Findings of auditory abnormalities in children with autism spectrum disorder (ASD) include delayed superior temporal gyrus auditory responses, pre- and post-stimulus superior temporal gyrus (STG) auditory oscillatory abnormalities, and atypical hemispheric lateralization. These abnormalities are likely associated with abnormal brain maturation. To better understand changes in brain activity as a function of age, the present study investigated associations between age and STG auditory time-domain and time-frequency neural activity.While 306-channel magnetoencephalography (MEG) data were recorded, 500- and 1000-Hz tones of 300-ms duration were binaurally presented. Evaluable data were obtained from 63 typically developing children (TDC) (6 to 14 years old) and 52 children with ASD (6 to 14 years old). T1-weighted structural MRI was obtained, and a source model created using single dipoles anatomically constrained to each participant’s left and right STG. Using this source model, left and right 50-ms (M50), 100-ms (M100), and 200-ms (M200) time-domain and time-frequency measures  and inter-trial coherence (ITC)) were obtained.t tests showed a right STG M100 latency delay in ASD versus TDC . In the left and right STG, time-frequency analyses showed a greater pre- to post-stimulus increase in 4- to 16-Hz TP for both tones in ASD versus TDC after 150 ms. In the right STG, greater post-stimulus 4- to 16-Hz ITC for both tones was observed in TDC versus ASD after 200 ms. Analyses of age effects suggested M200 group differences that were due to a maturational delay in ASD, with left and right M200 decreasing with age in TDC but significantly less so in ASD. Additional evidence indicating delayed maturation of auditory cortex in ASD included atypical hemispheric functional asymmetries, including a right versus left M100 latency advantage in TDC but not ASD, and a stronger left than right M50 response in TDC but not ASD.Paired Present findings indicated maturational abnormalities in the development of primary/secondary auditory areas in children with ASD. It is hypothesized that a longitudinal investigation of the maturation of auditory network activity will indicate delayed development of each component of the auditory processing system in ASD.The online version of this article (doi:10.1186/s13229-015-0065-5) contains supplementary material, which is available to authorized users. Autism spectrum disorder (ASD) is a set of developmental disorders characterized by difficulties in social communication and social interaction and restricted, repetitive patterns of behavior, interests, or activities. Electroencephalography (EEG) and magnetoencephalography (MEG) studies consistently report auditory abnormalities in children with ASD. Findings include delayed superior temporal gyrus (STG) auditory responses –4, reducIn adults, N100 (EEG) and M100 (MEG) are the most prominent deflections of the auditory event-related potential (EEG) or field (MEG), evolving with a peak latency of about 100 ms after stimulus onset . NaaThe maturation of primary/secondary auditory cortex involves a complex interplay of auditory event-related components. In young children, the P/M50 is readily evoked  and withDelayed auditory responses have been observed in ASD. As an example, in an initial study from our laboratory, the right STG M100 auditory response was delayed by approximately 10 ms in children with ASD versus typically developing children (TDC) . This laNeural networks exhibit oscillatory activity over a wide range of frequencies: from delta-band activity (0 to 4 Hz) to at least gamma-band activity (approximately 30 to 50 Hz). Oscillatory activity observed in EEG/MEG recordings reflects synchronized neural activity that occurs over relatively short time periods (milliseconds). Oscillatory activity within specific frequency bands is one of the most promising candidate mechanisms associated with information processing  as The present study comprehensively investigated the maturation of auditory cortical responses in TDC and in children with ASD, examining auditory time-domain and time-frequency activity.N = 6), no M50 and M100 response in either the left or right hemisphere (N = 1), and no structural magnetic resonance imaging (MRI) (N = 4). Evaluable MEG data were obtained from 63 TDC children  and 52 children with ASD . Demographics are reported in Table Of 126 examined participants, MEG data were excluded from five TDC and six children with ASD due to noise from dental metal artifacts (All participants were selected according to the following criteria: (1) no history of traumatic brain injury or other significant medical or neurological abnormality, (2) no active psychosis, (3) no MRI contraindications, and (4) no known drug or alcohol use prior to any study procedure. Members of the TDC group were evaluated by licensed clinical psychologists who ruled out the presence of DSM-IV-TR Axis I disorders based on clinical judgment, review of the child’s medical history form, and parent interview. Current diagnosis of ASD was confirmed by expert clinical judgment based on NIH CPEA guidelines, including the Autism Diagnostic Observation Schedule—Generic  and AutiIn the ASD group, 17 participants were being treated with ADHD medications , 3 participants treated with second-generation antipsychotics , 12 participants with antidepressants , and 1 participant with anxiolytics (Buspirone). No participants in the TDC group were taking psychotropic medications. The study was approved by the Children’s Hospital of Philadelphia IRB, and all participants’ families gave written consent.MEG data were recorded using a 306-channel Vector View system  with a sampling rate of 1000 Hz and a band-pass filter of 0.1 to 330 Hz. Electrooculogram (EOG)  and electrocardiogram (ECG) were also obtained. The participants’ head position was monitored using four head position indicator (HPI) coils attached to the scalp.Stimuli consisted of 500- and 1000-Hz sinusoidal tones presented using Eprime v1.1. Tones were presented via a sound pressure transducer and sound conduction tubing to the participant’s peripheral auditory canal via ear tip inserts . Prior to data acquisition, 1000-Hz tones of 300-ms duration and 10-ms rise time were presented binaurally and incrementally until reaching auditory threshold for each ear. Tones were presented at 45-dB sensation level above threshold. Each trial consisted of a 500- or 1000-Hz tone (300-ms duration) plus a 1000-ms (±100) inter-trial interval. A total of 125 tones per condition were presented. The 500- and 1000-Hz tones were separately analyzed. To minimize fatigue, during the auditory test, participants viewed (but did not listen to) a movie projected onto a screen.ps > 0.05.Following the MEG task, MEG data were corrected for head motion using MaxMove, and Maxfilter was used for noise reduction using a signal space separation method with a temporal extension (tSSS ). After 3).After the MEG session, structural magnetic resonance imaging (sMRI) provided T1-weighted, 3D MPRAGE anatomical images for source localization  as well as an additional 200+ points on the scalp and face were digitized for each participant using the probe position identification (PPI) system , and a transformation matrix that involved rotation and translation between the MEG and sMRI coordinate systems was obtained via a least-squares match of the PPI points to the surface of the scalp and face.For all participants, measures were obtained for the left and right 50-ms (M50), 100-ms (M100), and 200-ms (M200) response. The primary generator of the M50, M100, and M200 is well-modeled by a single dipole in the left and right Heschl’s gyrus and surrounding regions , 36–38. Dipole orientations were obtained after applying a 2  to 55-Hz  band-pass filter. The presence of a M50 (35 to 125 ms) and M100 (80 to 195 ms) response was determined based on amplitude (greater than baseline), latency, and hemisphere ingoing and outgoing flux topography . These slightly extended M50 and M100 latency ranges allowed capturing responses in younger children. M200 was operationally defined as the response showing a magnetic field topography similar to M100 but occurring after M100. As reported below, in many participants, a M100 response was not observed. In such instances, the M200 was defined as a response occurring later than 200 ms and with the appropriate magnetic field topography. Of note, as detailed in Edgar et al. [When a M50 or M100 response was observed, left and right M50 (35–125 ms) and M100 (80–195 ms) amplitude was measured at the signal maxima and latency the time at