised the manuscript, and approved the version to be published.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.


Preliminary studies suggest that sports-related concussion (SRC) is associated with alterations in cerebral blood flow (CBF) regulation. Here, we use advanced magnetic resonance imaging (MRI) techniques to measure CBF and cerebrovascular responsiveness (CVR) in individual SRC patients and healthy control subjects.2 during blood oxygenation level-dependent (BOLD) MRI. Group differences in global mean resting CBF were examined. Voxel-by-voxel group and individual differences in regional CVR were examined using statistical parametric mapping (SPM). Leave-one-out receiver operating characteristic curve analysis was used to evaluate the utility of brain MRI CO2 stress testing biomarkers to correctly discriminate between SRC patients and healthy control subjects.15 SRC patients  and 27 healthy control subjects  underwent anatomical MRI, pseudo-continuous arterial spin labeling (pCASL) MRI and model-based prospective end-tidal targeting (MPET) of CO2 stimulus and O2 targeting during BOLD MRI. Significant group and patient-specific differences in CVR were observed with SRC patients demonstrating a predominant pattern of increased CVR. Leave-one-out ROC analysis for voxels demonstrating a significant increase in CVR was found to reliably discriminate between SRC patients and healthy control subjects . The optimal cutoff for increased CVR declarative for SRC was 1,899 voxels resulting in a sensitivity of 0.867 and a specificity of 0.778 for this specific ROC analysis. There was no correlation between abnormal voxel counts and Postconcussion Symptom Scale scores among SRC patients.All studies were tolerated with no complications. Traumatic structural findings were identified in one SRC patient. No significant group differences in global mean resting CBF were observed. There were no significant differences in the CO2 stress testing in individual patients.Acute and subacute SRCs are associated with alterations in CVR that can be reliably detected by brain MRI CO Expert consensus has historically viewed sports-related concussion (SRC) as an injury resulting in clinical features that arise from transient disturbances in brain functioning occurring in the absence of structural brain injury . Clinica2 during blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI), we have previously demonstrated patient-specific qualitative and quantitative regional alterations in CVR among adolescent postconcussion syndrome patients while global mean resting CBF remained within normal limits (Among the processes implicated in the pathogenesis of acute concussion and traumatic brain injury (TBI) are disturbances in cerebrovascular physiology that result in a mismatch between the metabolic demands of the injured brain and the delivery of cerebral blood flow (CBF) –13. One l limits . Althougl limits –21.In this study, we examined group differences in global mean resting CBF and CVR in healthy control subjects and adolescent acute and subacute SRC patients. Using a leave-one-out receiver operating characteristic (ROC) analysis we examined individual differences in CVR among SRC patients compared to an institutional atlas of healthy adolescent control subjects assessed using the same neuroimaging assessment technique.We conducted a case–control study of adolescent SRC patients that were compared to an institutional atlas of healthy control subjects that includes subjects used in previous studies conducted by our group , 22. AdoIn general, SRC patients were deemed clinically recovered when they were asymptomatic at rest, were back to full-time school, had a normal neurological examination, and successfully completed the International Consensus on Concussion in Sport return-to-play guidelines . Some paInstitutional research ethics approval at the University of Manitoba was obtained for this study. Informed patient, and where applicable, parental consent was obtained for all participants prior to participating in the study. On the day of neuroimaging, all subjects completed the Postconcussion Symptom Scale (PCSS), a concussion symptom inventory consisting of 22 symptoms that are rated on a 7-point (0–6) Likert scale with a maximum score of (6 × 22) = 132 .Prior to neuroimaging assessment, all study subjects were exposed to a short trial of the breathing sequence to familiarize them with the changes in breathing that would occur while in the MRI scanner. During MRI assessment, subjects underwent non-invasive heart rate, pulse oximetry, and blood pressure monitoring. Subjects were also given a hand-held call button that allowed them to voluntarily terminate the test at any time. Neuroimaging assessment in this study consisted of (1) anatomical imaging; (2) BOLD CVR mapping; and (3) global resting CBF measurements that were all obtained in same order.2 targeting. BOLD MRI data were acquired using a T2*-weighted single-shot gradient echo pulse sequence with echoplanar readout . Global mean resting CBF was assessed using pseudo-continuous arterial spin labeling (pCASL) that included an initial M0 scan—Siemens ep2d_pCASL—echo planar readout . The formal pCASL sequence then followed consisting of an echo planar readout . Imaging duration was for 3 min. The first two labeled–non-labeled pairs were discarded.Images were acquired using a Siemens Verio 3.0 T MR scanner with a 12-channel phased-array head coil. Anatomical imaging was acquired without manipulation of end-tidal gases using a sagittal 3D T1 MPRAGE  and axial gradient recalled echo planar (GRE) sequences to screen for cerebral microhemorrhages as well as GRE B0-field mapping. CVR was assessed using continuous BOLD MRI during MPET CO2 and O2via a computer-controlled gas blender  connected to a rebreathing circuit and mask secured to the subject’s face. This device allows MPET targeting of PETCO2 under isoxic conditions while providing continuous breath-by-breath measurement of PETCO2 and PETO2. Previous work has demonstrated that measurement of PETCO2 is an accurate surrogate for PaCO2 during MPET CO2 targeting (ETCO2 (120 s), hypercapnia (5 mmHg above baseline for 120 s), baseline PETCO2 (30 s), hypercapnia (5 mmHg above baseline for 120 s), baseline PETCO2 (30 s), hypercapnia (5 mmHg above baseline for 120 s), and baseline PETCO2 (120 s). During this sequence a stable PETO2 of 115 mmHg was targeted. A representative illustration of the breathing sequence is provided in Figure The vasodilatory stimulus used for CVR mapping in this study consisted of precise delivery of COargeting . The breg  toolbox and custom written in-house MatLab scripts. The BOLD data were interpolated to the MPRAGE voxel dimensions. The fMRI model specification was a two-pass process. First, the data were modeled using the finite impulse response package in SPM. The MPET triple stimulus square-wave sequence was assessed with a zero offset. An event-related response was calculated by examining the whole brain response to the COFurther preprocessing was conducted using a motion correction file generated with realignment as regressors in the model. First-level CVR analyses were undertaken using the time-corrected analysis, and the contrast images generated were then used in the second level CVR analysis. In order to account for BOLD EPI signal inhomogeneities at the skull base and within the choroid plexus and periventricular white matter , individhttp://www.cfn.upenn.edu/~zewang/ASLtbx.php).An ASL