which this maximum occurred, within each latency interval. Examination of M200 source waveforms indicated that for most participants, the M200 response occurred between 200 and 325 ms and that for many participants, the M200 response was broad  and often not very “peaked”. Due to this feature, M200 latency was not scored. For M200 amplitude, for each participant, in the 200- to 325-ms window, a source strength measure integrated over the full-width at half-max (FWHM) of the M200 response rather than selecting a single timepoint amplitude measure. For left and right STG M50, M100, and M200, the amplitude measure was obtained after baseline correction (−400 to −25 ms).The calculation of single-trial phase and magnitude for the left and right sources used procedures outlined in Hoechstetter et al.  where inTotal power and phase-locking measures were extracted from the single-trial complex time-frequency matrix. Total power was calculated by averaging the time-frequency spectra of each MEG epoch. When baseline power is subtracted, post-stimulus total power (TP) assesses the post-stimulus increase in oscillatory activity. A measure of phase-locking referred to as ITC was also computed. ITC is a normalized measure assessing the trial-to-trial similarity of oscillatory activity, with ITC = 1 reflecting no phase variability and ITC = 0 reflecting maximal phase variability across trials. Time-frequency measures were obtained using the M50, M100, and M200 source models.t test group comparisons, family-wise error was controlled using cluster size thresholds derived from Monte Carlo simulations  package (AFNI AlphaSim) and clustering performed with custom MatLab software.For the TP and ITC time-frequency .g., see , 28). ThFor all following analyses, participants with values more than 2.5 standard deviations were excluded .As shown in Table ps >0.05). For the 500- and 1000-Hz tones, M200 GOF was slightly lower in ASD (500 Hz = 76 % and 1000 Hz = 74 %) versus TDC . As described below, the slightly lower M200 GOF in ASD was likely due to more extended and less synchronous M200 responses in ASD versus TDC.For the 500- and 1000-Hz tones, bilateral M50 and M100 goodness-of-fit  did not differ between ASD and TDC . As shown in Table ps <0.001). Analyses indicated similar age dependence of the presence of a M100 response in TDC versus ASD. The presence of a M200 response is not reported in Table As shown in Table ANOVAs examined hemisphere, frequency, and group latency differences in the participants with M50, M100, or M200 responses.F = 16.49, p < 0.001, showed earlier M50 responses in the right versus left STG. A main effect of frequency, F = 67.01, p < 0.001, showed earlier M50 responses for 1000-Hz versus 500-Hz tones.A main effect of hemisphere, F = 3.02, p = 0.09, showed earlier right than left response latencies in TDC (p < 0.001) and similar right and left response latencies in ASD (p > 0.05).A trending group × hemisphere interaction, t tests showed the expected right hemisphere M100 latency delay in ASD versus TDC .Table ANOVAs examined hemisphere, frequency, and group amplitude differences in the participants with M50, M100, or M200 responses.F = 6.23, p < 0.01, indicated stronger left than right M50 responses in TDC but not ASD (p < 0.01). A hemisphere × frequency interaction, F = 5.34, p < 0.05, indicated stronger 1000- than 500-Hz responses in the right (p < 0.001) and similarly strong 1000- and 500-Hz responses in the left (p > 0.05).A group × hemisphere interaction, F = 4.82, p < 0.05, indicated stronger M100 responses to 1000- than 500-Hz tones.A main effect of frequency, F = 30.12, p < 0.001, indicated stronger M200 responses in the right versus left. A trending main effect of group, F = 2.68, p = 0.10, indicated stronger M200 responses in TDC versus ASD. Figure A main effect of hemisphere, F,108 = 30Given absent M100 responses in many participants, time-frequency analyses were not performed for M100 sources. Given M200 amplitude group differences bilaterally, to reduce the number of tests, time-frequency analyses were performed only for M200 sources  = 3.81, p < 0.05, indicated greater TP for the 500- versus 1000-Hz tones. Simple effect analyses of a hemisphere by group interaction, F = 7.60, p < 0.01, showed greater left STG TP in ASD versus TDC (p < 0.05) and greater right than left TP in TDC (p < 0.01) but not ASD (p > 0.05). For ITC, simple effect analyses of a hemisphere by group interaction, F = 15.43, p < 0.001, showed greater right STG ITC in TDC versus ASD (p < 0.05) as well as greater right than left ITC in TDC (p < 0.001) but not ASD (p > 0.05). Given no group × condition interactions for TP or ITC, to reduce the number of tests, all further analyses were performed collapsing across 500- and 1000-Hz conditions. Table To further explore the post-stimulus low-frequency group differences, ANOVAs examined hemisphere, condition , and group low-frequency differences using a single measure per hemisphere identified from the family-wise corrected statistics maps, computed for TP as the average in a 150- to 400-ms and 4- to 16-Hz region of interest (ROI), and computed for ITC as the average in a 200- to 300-ms and 4- to 16-Hz ROI. For TP, a main effect of condition, Given the pre-stimulus TDC and ASD group findings reported in Edgar et al. , a pre-sF = 50.16, p < 0.01, indicated that less pre-stimulus activity was associated with increased post-stimulus TP in both groups (r = 0.21). In the left, a significant second block main effect, F = 3.87, p < 0.05, indicated that left TP post-stimulus group differences remained even after removing variance associated with pre-stimulus activity. In the left, a significant first block main effect, F = 12.29, p < 0.001, indicated that less pre-stimulus activity was associated with increased ITC in both groups (r = 0.32). The left STG findings remained even after removing variance in post-stimulus activity associated with age. Age + pre-stimulus activity together explained 13 and 25 % of the variance in left TP and ITC, respectively. Right pre-stimulus activity was not associated with right TP or ITC. A significant second block main effect, F = 4.05, p < 0.05, indicated that right ITC post-stimulus group differences remained even after removing variance associated with pre-stimulus activity.Given previous findings that pre-stimulus activity predicts post-stimulus STG auditory processes , regressr2 = 0.24) versus ASD (r2 = 0.02), the interaction term was not significant, and as detailed in the “Figure ps >0.23) indicated that increased post-stimulus TP was associated with increased post-stimulus ITC in both groups.An additional set of analyses examined associations between post-stimulus TP and ITC (using the above defined M200 time-frequency ROIs). Given the above reported age, pre- and post-stimulus activity associations, for each hemisphere, a regression model examined whether post-stimulus TP predicted variance in post-stimulus ITC after removing variance associated with pre-stimulus activity and age . Post-stimulus TP predicted unique variance in left (23 %) and right (44 %) post-stimulus ITC. This positive association likely reflects the fact that (1) post-stimulus TP is computed as a percent change from pre-stimulus activity, with less pre-stimulus activity “allowing” a greater pre- to post-stimulus change in power and (2) phase is more accurately estimated a higher versus lower signal-to-noise and thus ITC more accurately estimated in those individuals with stronger versus weaker post-stimulus activity . Non-sigThe present study examined the maturation of STG auditory responses in ASD. Cross-sectional findings indicated an array of abnormalities in the development of primary/secondary auditory function in children with ASD, including delayed right STG M100 responses and a reduced rate of change (and perhaps even no change) in left and right STG M200 activity as a function of age. Additional evidence indicating delayed auditory cortex maturation in ASD included atypical hemispheric functional asymmetries, including a right versus left M100 latency advantage in TDC but not ASD, and a stronger left than right M50 response in TDC but not ASD. The pattern of findings suggests the need for longitudinal studies investigating the maturation of auditory network activity in ASD, with the hypothesis that results from these studies will demonstrate delayed development of each component of the auditory processing system.In the present study, a novel, and perhaps the most robust finding, was abnormal maturation of the networks supporting low-frequency (4 to 16 Hz) M200 neural