toolbox was used to undertake preprocessing of the pCASL data . For each individual second-level comparison, the images were masked using the combined individual mask described above versus the atlas output. Voxel counts calculated for each subject were based on the applied individual mask. For the control subjects each of their contrast images were removed in turn (leave one out) from the atlas to determine the second-level voxel counts for that control subject. We report on the p = 0.005 values for each patient and subject.Second-level analysis for the CVR data was completed among individual SRC patients and control subjects. Voxel-by-voxel comparisons on a group and individual basis for the SRC patients was conducted at the p = 0.005 greater than and less than voxel counts for each individual. The ROC curves were constructed comparing the leave one out counts for each control subject (SRC = 0) and the counts calculated for each SRC patient (SRC = 1). The Analyse-It statistical package for Excel 2010 was used to calculate the ROC AUC values for these voxel counts.Leave-one-out ROC analyses were undertaken to determine whether abnormal voxel counts derived from the second level CVR analysis could accurately discriminate between individual SRC patients and the leave-one-out results determined for each healthy control subject on a quantitative basis. Second-level analysis voxel counts for each individual  were determined based on the 2 targeting. Therefore, 27 healthy control subjects and 15 SRC patients were included in the final analysis. Two acute SRC patients included in a prior study of longitudinal CVR imaging were included  of healthy control subjects with one patient found to have a right frontal developmental venous anomaly. Anatomical imaging studies were normal in 11/15 (73%) of SRC patients. Abnormalities found among SRC patients included a left frontal developmental venous anomaly (one patient), pineal cyst (one patient), non-functioning pituitary microadenoma (one patient), and a traumatic microhemorrhage located in the splenium of the corpus callosum that was identified on imaging obtained following a previous concussion  was 0.223 ± 0.127 for the SRC patients and 0.139 ± 0.079 for the control subjects  and greater than  mean control group responses at the second-level analysis voxel counts at the p = 0.005 level is shown in Figure First- and second-level group comparisons of unmasked CVR data are summarized in Table 2 stress testing was found to reliably discriminate patients with and without SRC with an ROC AUC of 0.879 (p = 0.0001). At a cut-point of approximately 1,900 voxels for increased CVR derived from our second-level analysis at the 0.005 level, this biomarker resulted in a sensitivity of 87% and a specificity of 78% for correctly identifying patients with SRC. There was no discrimination seen for a decreased CVR response for these SRC patients (AUC = 0.468). Importantly, all neuroimaging studies were well tolerated with no complications.This study examined global mean resting CBF and global and regional CVR in adolescent SRC patients and healthy controls subjects. In a heterogeneous population of SRC patients who underwent neuroimaging assessment at various stages of acute and subacute injury, we observed significant group differences in CVR that occurred in the setting of normal global mean resting CBF. When compared to an atlas of healthy control subjects, we found that SRC patients demonstrated significant patient-specific alterations in CVR with a predominant pattern of increased CVR including those patients who were found to demonstrate exercise intolerance during graded aerobic treadmill testing and were diagnosed with autonomic/physiological PCD. Furthermore, a preliminary leave-one-out ROC analysis utilizing the quantitative biomarker of increased CVR response (voxels demonstrating an increased response to a controlled hypercapnic stimulus) generated from brain MRI CO2 challenge. In the SRC group, they observed increased CVR for all preselected regions of interests including significant increases within the median default mode network and the anterior cingulate. They also found that increased CVR in the hippocampus was positively correlated with increased functional connectivity between the hippocampus and precuneus in the SRC group but not in the healthy control group. The authors also found no group differences in resting mean CBF between groups similar to the results of our present study. Finally, in a previous study our group examined CVR in 17 healthy control subjects and 15 adolescent SRC patients who remained symptomatic during the chronic phase of injury (33–993 days postinjury)  . Using M2, and MPET CO2 targeting (2) over time (ETCO2 detected at the end of breath-holding (the measured stimulus) is not always equal to the PaCO2 ; and PETCO2 and PETO2 levels cannot be sampled throughout the breath-holding periods , a company affiliated with the University Health Network that developed the RespirAct™, a non-commercial research tool assembled by TRI to enable cerebrovascular reactivity studies. The other authors have no financial disclosures or conflicts of interest to declare.


In response to sub-lethal metabolic stress, adult human oligodendrocytes reduce overall energy production rate impacting the capacity to maintain myelination. Here, we directly compare the metabolic profiles of oligodendrocytes derived from adult rat brain with oligodendrocytes newly differentiated in vitro from oligodendrocyte progenitor cells obtained from the post natal rat brain, under both optimal culture and metabolic stress (low/no glucose) conditions. Oxygen consumption and extracellular acidification rates were measured using a Seahorse extracellular flux analyzer. Our findings indicate that under optimal conditions, adult rat oligodendrocytes preferentially use glycolysis whereas newly differentiated post natal rat oligodendrocytes, and the oligodendrocyte progenitor cells from which they are derived, mainly utilize oxidative phosphorylation to produce ATP. Metabolic stress increases the rate of ATP production via oxidative phosphorylation and significantly reduces glycolysis in adult oligodendrocytes. The rate of ATP production was relatively unchanged in newly differentiated post natal oligodendrocytes under these stress conditions, while it was significantly reduced in oligodendrocyte progenitor cells. Our study indicates that both age and maturation influence the metabolic profile under optimal and stressed conditions, emphasizing the need to consider these variables for in vitro studies that aim to model adult human disease.Within the microenvironment of multiple sclerosis lesions, oligodendrocytes are subject to metabolic stress reflecting effects of focal ischemia and inflammation. Previous studies have shown that under optimal conditions  Oligodendrocytes (OLs) are the myelinating cells in the CNS. Carbon 14 dating studies performed on post mortem human tissues indicate that OLs in the adult brain are long lived cells with low levels of turnover .in vitro optimal conditions have distinct metabolic properties compared to OLs derived from post natal rat brain and matured in vitro; the human cells primarily utilize glycolysis for ATP production instead of OXPHOS [in vitro are more resistant to metabolic stress (low nutrient and glucose) and to exposure to pro-inflammatory mediators (tumor necrosis factor) compared to their post natal counterparts in rat [in vitro, the cells showed a significant reduction in overall energy utilization, particularly in glycolytic ATP production.Myelinating OLs are highly specialized cells that require substantial amounts of energy to make and maintain their elaborate processes. Myelin maintenance depends on oxidative phosphorylation (OXPHOS) for ATP production ; howeverf OXPHOS . We furts in rat . These cs in rat . We docus in rat –10. At tin vitro from OPCs obtained from the post natal rat brain; the typical preparation used to assess OL response to injury in vitro. A further comparison was made between these newly differentiated OLs and the initial post natal OPCs.To assess how well the adult rat derived cells may model OLs in the adult human brain, the current study aimed to define the metabolic properties of existent OLs derived from the adult rat brain under optimal conditions. We then compared the response of these cells to metabolic and inflammatory insults with that of OLs newly differentiated Sprague-Dawley rats were obtained from Charles River Canada . The animals were euthanized with isoflurane exposure preceding the CO2 inhalation followed by cervical dislocation.Ethics statement: All procedures involving animals were performed in accordance with the Canadian Council on Animal Care’s guidelines for the use of animals in research. McGill University Animal Care Committee approved the use of animals under the protocol number 4330.OPCs and OLs were prepared from the brains of P2 Sprague-Dawley rats  as previously described  and plat5 cells per mL on poly-L-lysine coated chamber slides and cultured in the aforementioned differentiation media.OLs were prepared from the brains of adult Sprague-Dawley rats. Briefly, the meninges were removed from the brain. Tissue was enzymatically digested and placed on a linear 30% Percoll density gradient . The total cell fraction was cultured for 24 hours in non-coated flasks. The floating cell fraction were plated at a density of 2.5 × 10Selected cell cultures derived from both the post natal and adult brain tissue samples were immunostained with monoclonal antibody that detects OL lineage marker O4 (mid-late OL marker) for 30 minutes at 4°C then fixed with 4% paraformaldehyde for 10 minutes at 4°C, followed by blocking with HHG  for 10 minutes. Cultures were incubated with goat anti-mouse IgM Cy3 or FITC for 30 minutes at room temperature. Antibody isotype controls showed very low nonspecific staining (data not shown). Cells were co-stained overnight with Ki67 (detection of cell proliferation) , Olig2 (transcription factor expressed throughout the OL lineage) , platelet derived growth factor α (PDGFRα) (an OL progenitor cell marker)  or myelin basic protein (MBP) (a very late OL lineage marker) , or markers for non-OL lineage cells, namely astrocytes (GFAP)  and microglia (IbA1) . After washing, the cells were incubated with the corresponding secondary antibodies and final nuclear staining for Hoechst 33342  for 60 minutes at room temperature. The stains were visualized using epifluorescent microscopy  and Open Lab imaging software .Following the initial culture stages , cells were treated with either optimal , low glucose/DMEM (0.25g/L glucose) or no glucose/DMEM media for 24 hours. After 24 hours, cell death was assessed by TUNEL staining using a commercial kit . Cell nuclei were stained with Hoechst 33258  for 10 minutes at room temperature. Cells from these cultures were then evaluated in the Seahorse bio-analyzer.Prior to running samples in the Seahorse analyzer, cells from all experimental conditions  were placed in XF assay media (pH adjusted to 7.4) and equilibrated in the Seahorse incubator. As cell survival differed amongst cell types in response to the stress conditions, all Seahorse values were normalized based on total protein concentrations. The XFe96 plate was placed in the Seahorse analyzer  where 4 baseline cycles were performed consisting of a 3 min mix followed by 3 min measure. This was followed by the addition of oligomycin (0.5 μM injection volume) for 3 assay cycles. Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone  was then added for an additional three assay cycles, followed by rotenone (1 μM) plus antimycin A (2 μM) for another three assay cycles. All drugs were added by automatic pneumatic injection. Extracellular acidification rates were calculated by the addition of 2-deoxy-glucose (2-DG) (1M). All reagents were purchased from Sigma .The difference in oxygen consumption rate (OCR) between the basal level and following oligomycin addition were determined. This was then used to convert OCR measures to ATP production using a phosphate/oxygen ratio of 2.3 , 13. GlyA two-way ANOVA analysis was performed for most of the comparisons. An unpaired Student’s t-test was used for all comparisons depicted as bar graphs. All results are presented as the mean ± SEM. The number of experiments for each study is indicated in the results section. Probability values < 0.05 were considered statistically significant and are indicated in figure legends.in vitro under optimal and metabolic stress conditions and compare the response of these cells with newly differentiated OLs derived from OPCs isolated from the early (P2) post natal rat brain. Our goal was to assess how closely the rat derived cells model the previously observed characteristics of adult human OLs [Our primary aim in the current study was to characterize the metabolic properties of OLs isolated from the adult rat brain and maintained uman OLs .in vitro, and the initial post natal OPCs under optimal culture conditions are provided in in vitro and post natal OPCs under optimal conditions (N1) respectively. At day 8 in culture, a mean of ~89% of the in vitro differentiated post natal OL cultures are O4+ and ~75% of these cells are MBP+. At day 4 in vitro, ~90% of the cells in the post natal OPC cultures express O4 and only ~4% of these are MBP+.As summarized in in vitro  and extracellular acidification rate (ECAR) to determine ATP production by OXPHOS and glycolysis, respectively.OCR—For the purpose of this paper, the resting OCR refers to the OCR measurement before the addition of mitotoxins, which are mitochondrial inhibitors used to determine the mechanism of oxygen consumption using the Seahorse XF96 extracellular flux analyzer.in vitro is higher compared to adult OLs  or a more severe injury  for 24 hours using a previously established model (5).OCR–Our findings revealed that the resting OCR of adult OLs increased under LG conditions and were increased further still in NG . The OCRECAR–ECAR was not significantly altered in adult brain derived OLs in LG but was reduced in NG by two fold . As showin vitro  cells are being used. Our studies indicate the need to consider both age and maturation state of these cells for in vitro studies that aim to model adult human disease.Our study indicates the significance of both age and the differentiation state on the metabolic profiles of OLs. Age, as described here, includes both time since differentiation and the actual age of animal . Such distinct profiles will contribute to the role played by these cells in physiologic conditions related to establishing, maintaining, and/or restoring myelination and the response to stress conditions as encountered in the microenvironment of MS lesions. Studies using parabiotic animals indicate that age is a major contributor to the capacity for remyelination to occur . Our prein vitro . Most suS1 Fig(PZF)Click here for additional data file.