activity in ASD versus TDC. In particular, whereas in TDC, low-frequency M200 TP and ITC decreased as a function of age (resulting in a decreased M200 time-domain response over the course of development); these age-associated TP and ITC changes were less evident or were absent in ASD see Fig. . There wEEG studies examining typically developing children show that N2 amplitude decreases as a function of age , 47, 48.Also indicating delayed maturation of auditory cortex in ASD was the observation of an absence of hemisphere functional asymmetries that are typically observed in children. First, replicating a previous finding , the absOther auditory cortex hemisphere asymmetries often observed in typically developing children, however, were observed to be intact in ASD. As an example, a M100 response was absent in many TDC and ASD participants, with M100 responses more often present in the right than left hemisphere and with M100 responses more often present in older (>10 years old) than younger participants. The latter findings indicated that the older individuals more easily encoded the auditory stimuli, with present findings thus consistent with prior studies showing that 100-ms auditory generators require longer intervals to produce a response in children than adults given hypothesized longer refractory periods in children than adults , 54, 55.In the present study, the presence/absence of M100 was similar between TDC and ASD. Edgar et al. , howeverTwo other findings observed in both TDC and ASD suggested faster maturation of right than left STG auditory areas: earlier M50 responses in the right than left hemisphere and less right than left pre-stimulus activity. The left versus right STG M50 latency difference observed in the present study has been reported in previous studies examining children and adolescents . As an eGiven changes in auditory cortex activity as a function of age, it is hypothesized that the pattern of TDC versus ASD group differences in auditory cortex activity (including group differences in hemisphere asymmetries) will differ as a function of the age, with some auditory encoding impairments most clearly observed in younger children with ASD , and other impairments more evident in older children with ASD. A review of the literature supports this hypothesis. For example, although in the present study, the observation of a delayed right M100 response in ASD versus TDC replicates previous studies , 4, the A limitation of the present study is that analyses were cross-sectional rather than longitudinal. A longitudinal study examining changes in auditory processes as a function of age in TDC and in children with ASD 5 to ~12 years old would support and extend the present cross-sectional findings. Future studies examining auditory processes in infants and children also are of interest given that treatments seeking to normalize auditory processes in ASD will likely target younger rather than older populations in order to have a better chance at normalizing auditory processes. In these studies, use of a whole-head infant MEG system is preferred given that MEG sensors are closer to the head in infant versus adult MEG systems –63.Although a movie was shown during the task to keep participants comfortable and awake , another study limitation is that group differences attending to the movie (not assessed) could account for some study findings, such as decreased attention to the movie and increased attention to the tones in one group contributing to the M200 group differences. Studies assessing group differences in attention during such tasks (perhaps using eye tracking) are needed.r = 0.49, ASD r = 0.14, power = ~78 %; see also Additional file Finally, sample size was a slight limitation. In particular, although samples were moderately large, the present study was slightly underpowered for some analyses, especially for analyses involving interaction terms. As an example, as previously noted, although examination of the Fig. Present cross-sectional findings showed an array of abnormalities in the development of primary/secondary auditory areas in children with ASD. Longitudinal studies, examining left and right auditory encoding processes from infancy through late adolescence  are needed to fully understand auditory encoding abnormalities in ASD. It is hypothesized that investigation of the maturation of auditory network activity will indicate delayed development of each component of the auditory processing system in ASD.


Curcuminoid as a single agent significantly induced EBV reactivation in recombinant GC and NPC lines. The drug effects were dose- and time-dependent. Micromolar concentration of curcuminoid EF24 enhanced the CLVA effect in all cell systems except SNU719, a naturally infected EBVaGC cell that carries a more tightly latent viral genome. These findings indicated that EF24 has potential as EBV lytic activator and may serve as an adjuvant in CLVA treatment.Epstein-Barr virus (EBV) persists in nasopharyngeal (NPC) and gastric carcinomas (EBVaGC) in a tightly latent form. Cytolytic virus activation (CLVA) therapy employs gemcitabine and valproic acid (GCb+VPA) to reactivate latent EBV into the lytic phase and antiviral valganciclovir to enhance cell death and prevent virus production. CLVA treatment has proven safe in phase-I/II trials with promising clinical responses in patients with recurrent NPC. However, a major challenge is to maximize EBV lytic reactivation by CLVA. Curcumin, a dietary spice used in Asian countries, is known for its antitumor property and therapeutic potential. Novel curcuminoids that were developed to increase efficacy and bioavailability may serve as oral CLVA adjuvants. We investigated the potential of curcumin and its analogs (curcuminoids) to trigger the EBV lytic cycle in EBVaGC and NPC cells. EBV-reactivating effects were measured by immunoblot and immunofluorescence using monoclonal antibodies specific for EBV lytic proteins. Two of the hit compounds ( Epithelial malignancies associated with Epstein-Barr virus (EBV) infection include undifferentiated nasopharyngeal carcinoma (NPC) and a subset of gastric carcinoma (GC) . EBV is Most EBV-associated carcinomas contain viral DNA that persists in a latent state with only a limited set of viral genes being expressed. EBV nuclear antigen 1 (EBNA1), small RNAs EBER1 and 2, BARF1 protein, and BamHI-A rightward transcripts (BART) encoding 40 miRNAs are expressed in all carcinoma cells [Besides personalized T-cell based immunotherapy, to date, no effective virus-targeted treatment has been developed for NPC and EBVaGC ,5. RecenThe mechanisms of induction of EBV lytic replication from latency using chemical inducers such as histone deacetylase inhibitors (HDACi), DNA demethylating agents, aspirin, NF-κB inhibitory compounds and chemotherapeutic drugs that induce DNA-damage repair have been extensively investigated and recently reviewed in detail ,8,9. IndCurcuma longa possess various therapeutic properties including anti-oxidant, analgesic, anti-inflammatory and anti-cancer activities due to its effect on multiple biological pathways including the inhibition of NF-κB [Meanwhile, several human clinical trials have reported that curcumin, a polyphenolic compound known derived from of NF-κB ,13,14. Iof NF-κB . Severalof NF-κB ,21,22,23of NF-κB ,21,22,23PGV-0, PGV-1, PGV-5, THPGV-0, cyclohexanone 206, piperidinone EF24, thiopyranones 211, 219 and thiopyranone dioxides 41, 227  have been reported to possess cytotoxic, antiproliferative and anti-angiogenesis properties in tumor cells by inhibiting COX-2 and NF-κB signaling [EF24, a widely investigated curcuminoid with improved stability and bioavailability, has pleiotropic effects on inflammatory and oncogenic signaling pathways [EF24 induced apoptosis in leukemic cells [206 and thiopyranone 211 [41 > 227 > EF24, based on cell-based growth inhibitory concentrations (IC50). The apoptotic effects of 41 and 227 were attributed to activation of the unfolded protein response in response to heightened endoplasmic reticulum (ER) stress induced by these compounds [The cyclopentanones were obtained from the UGM-VU collection of curcuminoids and two members  and EBV-positive NPC cell lines  were used in this study. Natural EBV genome-carrying SNU-719 cells , natural EBV genome-carrying C666.1 NPC cells and recombinant AGS-BX1 cells were cultured as described before ,24,27. A206), thiopyranone , thiopyranone dioxide  and piperidinone (EF24) moieties [PGV0 and PGV1) demonstrated cytotoxic, anti-proliferative and anti-angiogenesis properties in tumor cells [Natural purified curcumin