TPMT gene and a panel of 15 other genes implicated in thiopurine toxicity were analysed using a gene based statistical test (SKAT-O test). Nine patients out of 100  had known TPMT mutations associated with deficient enzyme activity. A novel and a highly pathogenic TPMT variant not detectable through standard genotyping, was identified through NGS in an individual intolerant to thiopurines. Of the 14 patients intolerant to thiopurines, NGS identified deleterious TPMT variants in 5 individuals whereas the biochemical test identified 8 individuals as intolerant . SKAT-O test identified a significant association between MOCOS gene and TPMT activity (p = 0.0015), not previously reported. Although NGS has the ability to detect rare or novel variants not otherwise identified through standard genotyping, it demonstrates no clear advantage over the biochemical test in predicting toxicity in our modest cohort.The aim of our study was to assess the utility of next generation sequencing (NGS) for predicting toxicity and clinical response to thiopurine drugs in paediatric patients with inflammatory bowel disease. Exome data for 100 patients were assessed against biochemically measured TPMT enzyme activity, clinical response and adverse effects. The  Thiopurine drugs, which include azathioprine and 6-mercaptopurine (6-MP) have been effectively used in inflammatory bowel disease for more than 30 years. They are also widely used in the treatment of patients with neoplastic conditions, post-organ transplantation and a wide range of autoimmune and inflammatory conditions. However concerns over toxicity and adverse reactions frequently result in discontinuation of treatment and a switch to alternative therapies12TPMT gene is known to exhibit genetic heterogeneity resulting in wide inter-individual differences, both in terms of clinical efficacy and toxicity profiles based on the enzyme activity2TPMT allele with intermediate enzyme activity, whereas approximately 1 in 300 individuals are homozygous or compound heterozygous with a consequent low or absent TPMT activity3TPMT status in a given patient before commencement of thiopurine therapy; this is achieved by measuring TPMT enzyme activity in the circulating red blood cells or through genotyping known TPMT variants associated with enzyme deficiency28Thiopurine S-methyl transferase (TPMT) is a key enzyme involved in the metabolism of thiopurine drugs and functions by catalysing the S-methylation of aromatic and heterocyclic sulfhydryl groups2The rationale for assessing the TPMT status before commencement of thiopurine therapy is to minimise the risk of adverse effects whilst aiming for an optimal clinical response. Thiopurines are best avoided in individuals who are deficient or have extremely low TPMT activity and administered at a reduced dose if TPMT activity is intermediate or low normal29TPMT is the most crucial pharmaco-gene involved in the metabolism of thiopurines, previous studies have highlighted the role of other genes, whose products substantially alter drug metabolism and consequently impact clinical efficacy or toxicity11121314151617TPMT status (genotype or phenotype), could be explained by variation in genes encoding the other enzymes involved in thiopurine metabolism.Although In this study, we identify genes known to be involved in thiopurine metabolism and determine all coding mutations in these genes in a cohort of 100 children with IBD.TPMT gene and other genes implicated in thiopurine toxicity on TPMT enzyme activity through the application of a gene based statistical test (SKAT-O). The test was also conducted to investigate the association of these variants with thiopurine tolerance and clinical response.We assess the joint effect of rare and common variants within the Patients were identified through the paediatric gastroenterology service database based at the University Hospital Southampton (UHS), recruited from outpatient clinics and followed through their treatment. All children were diagnosed using the Porto diagnostic criteriaThe study was ethically approved by Southampton and South West Hampshire Research Ethics Committee (09/H0504/125). Informed consent was obtained from all participants before recruitment to the study. All methods were carried out in accordance with the approved and published guidelines.TPMT enzyme activity was measured using standard high performance liquid chromatographic technique20−3) and/or thrombocytopenia ; liver toxicity was alanine transaminase (ALT), gamma-glutamyl transpeptidase (GGT) or alkaline phosphatase more than twice their normal levels; acute pancreatitis was defined as significant abdominal pain within 3 months of starting thiopurines, accompanied by a serum amylase or lipase level of greater than twice their normal levels as per our local laboratory valuesBritish Society of Paediatric Gastroenterology, Hepatology and Nutrition (BSPGHAN) recommend initiation of treatment with thiopurines for maintenance of remission in individuals who relapse in less than 6 months, have 2 or more relapses per year following initial successful therapy and in all steroid-dependent patients. Practice with regards to initiation of treatment varies among clinicians, but is usually commenced and monitored as per the BSPGHAN guidelines9Assessment of clinical poor response or non-response was based on one of the following: (1) Inability to achieve clinical improvement as assessed by global clinical assessment after at least 6 months of thiopurine therapy; (2) Corticosteroid dependence after at least 3 months of thiopurine therapy; (3) Relapse within 6 months of therapy; (4) Use of biologics within 6 months of therapy with thiopurines and; (5) Disease progression needing surgery within 6 months of thiopurine therapy commencement.Intolerance to treatment was based on all of the following: (1) Occurrence of adverse side effects ; (2) Adverse effects unexplained by disease course or other concomitant co-morbidities; and (3) Partial or complete resolution of the observed adverse effects following discontinuation of therapy.TPMT genetic variants and other genes involved in thiopurine metabolism and toxicity were included. We applied the TPMT nomenclature committee website  that outlines all reported TPMT variantsA systematic search was conducted through ovidsp using MEDLINE and EMBASE from inception to the end of March 2015. Only studies in humans describing Genomic DNA was extracted from peripheral venous blood samples collected in EDTA, using the salting out method as previously described24http://evs.gs.washington.edu/EVS/], in 46 unrelated human subjects sequenced by Complete GenomicsWhole-exome capture was performed using Agilent SureSelect Human all Exon 51 Mb (versions 4 and 5) capture kit as previously describedThe burden of genetic variations within the genes was conducted using a gene-based statistical test Of the 100 patients, 78 initiated thiopurines as part of their clinical management while 22 maintained remission without recourse to this therapy. The median duration of follow up for the 78 patients commenced on thiopurines was 46 months (7–156) from diagnosis and 43 months (6–119) from starting therapy with thiopurines .TMPT and these genes was included in downstream analysis of: (1) biochemically assessed TMPT activity; (2) tolerance to thiopurines and; (3) response to treatment.A systematic search of genes implicated in TPMT metabolism and toxicity identified 15 genes with robust evidence from a review of over 3,000 articles  for whic−12)  . While aTPMT variants across our cohort  and likely to have a deleterious impact on enzyme function (GERP = 4.98). The patient was initiated on thiopurine treatment at a reduced dose (1 mg/kg) but developed severe persistent nausea and treatment was discontinued. This variant has since been catalogued as a functionally significant allele by the TPMT nomenclature committee  with a unique allele number TPMT*39.One novel non-synonymous variant (p.A73V) was identified in a female patient with Crohn’s disease and a biochemical activity level of 55 mU/L. One UC patient harboured a rare intronic variant (in heterozygote form) proximal to the exon 7 splice junction (c.420-4G > A), this patient had