was purchased from Sigma , whereas novel analogs curcumin compounds were kindly provided by Mei Lin Go at The National University of Singapore (NUS), Singapore and by Ritmaleni at The University of Gadjah Mada (UGM), Indonesia in collaboration with Henk Timmerman of the Vrije Universiteit Amsterdam, The Netherlands (UGM-VU). The chemical structure of curcuminoids is summarized in moieties . NUS commoieties . Penta Gdinone EF moietiesdinone EF moietiesor cells ,27,28,294 cells) were seeded in 96-well culture plates without drug and allowed to adhere for 24 h. Subsequently, t = 0 was measured using MTT and hit compounds  including positive lytic activators SAHA and GCb+VPA were added at different concentrations (in 2- or 5-fold). After 72 h, cells were lysed in DMSO and absorbance was determined at 540 nm in a plate reader [The cytotoxicity of hit compounds was evaluated by 3--2,5-diphenyl tetrazolium bromide  proliferation assay. Briefly, EBV-negative and -positive NPC and GC cells  and GCb+VPA was determined by trypan blue exclusion assay. EBV-positive GC and NPC cells grown to 70% confluence were treated with a combination of hit compound  and GCb+VPA or GCb+VPA alone for 48–96 h depending on cell types  and high (1.25 µM) concentrations of curcuminoids for 48–96 h A. TreatmFACS analysis was used to screen lytic inducing capacity of various curcuminoids at nanomolar concentrations. GFP intensity representing induced lytic cells was measured by FACSCalibur Flow Cytometer . A gate was set to determine the percentage of lytic cells by analyzing the GFP intensity of uninduced AGS-BX1 cells and after the CLVA treatment (GCb+VPA). Expression of GFP in the infected AGS-BX1 and HONE1-EBV cells following curcuminoid treatments visualized and counted under a fluorescence microscope .Immunofluorescence staining was performed to analyze EBV reactivation in naturally infected GC (SNU-719) and NPC C666.1) lines 66.1 line,29,31. C41, EF24) and GCb+VPA or GCb+VPA alone for 48–96 h. Treatment with SAHA and SB were included in the analysis. After treatment, the cells were pelleted, washed once with PBS and RNA were extracted using Trizol reagent . EBV mRNA lytic gene expression was compared to untreated (DMSO) controls and presented as fold increase. Data were determined from three independent experiments.EBV latent and lytic gene expression was quantified by a multiplexed RT-qPCR method using a plasmid pool representing each EBV lytic target gene as described in detail recently ,32. EBV-We have previously reported that the CLVA drug regimen , significantly and synergistically reactivates the latent virus in NPC tumor cells into the lytic replicative phase both in vitro and in vivo and applied this strategy in a clinical Phase-I/-II trial with promising clinical responses ,6,7. We 211, 219, 41, 227, EF24) gave weak lytic induction signals   showed EBV Zebra and EA-D activation with 1.25 µM being the minimal dose for lytic reactivation in HONE1-EBV cells  in GFP-modified AGS-BX1 cells carrying recombinant EBV with GFP under lytic (BXLF1) promotor control and measured the green fluorescent intensity of lytic cycle induction by FACS. This cell line is frequently used by others in studies analyzing EBV lytic activation in an epithelial cell background ,9,12,28. signals . Howeverntration B. Curcum (10 µM) C. PGVs cBV cells C. To conEF24 for 48 h revealed that EF24 is more potent than 41 and 227  and HONE1 (NPC) cells with recombinant EBV genome are widely used to study induction of the EBV lytic cycle in vitro ,12,28. W and 227 D. These 41, 227, EF24) induced the expression of Zebra and EA-D proteins 1.5–2 fold higher than in HONE1-EBV (EF24 induced EBV reactivation in AGS-BX1 cells at a comparable level to GCb+VPA (62–69%), whereas curcumin itself induced only 10% of AGS-BX1 cells into the lytic cycle  could induce EBV lytic cycle in natural EBV genome-carrying GC (SNU-719) and NPC (C666.1) tumor cell lines. We included GCb+VPA [PGV0 10 µM) induced EBV lytic activation in C666.1 NPC cells, but not in SNU-179 (data not shown). Compound 41 and 227, which induced strong EBV lytic cycle in AGS-BX1 and HONE1-EBV, did not show lytic induction effects in SNU-719  and EA-D (range from 12% to 18%)  C,D. SAHA to 18%) D and in  to 18%) E. Intere to 18%) C,E, whic to 18%) C,E. In s41 and EF24 in GC and NPC cells, we performed MTT assays at 48 h post-treatment. Compound 41 and EF24 demonstrated higher toxicity in GC  and GCb+VPA in GC and NPC cells. As 41 and EF24 significantly inhibit cell proliferation and reduce cell viability, we wonder whether these hit compounds in combination with GCb+VPA can enhance the cell killing. GC and NPC cells were treated with 1.25 µM concentration of hit compounds  and/or GCb+VPA. Treatment with SAHA and SB were included as positive controls in each cell line and cell viability was measured by MTT assay for 96 h. At 48–96 h post-treatment, 41 and EF24 synergistically enhanced the cell killing effect of GCb+VPA in GC  significantly reduce viability in AGS-BX1 , the EBV lytic protein and lytic mRNA expression were examined by immunoblot and real-time quantitative RT-qPCR ,30,32 inheir own A,D and 4proteins E. On thell death A–D but all death .We also examined the effect of pre-treatment with curcuminoids prior to GCb+VPA on EBV reactivation in recombinant AGS-BX1 cells. Following pre-treatment for 24 h and a further 24 h GCb+VPA incubation, cell extracts were collected for immunoblot analysis. Surprisingly, the induction of EBV lytic proteins (Zebra and EA-D) in response to CLVA (GCb+VPA) treatment was downregulated by pre-treatment with curcuminoids for 24 h . CurcumiEF24 and CLVA regimen. SAHA treatment was included as additional lytic induction control [EF24 to identify increased levels of both apoptotic markers [The variation of curcuminoids in activating EBV lytic cycle in different cell types indicates possible mechanistic differences in cell line-dependent lytic reactivation. As replication of EBV can be triggered by apoptosis , we cond control . Since t markers ,28.EF24, GCb+VPA or SAHA treatments. AGS-BX1 and HONE1-EBV cells treated with combined EF24, GCb+VPA did not show increased levels of both cleaved caspase-3 and PARP proteins. SAHA treatment showed a tendency to increase cleaved PARP protein levels in recombinant GC and NPC cell lines  with GCb+VPA resulted in a slight increase in immediate early (ZEBRA) and early (EA-D) protein levels in both the EBV-positive GC and NPC cells , thymidine kinase (TK), and small capsid protein (VCA-p18) in AGS-BX1 and C666.1 cells . Our datPC cells E–G. To dEF24 together with GCb+VPA increased mRNA levels relative to DMSO of immediate-early , early , and late lytic mRNAs (VCAp18 ~5-fold) 48 h after treatment, whereas a treatment with GCb+VPA alone increased the EBV lytic gene expression approximately 10-fold  as adjuvant for CLVA treatment increases EBV reactivation at the mRNA level above GCb+VPA alone , but the effects were less pronounced in the natural genome-carrying GC cell line SNU-719. Compounds 41 and EF24 did have anti-proliferative activity in SNU-719 cells and enhanced anti-proliferative activity of GCb+VPA. Furthermore, we observed that the anti-proliferative activity of these compounds was not EBV-specific, confirming prior studies in other cancer models [Most existing lytic inducing agents are frequently cell line specific or reactivate a limited number of cells ,11,12,30r models ,33,34. HThe mechanisms that limit the level and speed of virus reactivation in different cell types or between cells in the same culture population are not well understood ,11,32,3441, EF24) with GCb+VPA in various EBV-linked carcinoma model cell lines. We observed that the combination of EF24 and GCb+VPA most effectively enhanced the reactivation of EBV lytic cycle in EBV-associated carcinoma cell lines, with the exception of SNU-719 cells. Reactivation was accompanied by a reduction of tumor cell viability. SAHA proved more broadly effective in EBV reactivation and triggered EBV reactivation even in the SNU-719 cell line. The synergistic induction of EBV lytic cycle combined with enhanced cell death in NPC and GC cells carrying recombinant EBV genomes, reflect the enhanced susceptibility of the recombinant lines to lytic induction.Recently, we showed that GCb+VPA can trigger EBV lytic replication in NPC patients in vivo creating therapeutic sensitivity to antiviral treatment ,6,7. How41 or EF24) with GCb+VPA induced immediate-early and early lytic rather than late lytic gene expression. In agreement with previous publications, low or undetectable levels of full EBV late lytic gene expression upon EBV reactivation by chemical treatment are suggestive of abortive lytic replication [It is well accepted that lytic activation in itself may render the infected cells more susceptible to immune recognition and killing due to the expression of viral lytic-switch proteins, in particular Zebra ,9,30,31.lication ,42. Howelication ,6,7. EF24 with the CLVA regimen at clinically acceptable doses, may enhance EBV reactivation and provide an attractive and safe therapeutic strategy for EBV-associated malignancies. A similar beneficial effect may be expected when using SAHA (Vorinostat) or the recently identified HDAC inhibitor romidepsin in the CLVA treatment regimen [Taken together, the combination of a curcuminoid, in particular compound  regimen . Combine regimen ,45 and o regimen .41) and piperidinone (EF24) scaffolds that serve as putative EBV lytic activators in latently infected EBV-positive carcinomas. EF24 has the potential to act as an adjuvant to enhance EBV reactivation induced by the CLVA regimen (GCb+VPA) for treatment of EBV-associated gastric and nasopharyngeal carcinomas.In conclusion, our study identified two curcuminoids bearing the thiopyranone dioxide (


The Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus causally linked to a broad spectrum of both lymphoid and epithelial malignancies. In order to maintain its persistence in host cells and promote tumorigenesis, EBV must restrict its lytic cycle, which would ultimately lead to cell death, selectively express latent viral proteins, and establish an unlimited proliferative potential. The latter step depends on the maintenance of telomere length provided by telomerase. The viral oncoprotein LMP-1 activates TERT, the catalytic component of telomerase. In addition to its canonical role in stabilizing telomeres, TERT may promote EBV-driven tumorigenesis through extra-telomeric functions. TERT contributes toward preserving EBV latency; in fact, through the NOTCH2/BATF pathway, TERT negatively affects the expression of BZLF1, the master regulator of the EBV lytic cycle. In contrast, TERT inhibition triggers a complete EBV lytic cycle, leading to the death of EBV-infected cells. Interestingly, short-term TERT inhibition causes cell cycle arrest and apoptosis, partly by inducing telomere-independent activation of the ATM/ATR/TP53 pathway. Importantly, TERT inhibition also sensitizes EBV-positive tumor cells to antiviral therapy and enhances the pro-apoptotic effects of chemotherapeutic agents. We provide here an overview on how the extra-telomeric functions of TERT contribute to EBV-driven tumorigenesis. We also discuss the potential therapeutic approach of TERT inhibition in EBV-driven malignancies. The Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus infecting more than 90% of the world’s population. Primary infection with EBV is often asymptomatic, but it can also manifest as infectious mononucleosis. Although EBV may infect various cell types, such as epithelial cells and T or Natural Killer cells, it preferably infects B lymphocytes, in which it establishes a lifelong asymptomatic latent infection. In immunocompromised individuals, EBV may cause a wide range of cancers, of both hematopoietic and epithelial origin, including Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), post-transplant lymphoproliferative disorders (PTLD), AIDS-associated lymphomas, and nasopharyngeal and gastric carcinomas .Like other γ-herpesviruses, EBV has both lytic and latent cycles. Primary EBV infection occurs in the oropharynx, leading to productive lytic infection of B lymphocytes. EBV antigens promote immune recognition, inducing an EBV-specific immune response which controls viral infection in the immunocompetent host, and the viral lytic cycle triggers the death of the infected cells . In tumoBZLF1 and BRLF1 [While latency programs predominate in EBV-driven tumors, lytic reactivation may occur in a small fraction of infected cells, favoring the spread of the virus , 6. Lytind BRLF1 . As lytind BRLF1 –10. Trignd BRLF1 , 12. Thund BRLF1 –15.The establishment of EBV latency programs promotes cell proliferation, inhibits apoptosis, blocks viral lytic replication, and ensures accurate and equal partitioning of the episomal viral genome to daughter cells . HoweverTelomeres are specialized DNA structures located at the ends of chromosomes which are essential for stabilizing chromosomes by protecting them from end-to-end fusion and DNA degradation . In humaAlthough EBV-infected B cells exhibit higher proliferative activity than resting primary B lymphocytes, very few EBV-carrying B cells eventually progress to immortalization: most of them reach a proliferative crisis and end their lifespan after about 150 population-doubling levels, according to genetic factors, including telomere length. Soon after EBV infection, B lymphocytes may exhibit multiple signs of telomere dysfunction and ALT markers, including highly heterogeneous telomeres, appearance of extra-chromosomal telomeric DNA, accumulation of telomere-associated promyelocytic leukemia nuclear bodies, and telomeric-sister chromatid exchange . This phTelomerase is a ribonucleoprotein complex containing an internal RNA template  and a catalytic protein, TERT, with telomere-specific reverse transcriptase activity. TERT, which synthesizes de novo telomere sequences using TERC as a template, is the rate-limiting component of the telomerase complex, and its expression is correlated with telomerase activity. Although TERC has broad tissue distribution and is constitutively present in normal and tumor cells, the expression of TERT is usually repressed in normal somatic cells and is essential for unlimited cell growth, thus playing a critical role in tumor formation and progression .Regulation of telomerase operates at several levels: transcription, mRNA splicing, subcellular location of each component, and assembly of TERC and TERT in an active ribonucleoprotein. Transcription of the TERT gene is probably the key determinant in regulating telomerase activity, since TERT transcription is specifically up-regulated in cancer cells but silent in most normal ones. The TERT promoter reveals complex regulation dynamics, whereby multiple transcriptional regulatory elements play functional roles in different contexts, either individually or interactively. TERT contains recognition sequences for many important transcription factors such as TP53, P21, SP1, ETS, E2F, AP-1, HIF1A and MYC . Regulatvia the NF-κB and MAPK/ERK1/2 pathways [MYC is in a germ-line configuration. In these malignancies, LMP-1 probably plays an essential role in TERT activation.Studies aimed at defining the mechanism underlying EBV-induced telomerase activation have demonstrated that LMP-1, the major EBV oncoprotein, up-regulates telomerase activity both in epithelial cells , 34 and pathways . Of intepathways , in B cepathways . This isThe canonical explanation for the tumor-promoting role of telomerase is that it allows cells to escape the barrier to unlimited replicative potential caused by telomere attrition. Accumulating evidence suggests that, besides its canonical role in stabilizing telomeres, TERT also has other biological functions, including enhancement of cell proliferation, resistance to apoptosis, and regulation of DDR, –39. TERTThe extra-telomeric roles of TERT have also been described in EBV-driven lymphomagenesis. TERT plays a critical role in establishing EBV latency and preventing the EBV lytic cycle, thereby contributing to transformed phenotypes. In particular, high levels of endogenous TERT or ectopic TERT expression in TERT-negative EBV-infected B cells prevents the induction of the viral lytic cycle. By contrast, TERT silencing by specific siRNA or short-hairpin (sh)RNA induces the expression of BZLF1, EA-D and gp350 EBV lytic genes, and triggers a complete lytic cycle. This occurs in both EBV-immortalized and fully transformed B cells, thus supporting the concept that TERT is a critical regulator of the balance between viral latent and lytic cycles , 21. TheStudies aimed at defining the mechanism(s) by means of which TERT prevents the viral lytic cycle have demonstrated the involvement of the NOTCH2/BATF pathway. BATF is a transcription factor expressed in hematopoietic tissues and in B cells infected with EBV –48. In LMore recently, the impact of TERT on EBV infection and viral gene expression has also been studied in epithelial cells . GastricTelomerase inhibitors remain an attractive approach to target cancer cells, given the specificity of TERT expression in tumor cells. However, in