a biochemical activity level of 32 mU/L, was administered thiopurine treatment which was tolerated and subsequently demonstrated therapeutic response. The MaxEnt score (0.95) for this splice junction variant predicts minimal impact on splicing within the gene. While this is positively consistent with drug tolerance, it does not explain the relatively low biochemical activity level observed in this patient.Finally, we observed a common synonymous variant (I158I) at high frequency (37 heterozygotes and 57 homozygotes). Mean biochemical activity level is 67.1 mU/L (32–99) and 66 mU/L (152-145) across the subgroup of individuals carrying the variant in heterozygous and homozygous state respectively. The very common frequency of this variant and the fact it does not alter amino acid composition is consistent with silent variation having no impact on function.HPRT1, GSTM1, FSLT5 and MTHFR).We interrogated exome data across fifteen additional genes implicated in thiopurine metabolism and toxicity. We observed mutations across 11 genes  and TPMT (p = 0.0017) gene with TPMT biochemical activity levels. The test also detected a nominal association between GMPS and drug tolerance (p = 0.0212) as well as variations within IL6ST (p = 0.0084) and ABBC4 (p = 0.0452) and drug response . The clinical data of individuals intolerant to thiopurines, is shown in Prediction of thiopurine toxicity showed a specificity of 93.75% through detection of toxicity . HoweverTPMT genotyping alone or in combination with TPMT phenotype is insufficient to predict tolerance to thiopurine drugsTPMT variants associated with deficient enzyme activity have been describedTPMT mutations (9% compared to 11% reported in previous studies)TPMT mutations. Our results indicate that although prediction of thiopurine toxicity through NGS has a higher specificity compared to the biochemical test, the sensitivity of both methods is clinically suboptimal (35.7% and 57.14% respectively). Among the 14 individuals intolerant to thiopurines, all the 5 patients who harboured deleterious TPMT variants would also have been predicted as potentially intolerant to thiopurines through the biochemical test. Furthermore, all the nine individuals with deleterious variants within the TPMT gene across the cohort of 100 patients would also have been identified as potentially intolerant as all these nine individuals had TPMT enzyme activity levels within the intermediate range. Hence based on first principles, NGS did not have a clear advantage over the biochemical test in predicting thiopurine toxicity.It is well established that in a small percentage of patients, TPMT in an individual who had TPMT enzyme activity level of 55 mU/L, but developed severe gastrointestinal toxicity despite a reduced dose. This suggests that thiopurine toxicity can develop in individuals harbouring rare or yet unknown variants, not detected through standard genotyping. As a widely used practical approach, TPMT genotyping of known variants is considered only when biochemical tests suggest a deficiency or if a patient has recently been transfused. However, a normal genotype for known variants cannot exclude the possibility of rare variation causing TPMT deficiency and development of adverse effectsTPMT genotyping.We identified a highly pathogenic novel variant in MOCOS gene and TPMT enzyme activity (p = 0.0015). Molybdenum cofactor sulfurase (MOCOS) is a protein-coding gene located on 18q12, which sulfurates the molybdenum cofactor in XDH and AOX1, key enzymes involved in the degradation of thiopurinesMOCOS gene in thiopurine metabolism with possible impact on clinical outcomes in patients with mutations, however an association between MOCOS and TPMT enzyme activity has not been explored. This is the first study to identify a significant role for this gene with variations causing alterations in biochemical enzyme activity. Further work is required to determine how MOCOS influences TPMT function.The SKAT-O test identified a significant association between variations within the GMPS and drug tolerance (p = 0.0212). GMPS is involved in the phosphorylation of 6-TIMP (6-thioinosine monophosphate) to thioguanine nucleotides, which is an important step for thiopurines to exert their cytotoxic effects. Further mechanistic studies will be required to elucidate the molecular mechanisms and clearly define the role of these genes involved in the thiopurine metabolic pathway.We also detected a nominal association between A limitation of this study is that only children who had undergone exome analysis with concurrent TPMT values were selected for the study. While approximately half of our cohort patients had TPMT activity in the intermediate range, only 20% of control samples had TPMT activity in the intermediate category. Exome sequencing was preferentially conducted on children with the most severe disease phenotype, which would have enriched for patients with poor response to first-line treatments. Secondly, the selected population is predominantly Caucasian and a relatively small cohort, thereby limiting the general applicability of the results. Following a systematic search of literature to identify genes implicated in thiopurine toxicity, a panel of 15 genes was prioritised for assessment. It is it is possible that potentially pathogenic variants in other genes may have been missed through this approach. However extension of the list of genes examined would compromise power in the highest priority candidate genes.Although there is no clear advantage of NGS over the biochemical test in predicting toxicity, our study demonstrates the strength of NGS as a powerful tool in identifying pathogenic variants in patients not detected through standard genotyping. As high throughput sequencing becomes more accessible and affordable, a more objective approach to assessing pharmaco-genomic variation in all genes involved in the drug metabolism pathway may be indicated in selected patients to guide treatment strategies. Replication in larger studies would be required for a comprehensive curation of candidate genes, possibly paving the way for a targeted gene panel as a reliable predictor of toxicity.How to cite this article: Coelho, T. et al. Genes implicated in thiopurine-induced toxicity: Comparing TPMT enzyme activity with clinical phenotype and exome data in a paediatric IBD cohort. Sci. Rep.6, 34658; doi: 10.1038/srep34658 (2016).


Chronic myocardial infarction (MI) triggers pathological remodeling in the heart and cardiac nervous system. Abnormal function of the autonomic nervous system (ANS), including stellate ganglia (SG) and dorsal root ganglia (DRG) contribute to increased sympathoexcitation, cardiac dysfunction and arrythmogenesis. ANS modulation is a therapeutic target for arrhythmia associated with cardiac injury. However, the molecular mechanism involved in the pathological remodeling in ANS following cardiac injury remains to be established.In this study, we performed transcriptome analysis by RNA-sequencing in thoracic SG and (T1-T4) DRG obtained from Yorkshire pigs following either acute (3 to 5 hours) or chronic (8 weeks) myocardial infarction. By differential expression and weighted gene co-expression network analysis (WGCNA), we identified significant transcriptome changes and specific gene modules in the ANS tissues in response to myocardial infarction at either acute or chronic phases. Both differential expressed genes and the member genes of the WGCNA gene module associated with post-infarct condition were significantly enriched for inflammatory signaling and apoptotic cell death. Targeted validation analysis supported a significant induction of inflammatory and apoptotic signal in both SG and DRG following myocardial infarction, along with cellular evidence of apoptosis induction based on TUNEL analysis. Importantly, these molecular changes were observed specifically in the thoracic segments but not in their counterparts obtained from lumbar sections.Myocardial injury leads to time-dependent global changes in gene expression in the innervating ANS. Induction of inflammatory gene expression and loss of neuron cell viability in SG and DRG are potential novel mechanisms contributing to abnormal ANS function which can promote cardiac arrhythmia and pathological remodeling in myocardium. Myocardial ischemia induces changes in the neurohumoral control systems, including sympathoexcitation, that contribute to autonomic nervous system (ANS) imbalance and myocardial remodeling, 2 3]. . 3]. CarIn the diseased heart, there is evidence that abnormal autonomic function can lead to and exacerbate congestive heart failure, ventricular arrhythmias, and sudden cardiac death , 2 3]. . 3]. CliCompared to the comprehensive focus on the molecular studies in the myocardium, our knowledge of the pathological remodeling in the cardiac ANS has been limited to few protein markers. MyocardYorkshire pigs (n = 9) were used in this study. All pigs were juvenile males and weighing 48 ± 4 kg at the time of terminal study. Animals undergoing infarction were infarcted at 30 ± 2kg in order to ensure same approximate size as control and acute ischemia animals by the time of tissue harvest. All animal experimental protocols were devised in accordance with guidelines set by the University of California Institutional Animal Care and Use Committee and the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” and approved by the University of California Los Angeles Animal Research Committee (ARC). Animals were housed in the University of California Division of Laboratory Animal Medicine facility. Seven days before the beginning of the experiment, the animals were transported from a farm to the local animal facility where they were individually housed in stalls under conditions of natural light and room temperature. All animals were fed with a commercial grain mixture and tap water ad libitum until 24 hours before surgery. This study followed The ARRIVE Guidelines Checklist for Animal Research: Reporting In Vivo Experiments .Animals were randomly assigned to control (n = 3), acute ischemia (n = 3), or chronic infarct with healed left ventricular anteroapical infarct (n = 3). Neural tissue was harvested from the left and right stellate ganglion as well as, the left and right dorsal root ganglion from thoracic (T1-T4) and lumbar spinal regions (L1-L4). For terminal tissue harvest, animals were sedated with intramuscular telazol (4-6mg/kg), intubated and mechanically ventilated. General anesthesia was maintained with inhaled isoflurane (1.5–2.5%) and intravenous boluses of fentanyl  during surgical prep. Heart rate and surface 12-lead ECG were monitored throughout using standard limb lead electrocardiogram. The femoral artery was catheterized to monitor the arterial blood pressure and the femoral vein was catheterized for intravenous saline infusion (10 mL/kg). Hourly arterial blood gas was tested and adjustments via ventilation or infusion of sodium bicarbonate were performed as necessary to maintain acid-base homeostasis. In the supine position, animals underwent median sternotomy to expose the heart as well as the left and right stellate ganglion. Animals were then placed prone and a dorsal spinal laminectomy was performed to expose the T1-T4 and L1-L4 dorsal roots bilaterally. Bilateral stellate ganglion, thoracic dorsal root ganglion, and lumbar dorsal root ganglion tissue samples were obtained. Animals were euthanized by terminal cardiac arrhythmia under deep anesthesia. All experiments were performed during the day and tissue collection was obtained between 2 and 3pm.Myocardial infarction was created as previously described. Brieflynd diagonal branch of the left anterior descending (LAD) coronary artery. Ischemia was induced by coronary artery ligation for 15 minute followed by reperfusion for 3 hours following coronary artery ligation removal. During coronary artery ligation, ischemia was confirmed by the presence of ST-segment elevations (0.1mv above baseline) in ≥ 5 contiguous limb and precordial leads as well as by direct visualization of the heart in the open chest. During ischemia regional hypokinesis of the left ventricular anteroapical walls distal to the ligation site was observed within 1 minute of coronary artery ligation.In an open chest, a 4–0 prolene suture was placed around the 2RNA was extracted from T1-T4 dorsal root ganglia and stellate ganglia in different treatment groups of Yorkshire pigs  using Trizol reagent (Thermo Fisher Scientific) according to the manufacture’s protocol. RNA sequencing was performed as previously described , 34.Transcriptomes were analyzed using the tuxedo suite of tools. Briefly, bases with poor quality scores were eliminated from the analysis using the fastq_quality_trimmer tool, and reads with fewer than 50 remaining bases were removed from the analysis. Remaining reads for each sample were mapped to the Sscrofa10.2 genome using Tophat 2.1.1 and counted, annotated and compared using cufflinks 2.2.1. Hierarchical Clustering was used to determine that one sample (204 LSG) was an outlier with no similarity to any other sample , and it WGCNA is a well-documented and commonly used algorithm for determining clusters of co-expressed genes which share functional roles and are linked to phenotypic traits of interest. Transcriptomes were filtered for all genes which were expressed (FPKM >1) and varying (CV >5%) across the 31 high-quality genomes (9589 genes). A correlation matrix was generated comparing each gene to the expression of each other gene, and, per the recommendations of the package, this matrix was transformed into an approximately scale-free (fit = 60%) network by raising the correlation network to a power (7). Topological overlap between each pair of genes is determined by the following equation:The eigengene, the first principle component of each module, is calculated, and modules with high similarity (R>0.75) are merged to identify the final set of modules of interest.Module lists were given to the GeneAnalysis package (ga.genecards.org) which uses a binomial distribution to test the null hypothesis that the genes within the module are not overrepresented in a given category.TUNEL staining was performed in healthy control, acute myocardial ischemia and chronic myocardial infarct treated stellate ganglia tissue sections using Click-iT Plus TUNEL Assay, Alexa Fluor 488 dye  according to the manufacture’s protocol. Nucleus was visualized by Hoechst 33342 .Immune-staining was performed in healthy control (n = 3), acute myocardial ischemia (n = 3) and chronic myocardial infarct (n = 3) treated stellate ganglia tissue section. Briefly, the tissue sections was deparaffinized and the antigen was unmasked using citric acid before blocking with blocking buffer (1*PBS/1%BSA/0.3%Triton X-100) at room temperature for one hour followed by incubation with tyrosine hydroxylase antibody  at 4 degree over night. The tissue sections were then rinsed with PBS for 3 times before incubating with anti-rabbit secondary antibody  for one hour at room temperature before staining with Hoechst 33342 .1μg RNA was used for first-strand cDNA synthesis using Random Primer and ProtoScript II Reverse Transcriptase  according to manufacturer’s instruction. Real-time PCR  was perfData are expressed as mean± SD. For comparison between two groups, differences were analyzed by 2-tailed Student’s t test. For comparison of multiple groups, differences were analyzed by 1-way ANOVA. Associations between binary phenotypic variables and eigengenes were calculated using the Wilcoxson Rank Sum test. P values ≤ 0.05 were considered as significant.The left and right stellate ganglia (SG) and T1-T4 dorsal root ganglia (DRG) tissues were collected from untreated healthy Control, Acute MI (3–5 hours from MI), Chronic MI (8 weeks after MI) pigs. There were no adverse events or unexpected animal mortality with acute ischemia or chronic infarct protocols. From these tissues, total RNAs were prepared and T1-T4 left vs. right DRG samples were pooled. A total of 8 pigs were analyzed including Control (n = 3), from Acute Ischemia (n = 3), and from chronic MI group (n = 2), respectively . For eacIn order to demonstrate the extent of transcriptome remodeling in SG and DRG following acute ischemia or chronic MI, we performed principal component analysis among the SG and DRG samples . While mIn order to uncover biological relevance of the transcriptome