theory, the time to antineoplastic effectiveness of telomerase inhibitors depends on the original length of the telomeres in cancer cells and, apparently, these agents are effective in halting tumor growth only after the cancer cells have shortened their telomeres. This aspect acquires particular importance in the context EBV-carrying malignancies as it has been demonstrated that EBV-positive BL cell lines show longer telomeres compared to EBV-negative BLs  and, durIn this scenario, the evidence of extra-telomeric functions of TERT in cellular kinetics and resistance to apoptosis has recently opened the door to potential telomere length-independent therapeutic effects. In EBV-driven malignancies, besides sustaining the latency program required for the EBV-driven transformation, TERT may promote EBV tumor progression by enhancing the kinetics of cell proliferation. In fact, EBV-infected B cells with sustained telomerase activity grow faster than telomerase-negative cells . AccordiAs mentioned previously, the first mechanism by means of which short-term inhibition of TERT may induce cell death of EBV-transformed cells is induction of the EBV lytic cycle. Inhibition of TERT leads to down-regulation of BATF and up-regulation of BZLF1, the main regulator of the viral lytic cycle. Notably, cell death induced by TERT inhibition in EBV-positive cells does not depend only on the induction of the EBV lytic cycle: inhibition of TERT with short hairpin (sh)RNA in both EBV-positive and EBV-negative BL cell lines induces apoptosis via a AKT1/FOXO3/NOXA pathway . In partIn EBV-positive tumor cells, the lytic cell cycle induced by TERT inhibition may be exploited to sensitize cells to antiviral drugs such as GCV. GCV is an antiviral prodrug activated by EBV lytic protein kinase , 11. PhoMost recent data show that LCLs and both EBV-positive and EBV-negative BL cells short-term treated with BIBR1532 (BIBR), a chemical compound which selectively inhibits the catalytic activity of TERT . These eIn addition, treatment of LCL with BIBR in combination with Fludarabine or Cyclophosphamide, two agents frequently employed to treat B-cell malignancies, induces a significant increase in specific cell death compared with results seen after treatment with chemotherapeutic agents alone. These results may lead to the substantial clinical application of TERT inhibitors in combination with standard chemotherapeutic protocols to treat EBV-positive B-cell malignancies .via the NOTCH2/BATF/BZLF1 pathway (Fig. via the AKT1/FOXO3/NOXA and ATM/ATR/TP53 pathways. Notably, cell cycle arrest and the pro-apoptotic effects of short-term TERT inhibition are independent of telomere length. Thus, in both EBV-driven and virus-unrelated B-cell malignancies inhibition of TERT seems to be an effective approach in inducing cell death, regardless of telomere length. In vitro experiments also demonstrate that the therapeutic approach based on inhibition of TERT enhances the pro-apoptotic and anti-proliferative effects of chemotherapeutic agents in EBV-transformed cells. Further studies of primary tumor cells from patients with EBV-driven malignancies and suitable animal models will pave the way for a solidly based pre-clinical rationale for including TERT inhibitors in chemotherapy protocols for treating these malignancies.Since a latent program is required to promote EBV tumorigenesis, whereas the lytic cycle induces cell death, the finding that TERT, besides maintaining telomere integrity, also plays a critical role in the establishment of EBV latency and in preventing the EBV lytic cycle Fig. , supportway Fig. , short-t


Epstein-Barr virus (EBV) replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.  •Unbiased global analysis of host and EBV proteome remodeling in lytic replication•Temporal profiles of >8,000 host and 69 viral proteins, using type I and II EBV•Both EBV types target the B cell receptor complex for degradation•Conserved EBV and HCMV lytic cycle host targets are identified Ersing et al. present a temporal proteomic map of EBV B cell lytic replication. Tandem-mass-tag-based proteomics uncover extensive remodeling of the human proteome by EBV, conserved across the two major EBV strains. Cell-cycle, innate, and adaptive immune pathways are modulated, complement is upregulated, and the B cell receptor is degraded by infection. Epstein-Barr virus (EBV) is a gamma-herpesvirus that establishes persistent infection in >95% of adults worldwide. Two distinct strains of EBV have been identified, referred to as type I and II . FollowiEBV is the etiologic agent of infectious mononucleosis and is closely linked to the pathogenesis of multiple human malignancies, with 200,000 EBV-associated cancers reported annually . Lytic vUpon lytic reactivation, EBV genes are sequentially expressed in immediate-early (IE), early  and late (L) phases. The immediate early transcription factors ZTA (encoded by BZLF1) and RTA (encoded by BRLF1) jointly trigger the EBV lytic cycle. EBV early genes are synergistically induced by ZTA and RTA and encode the viral polymerase and replication machinery. Late viral genes encode structural proteins that encapsidate and mediate release of infectious virions .mRNA expression profiling has provided important information on the kinetics of viral gene expression upon lytic cycle induction in Burkitt lymphoma cell lines . LikewisWe used tandem-mass-tag (TMT)-based MS3 mass spectrometry to perform quantitative temporal proteomic analysis of EBV replication in human Burkitt lymphoma B cells latently infected by type II EBV, prior to and at four time points after induction of lytic replication . Selecti+ Burkitt lymphoma cell line P3HR1 to quantify changes in PM and whole cell lysate (WCL) protein expression by 10-plex TMT and MS3 mass spectrometry at three reference time points after induction of lytic EBV replication , including 6,307 proteins in all three replicates. We additionally quantified 69 EBV proteins . In experiment PM1, 550 PM proteins were quantified B. We obs+ Akata cells are highly permissive for EBV lytic replication in response to anti-immunoglobulin G (IgG) cross-linking  . DAVID sPrior proteomic analyses of EBV lytic protein expression have identified 16 or 44 EBV proteins at single time points of viral reactivation . A partiWe quantified 63 canonical EBV-encoded proteins in all three P3HR1 WCL samples. We used a “proteomic ruler” approach  to estim+ cells, we searched our data against a six-frame translation of the P3HR1 genome  and immunofluorescence, suggests that both PM and intracellular BCR pools are targeted. Further studies are required to identify whether soluble Ig isoforms are also targeted, and to characterize the ubiquitin-proteasome pathway by which EBV downmodulates these BCR pools. One candidate is EBV BDLF3, an early factor that targets cell-surface and intracellular MHC class I molecules for ubiquitin-mediated proteasome degradation .Targeting of the BCR during EBV B cell lytic replication may be conserved across EBV strains, since we observed a similar degree of Ig loss in Akata and P3HR1 B cells, which harbor type I and II EBV, respectively. We hypothesize that EBV targets the BCR to facilitate EBV B cell replication for one of the following reasons. First, a subset of lytic B cells may produce Ig reactive to antigen present on virions. BCR degradation would then enable EBV to avoid becoming trapped by binding to these antibodies within the secretory pathway or at the cell surface. Second, EBV lytic replication is thought to occur in tonsillar plasma cells, which are loaded with Igs. Plasma cells secrete Igs at the rate of approximately 2,000 molecules per second . Ig degrAdditional pathways were selectively downregulated during lytic viral reactivation, including multiple components of the cell-cycle machinery. Protein downregulation in some cases may involve the EBV host shutoff protein BGLF5, which was expressed early in infection B. As BGLMultiple aspects of the EBV life cycle are intimately linked with the complement system. The EBV entry protein gp350 has homology with the C3 complement component cleavage product C3dg . gp350 aEBV establishes latent infection upon B cell entry, and the tonsillar plasma cell population harbors lytic EBV in ex vivo studies. These observations support a model in which plasma cell differentiation provides a cue that stimulates EBV replication. Interestingly, our findings suggest that EBV lytic replication may likewise facilitate plasma cell differentiation, through suppression of TFs that maintain germinal center state and through upregulation of factors that promote plasma cell differentiation. Key germinal center B cell TFs downmodulated by EBV lytic replication included BCL6, ETS1, IRF8, ID3, and MYC, whereas the antibody secreting cell-inducing TFs IRF4, ZBTB20, FOS, and FosB were strongly upregulated. Thus, rather than passively activating as a bystander of plasma cell differentiation, EBV may play a more active role in remodeling the B cell environment to favor lytic replication.To persist in an infected individual lifelong, herpesviruses have developed multiple strategies to modulate innate and adaptive immunity. One benefit is that we can use the overlap between proteins targeted by different herpesviruses to discover molecules particularly important in host defense. By comparing EBV, CMV, and KSHV, we identified downregulation of several members of the same protein family including Neuroligins and human leukocyte antigen (HLA) molecules. EBV and CMV both targeted Protocadherin γC3 for downregulation suggesting that this molecule, other members of the same family, and the Neuroligins might be activating NK ligands, as we previously demonstrated for Protocadherin FAT1 .