reprogramming observed in the ganglia samples, we performed unsupervised Weighted Gene Co-expression Network Analysis (WGCNA) from differentially expressed genes in order to identify gene modules based on shared expression profile among the 31 RNA-seq samples. As shown in To further validate the involvement of apoptotic cell death in the autonomic nervous circuit following MI, we performed histological analysis in the stellate ganglia tissue obtained from Control, acute ischemia and chronic MI groups. from infarcted pigs relative to the Controls. However, these genes were not altered in the corresponding neurons from lumbar DRGs of the same animals  and dorsal root ganglia (DRG). Our major findings include; 1) global change in gene expression in stellate ganglia and dorsal root ganglia is seen in both acute myocardial ischemia and following chronic myocardial infarction, 2) using weighted gene co-expression network analysis (WGCNA) tool, we also identified specific gene modules expressed in the ganglia that are significantly associated with MI injury in heart, 3) both gene modules and differentially expressed genes are significantly enriched with inflammation and cell death regulators. To our knowledge, this is the first unbiased approach to profile and characterize global molecular changes in ANS following cardiac injury. In addition to the previously known changes in individual genes, such as NPY , our resIn this study, we observed strong induction of inflammatory genes after acute ischemia and highly elevated apoptotic activity in the extrathoracic SG after chronic myocardial infarction. These data suggest a potential pathogenic process where cardiac injury induces early onset of inflammatory gene expression in ANS neurons, which is followed by apoptotic cell death and abnormal activities. Our gene expression findings of neural remodeling in the SG following myocardial injury are supported by previous studies in both animal and clinical models., 36–38 HOur study expands on the results from previous investigations that have demonstrated neuronal cellular changes after ischemia. In this study, we describe the global genetic changes which are thought to be underlying the observed neural remodeling with inflammatory processes being activated in early ischemia followed by apoptosis and abnormal cell function after chronic infarction. Inflammation has recently been implicated in the pathogenesis of heart failure and as a target for developing potential therapies for acute or chronic heart diseases. The sympathetic and parasympathetic nervous system have been reported as targets for treating inflammatory states like myocarditis. AlthougWhile neural remodeling in the intrathoracic sympathetic ganglia (SG) following myocardial ischemia has been hypothesized to be caused by local mechanisms such as retrograde axonal transport of proteins from infarcted myocardium , mechaniWe found changes in gene expression localized to thoracic spinal cord DRG only, with no change in gene expression in lumbar DRG, after myocardial ischemia or infarction. Significant changes in the expression of several neural and immune modulators  and apoptotic genes  were observed in the thoracic DRG following MI, but not in the lumbar DRG from the same post-MI animals. The fact that both inflammation and cell death signals were only observed in the ganglia tissues from thoracic DRG but not from the lumbar DRG suggests that these changes are specifically related to sympathetic ANS circuit innervating heart rather than systemic effects associated with MI. These finding suggest that cardiac arrhythmias following myocardial injury may in part be due to neural remodeling occurring throughout neural circuits in the intrinsic and extrinsic cardiac nervous system, thus leading to ANS imbalance and increased sympatho-excitation.Our current transcriptome profiling was limited by the tissue heterogeneity and the cell-autonomous mechanism associated with the observed induction in inflammation and cell death. Due to the small sizes, the collected tissues may have been contaminated by surrounding muscle or fat tissues which may have contributed to heterogeneity observed in the gene expression profile. In addition, the histological analysis did not include sufficient information to determine the cellular type responsible for inflammatory gene expression or apoptotic signal. Further studies with more resolution and cellular identification information will be needed to fully illustrate the mechanistic contribution and therapeutic potential for our findings.Myocardial injury leads to time-dependent global changes in gene expression in the innervating ANS. Induction of inflammatory gene expression and loss of neuron cell viability in SG and DRG are potential novel mechanisms contributing to abnormal ANS function that can promote cardiac arrhythmias and pathological remodeling in myocardium. These findings lead to obvious questions for future investigations. Are inflammation and neural cell death associated with sympathetic overdrive? Do neuromodulation therapies ameliorate ANS remodeling and attenuate cardiac arrhythmias or remodeling following injury? Greater understanding of the molecular changes in the ANS following cardiac injury will allow deeper insights into the mechanisms of cardiac arrhythmias and help in the optimization of neuraxial therapies.S1 File(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Recent studies have enhanced our understanding of the linkage of oxygenation and metazoan evolution in Early Cambrian time. However, little of this work has addressed the apparent lag of animal diversification and atmospheric oxygenation during this critical period of Earth history. This study utilizes the geochemical proxy and N isotope record of the Ediacaran–Cambrian boundary preserved in intra-shelf basin, slope, and slope basin deposits of the Yangtze Sea to assess the ocean redox state during the Early Cambrian metazoan radiation. Though ferruginous conditions appear to have prevailed through the water column during this time, episodes of local bottom-water anoxia extending into the photic-zone impacted the slope belt of the basin. Heterogenous oceanic redox conditions are expressed by trace element concentrations and Fe speciation, and spatial variation of N isotopes. We propose that the coupling of ocean chemistry and Early Cambrian animal diversification was not a simple cause-and-effect relationship, but rather a complex interaction. Specifically, it is likely that animal diversification expanded not only temporally but also spatially from the shallow shelf to deep-water environments in tandem with progressive oxygenation of the extensive continental margin. Though these biological events have been attributed to environmental modifications, notably oxygenation of the global ocean, specifics of the relationship between the emergence and diversification of metazoans and atmospheric-oceanic oxygenation remain unresolved. Large-scale investigations have focused on the nature of geochemical constraints on atmospheric oxygen levels through geological time7 and (minimum) oxygen requirements for the stimulation and maintenance of metazoan evolution11. Conflicting conclusions of the necessity of oxygenation to the biological evolution of metazoans have been reported. Indeed, some have argued that increasing atmospheric oxygen levels might not have triggered the appearance of metazoans12. However, increasing concentrations of dissolved oxygen would have been required to sustain the subsequent diversification of metazoans and the establishment of a Phanerozoic-type ecosystem. Other studies have postulated that the activity of benthic faunas improved the chemical and physical conditions of their habitats by bioturbation and related bioirregation since the Fortunian stage13. Finally, although inferred atmospheric oxygen levels suggest that the global ocean may have been oxygenated by Late Ediacaran time, anoxic (perhaps intermittently euxinic) waters appear to have persisted in some areas of the Early Cambrian ocean.The Cambrian explosion of diverse bilaterian clades is considered to have proceeded in three phases preserved in the fossil record: (1) the first phase is recognized by the appearance of basal metazoan phyla in Late Ediacaran time; (2) the second phase begins with the first occurrence of biomineralization of plates, spines and shells, and is recorded by the widespread occurrence of small shelly fossils (SSFs); (3) the final, perhaps the most profound, phase is defined by the emergence of the three supraphylogenetic clades during Cambrian stage 398/95Mo from +1.7‰ at ~540 Ma to +2.3‰ (similar to modern seawater composition) by ~520 Ma suggests that bottom waters were fully oxygenated by Cambrian stage 314. Fe speciation and S isotope data reflect the existence of a mostly ferruginous water column above a zone of limited bacterial sulfate reduction (BSR) in continental shelf and deep waters until Cambrian stage 3 in the Yangtze Sea17. Benthic redox conditions likely fluctuated frequently in intra-shelf basin areas of the Yangtze Sea as suggested by documented authigenic Mo-U co-variation18. Cerium anomalies documented from phosphate nodules recovered from basal Cambrian strata are consistent formation beneath an oxygenated water column19, yet the existence of photic-zone anoxia is suggested by largely negative sedimentary N isotope values described from Ediacaran-Cambrian deposits of the Yangtze platform21. Thus, various geochemical proxies, some of them conflicting, suggest that redox history of Yangtze Sea was quite debatable at the time of the Cambrian explosion.A variety of geochemical approaches have been employed to characterize oceanic redox conditions during the Ediacaran–Cambrian boundary interval. Increasing δHerein, we address water column geochemistry of the Yangtze Sea during the Ediacaran-Cambrian transition using a combination of independent redox proxies, including N isotopes, trace element concentrations (mainly Mo and U), and Fe speciation. Application of this approach to the investigated sections, which were selected based on their inferred paleoceanographic positions within the Yangtze Sea Fig. , elucida25. Sedimentary δ15N values of investigated samples range from −7.5‰ to +5.9‰, showing the greatest variation for all studied sections near the Ediacaran-Cambrian boundary  results in a reduction of δ15N values of accumulating sediment. Especially, marine sediments deposited during the OAEs may record striking 15N-depletion, with δ15N values commonly <−2‰ interpreted to be an expression of strongly reducing water conditions29. Sharply negative δ15N deviations (by 2–4‰) from normal marine N cycling, then, can be considered as the evidence of photic-zone anoxia perhaps induced by a chemocline upward excursion29.Principal biogeochemical processes that influence N isotope composition include N-fixation by diazotrophic cyanobacteria, denitrification in the water column or within sediment and anaerobic ammonium oxidation (anammox)29. The diagenetic loss of N from organic matter would be expected to yield C/N atomic ratios in excess of the 6.6 value of the Redfield ratio. Though the average C/N ratio of studied samples reaches 43.5, the weak correlation of δ15N and C/N (R2 = 0.0284) is not consistent with an interpretation involving strong diagenetic alteration of organic matter. Indeed, the weakly negative correlation (R2 = 0.3924) of δ15N and C/N values of the Nangao section suggests minor effect of diagenetic alteration on N isotopes  are analogical to those of the present study , suggesting that Cr enrichment principally by scavenging of particle reactive Cr (III) phases under strongly reducing conditions. Relatively low Cr/Ti ratios documented from the upper Niutitang Formation  reflects less reducing conditions. Accelerated uptake of redox-sensitive metals and the coeval development of strong negative excursions in each of the investigated sections suggest the establishment of widespread reducing conditions. However, anoxic conditions appear to have persisted in slope regions into interval 3 as recorded by elevated Cr/Ti ratios and enrichment of Mo, U and V  associated with intervals 1 and 2. The Fe proxy indicates that the global ocean was dominated by ferrugenous water through the late Ediacaran to interval 136. Accelerated upwelling at this time enhanced surface water productivity18 thereby increasing the flux of organic matter to the ocean floor, which further reduced bottom-water conditions. The shoaling of anoxic water mass into the photic zone caused primary producers to consume15N-depleted NH4+ resulting in the distinctly negative δ15N values documented from the studied deposits. It appears that enrichment of redox-sensitive trace metals lagged behind the isotopic response, gradually increasing as euxinic bottom water conditions were established. As modern oceans indicate, the residence times of those geochemical indices are ~3 kyr for N isotope, ~780 kyr for Mo and ~480 kyr for U, respectively. Thus a reason for the lag is proposed that isotopic evidences respond to oceanic changes quicker than trace metals. Probably, global ocean began to yield the oceanic anoxia earlier than the local ocean that experienced reducing conditions and consequent accumulation of redox-sensitive metals. However, this lag is not recognized in the slope belt, including the Nangao section. It is plausible that a rapidly rising sea level caused anoxic bottom water to shoal into the photic-zone producing the negative isotope excursion. Enrichment of trace metals appears to have accelerated across the basin at this time34. Following this event and the related negative δ15N excursion, N cycling in the intra-shelf basin (Zhongnan section) returned to a more normal state with the δ15N values varying between 0‰ and 2‰. However, euxinic water persisted in the slope and deep-water basin, with unscheduled shoaling, as indicated by negative δ15N values (mostly <−1‰) and high concentrations of Mo in sediments. Despite the branching in time probably resulted, both the N cycling and trace-metal enrichment respond to the fluctuating oceanic redox state and reflect evidently in their sedimentary evidences.The documented temporal association of stable N isotopic excursions and periods of redox-sensitive trace metal enrichment is consistent with what is known of the oceanic redox structure, but its timing may vary across the basin. Interestingly, the onset of negative δ36. During this time, though, the water column of the shallow shelf remained oxygenated, accumulating widespread carbonate sediment as exemplified by the Xiaotan section in the eastern Yunnan province and studied sections in the Three Gorge Area . However, these conditions may have persisted for only several million years before the ocean, except for the mixed surface layer, returned to anoxic and ferruginous conditions that persisted well into the Early Cambrianrea Fig. 20, 37. I80 Ma. Ho5. Recent studies25, including results of the present investigation, have presented secular coupling records of N isotopes and inferred atmospheric oxygen levels for the Ediacaran-Cambrian boundary  values were calculated according to the formula, δ15N = ((15N/14Nsample)/(15N/14Nair) − 1) * 1000‰.Samples selected for N analysis were first analyzed for the total nitrogen (TN) content. Only those samples in which TN > 0.012 mg  were deemed capable of providing reliable nitrogen isotope results. These samples were analyzed on a Finnigan MAT-253 stable isotope mass spectrometer. Nitrogen isotope (δsample/(X/Al)PAAS, where X is the concentration of an element of interest and PAAS refers to post-Archean average shale compositions of Taylor and McLennan39. Authigenic trace element concentrations were calculated as follows: Xauth = Xsample − (X/Al)detr * Alsample, where detrital element concentrations are those of the average upper continental crust of McLennan40.Samples analyzed for N isotopes were also analyzed for major and trace element concentrations. Major element analysis was carried out using an Axios mAX X-ray fluorescence spectrometer with results reported as weight percent of oxides. Trace elements were measured by on ELEMENT XR ICP-MS instrument. Analytical uncertainties for analyzed elements was less than 5%. Enrichment factors (EF) were calculated as per the following; X (EF) = (X/Al)Supplementary InformationDataset 1