– cells compared to gp350+ cells, depending on the time point studied after induction .TMT-based analysis was performed as described previously . AlkylatWe performed mass spectrometry as described previously using an Orbitrap Fusion or Orbitrap Lumos  and quanFurther information and reagent requests may be directed to the corresponding authors.I.E., M.P.W., and B.E.G. designed the experiments. I.E., L.N., J.A.P., Y.N., and M.P.W. performed proteomics experiments; I.E., L.W.W., C.W.A., and C.J. performed biochemical experiments; and I.E., L.N., M.P.W., and B.E.G wrote the manuscript. L.N., L.S., J.A.P., S.P.G., and M.P.W. analyzed the proteomics data, and N.E.G. provided EBV genome sequence data and bioinformatic support. Y.M. and N.E.G. performed additional bioinformatic analyses; E.K. provided helpful scientific discussions; and I.E., L.N., S.P.G., M.P.W., E.K., and B.E.G. edited the manuscript. M.P.W. and B.E.G. supervised all research.


We investigate the dynamics of two-dimensional soft vesicles filled with chiral active particles by employing the overdamped Langevin dynamics simulation. The unidirectional rotation is observed for soft vesicles, and the rotational angular velocity of vesicles depends mainly on the area fraction (ρ) and angular velocity (ω) of chiral active particles. There exists an optimal parameter for ω at which the rotational angular velocity of vesicle takes its maximal value. Meanwhile, at low concentration the continuity of curvature is destroyed seriously by chiral active particles, especially for large ω, and at high concentration the chiral active particles cover the vesicle almost uniformly. In addition, the center-of-mass mean square displacement for vesicles is accompanied by oscillations at short timescales, and the oscillation period of diffusion for vesicles is consistent with the rotation period of chiral active particles. The diffusion coefficient of vesicle decreases monotonously with increasing the angular velocity ω of chiral active particles. Our investigation can provide a few designs for nanofabricated devices that can be driven in a unidirectional rotation by chiral active particles or could be used as drug-delivery agent. A great many biological and physical systems can be referred to as active matter systems, including molecular motors6, swimming bacteria8, self-propelled colloids11, motile cells13, and macroscopic animals15. Previous works have demonstrated that active particles which perform spontaneous and sustained motion are fundamentally different from passive particles in thermal equilibrium systems. In other words, active matter systems move actively by gaining energy from an external source under non-equilibrium conditions. Hence, active particles with suitably designed constructions are able to convert energy into desired control of function, which have wide potential applications in a diversity of fields, such as drug delivery in medicine17. More details about microswimmers, propulsion mechanism and application prospects can be found in excellent review articles19.Active matter is a rapidly growing subject which has been studied theoretically and experimentally over the past few years22, confirming that asymmetric environments can rectify the random motion of self-propelled particles and extract energy accordingly. That is, external confinement has a crucial impact on the motion of active particles. In addition, a great number of biological systems are confined in finite regions, leading to novel phenomena such as swarming25, swimming in a spiral vortex26, accumulating at walls28, and so on. Meanwhile, recent studies have also paid more attention to flexible boundaries enclosing active swimmers instead of rigid walls. For instance, the interesting case of active Brownian particles constrained within a deformable boundary has been deeply investigated, exhibiting obvious shape and displacement fluctuations in soft vesicles filled with active particles30.In recent years, there are tremendous researches from experiments and computer simulations focused on the nanofabricated objects immersed in a bath of randomly swimming bacteria31. However, asymmetries in self-propulsion mechanism can generate a more complex behaviour, the direction of motion and that of the force are no longer aligned and the active particles tend to execute circular motion. Such chiral active particles have attracted mounting interest over the last few years35. Microscopy images show that non-tumbling E. coli bacteria swim in circular trajectories near planar glass surfaces34, indicating the motion of bacteria close to surfaces differs from the run-and-tumble motion in free solution. Active colloidal cell driven by gear-like spinners has also been observed35, which mainly focus on the shape control and compartmentalization of cells. Here, we choose spherical self-propelled and self-rotated particles without any special design as chiral active particles, and reveal more general organization principles of this class of active particles confined by a vesicle.As mentioned above, dynamics of self-propelled particles has captured the growing interest of various groups. In the models of self-propelled particles, it is assumed that the active particles possess uniaxial symmetry and that the direction of the self-propulsion is along the axis of symmetry of the particle. Thus active particles tend to orient along the active force and move in a straight line26. In fact, short-range forces and noise dominate the interactions between swimming organisms, and the influence of hydrodynamic interactions is negligible36. Only for swimmers in close contact with a surface, the hydrodynamic effects become important, favouring long residences time along the walls. However, this effect is given also by self-propulsion36. Therefore, hydrodynamic interactions should not change the phenomenology qualitatively, and the active particles obey overdamped Langevin dynamics without the hydrodynamic interactions42. That is, we employ the overdamped Langevin dynamics simulations to study the dynamical behaviours of soft vesicles filled by spherical self-propelled and self-rotated particles. And the aim of this work is to explore how the additional angular velocity of chiral active Brownian particles affects the dynamics of soft vesicles. Owing to the presence of angular velocity for chiral active particles, active particles collide with the vesicle edge and consequently drive the vesicles into unidirectional rotation. The rotational angular velocity of vesicles depends mainly on the area fraction (ρ) as well as the angular velocity (ω) of chiral active particles. Meanwhile, the mean square displacement of the center of mass of vesicles filled with chiral active particles shows that the diffusion of vesicles is accompanied by the oscillation at short timescales, and its oscillation period is consistent with the rotation period of chiral active particles.Active particles use a wide variety of mechanisms to achieve self-propulsion in liquid environments, and swimming at the macroscopic scale heavily relies on the inertia of the surrounding fluid. Hydrodynamic interactions between active particles and their detached vertical structures are the basis of higher swimming (or flying) efficiencyσ. Each chiral active particle moves with a translational velocity V0 for self-propulsion and a counterclockwise angular velocity ω for self-rotation, performing a circular active Brownian motion. The overdamped dynamics is governed by Langevin equations for the position ri and the orientation θi of the polar axis θi, sinθi) of the center of the i-th active particle inside the vesicle42γ is the friction coefficient, V0 is the self-propulsion velocity of chiral active particles, and U is the configurational energy. D0 and Dθ denote the translational and rotational diffusion coefficients respectively. There exist the relations of γ = kBT/D0 and Dθ = 3D0/σ2 42. ξiT(t) and ξiR(t) are Gaussian white noise with zero mean and satisfy < ξiαT(t)ξjβT(s) > = δijδαβδ(t-s)  and < ξiR(t)ξjR(s) > = δijδ(t-s), respectively43. Here < …. > donates an ensemble average over the distribution of the noise, and δ is the Dirac delta function.Molecular dynamics simulations are employed to study the dynamical behaviors of vesicles filled with chiral active particles. Chiral active particles are modelled as Lennard-Jones (LJ) spheres with a bead diameter of ULJ is used for chiral active particles as the configurational energy U in eqn  in two-dimensional vesicle model. The equation of motion can also be described as eqn )−1σ and the area fraction of chiral active particles for the initial vesicle is determined by ρ = Nσ2/(4R02)29. Here N is the number of chiral active particles within the vesicle, and for example, N = 1596 for L = 150 and ρ = 0.7. MD simulations are carried out by performing Langevin dynamics with the open source software, LAMMPS45. We nondimensionalize the equations of motion using σ and ε as basic units of length and energy, and τ0 = σ2/D0 as the unit of time. And the time step is set to be τ = 0.0001 τ0. Meanwhile, the parameters are chosen to be D0 = 0.01, Dθ = 0.03, γ = 100, and V0 = 0.5. We have performed the simulations with various vesicle perimeters of L = 50, 100, and 150, and various area fractions of active particles from ρ = 0.05 to 0.7. Moreover, the angular velocities of chiral active particles are selected from ω = 0 to 1.0. The total simulation time for each run in our results is not less than 2 × 108τ. In addition, all the simulation snapshots are captured using the Visual Molecular Dynamics (VMD) package46.The vesicle moves in the x-y plane of 200σ × 200σ with periodic boundary conditions. The initial configuration of the vesicle is a circle of radius Rt. Here L = 50. The straight back lines are linear fitting curves and the slopes of these lines are also given in Fig. i(t) for i-th monomer of the vesicle at time t. The average rotation angle β(t) for the vesicle is defined asi(t) means the cumulative rotation angle of i-monomer at time t. Figure t, and the slope of line increases monotonically with ρ increasing. However, there is an exception for the case of ρ = 0, in which the cumulative rotation angle oscillates slightly near zero under thermal interference of a vesicle without any chiral active particles which can drive the vesicle to rotate counterclockwise. Moreover, we also study the dependence of cumulative angular angle β on time t with various angular velocities of chiral active particles, and the results are shown in Fig. In our simulation, the chiral active particles perform the counterclockwise motion with ω > 0. These active particles can resemble molecular motors, and the collision with the vesicle membrane results in the rotational motion of vesicle in the same direction. Figure op at which ω’ reaches its maximum value. Figure op always appears about ωop ≈ 0.04, which seems to be independent of the area fraction ρ and the vesicle perimeter L. In order to explain the novel non-monotonic behaviour of ω’ in Fig. S > of chiral active particles stacked to the vesicle membrane, and the results are shown in Fig. 40. The larger the angular velocity ω is, the smaller the radius of circular trajectory is40. Since the occurrence of collision with the membrane is rare for chiral active particles with large ω, the rotation of the vesicle is very slow for large ω, especially at a lower area fraction of ρ = 0.2. We also measure the average tangential force < Fτ > per monomer of the membrane with different angular velocities for various area fractions and various vesicle perimeters respectively, and the results are given in Fig. τ > in Fig. op ≈ 0.04). Therefore, the average tangential force < Fτ > plays an important role in the appearance of optimum angular velocity. By means of the schematic illustrations in Fig. 47. For self-propelled active particles, the asymmetry boundary is a basic ingredient, as observed in many other thermal ratchet mechanisms48, and self-starting micromotors with asymmetric boundary can be observed in a bacterial bath47. However, at a moderate angular velocity of ω = 0.04, there exists the higher collision probability with the membrane for chiral active particles because the radius of circular trajectory for chiral active particles is very large and the chiral active particles usually are aggregated near the membrane, as shown in Fig. τ > or rotation angular velocity ω’ only exists for high collision synchronization and high collision probability. As for the case of ω = 1.0, since the radius of circular trajectory for chiral active particles is fairly small, the collisions with the membrane occur rarely. Consequently, although the collision also produces the counterclockwise tangential force  and different angular velocities (ω) in Fig. et al.29. Four detailed configurations of vesicle are also shown in Fig. 0, and probability distributions P(κ) are normalized. Here, κ0 represents the curvature of initial circular configuration, and κ is the local curvature29. The reduced local curvatures follow Gaussian distributions centered near κ/κ0 ≈ 1 with decreasing widths, as the area fraction ρ increases gradually. The chiral active particles are distributed homogeneously for ω = 1.0, and they slowly stretch the membrane with increasing ρ. Combining with Fig. 0.To analyze the deformation of vesicle shape, we quantify the asphericity of vesicle (Δ)Rg–R0)/Rg as a function of the actual area fraction ρR02/Rg2 with different vesicle perimeters. For active particles with ω = 0.0, (Rg–R0)/Rg scale linearly with the packing fraction, especially for L = 100. Deviations of the (Rg–R0)/Rg from the linear regime are visible at high area fraction ρ due to the excluded volume effects51. However, for vesicles filled with chiral active particles, most of the quantities (Rg–R0)/Rg are less than zero at the low packing fractions. In fact, (Rg–R0)/Rg is proportional to the average pressure exerted by the active particles51. We also measure the average pressure < Pn > exerted by the active particles on the membrane, and the results are shown in Fig. g and produces a negative value of (Rg–R0)/Rg for ω = 1.0, see the inset of Fig. Figure 53.cm(t) and ycm(t) are the coordinates of the center of mass of vesicles filled with chiral active particles. Soft vesicles perform a random walk under the action of collision arising from chiral active particles. Since active particles and monomers of vesicle have the same size and mobility, the velocity of the center of mass 29μ is the mobility. The center of vesicle moves as a body of reduced mobility μ/(N + L) under the action of the total force on the active particles. The corresponding velocity-velocity correlation function is given by29Dynamical behaviors of vesicles are investigated through calculating the mean square displacement (MSD) of the center of mass of vesicles filled with chiral active particles, which is given byFor chiral active particles with an angular velocity of ω, forces are uncorrelated and the corresponding force-force correlation function is29. Here, we also neglect the translational noise for the direct comparisons with the theoretical results in our simulation. The mean square displacement (MSD) of the center of mass of vesicles is calculated by a double time integration of eqn (13)29If ω = 0, eqn  becomes A, D and cos φ0 are given byThe parameter 3(t) follows the expression very well,Comparing with the theoretical results given by eqn , we findA and cosφ0 are given by eqns A and cosD and Deff is made in Fig. D and Deff are obvious. However, Deff is in good agreement with the theoretical results of D for ω < 0.2. In fact, for ω = 0, eqn  is close to 0. Of course, the corresponding force-force correlation function of eqn 59. When active particles touches the membrane, although all interactions between active particles and monomers of the membrane are center to center, the collision angle α between the active particles and the monomers of the membrane isn’t always equal to π/2 because of the counterclockwise rotation for chiral active particles. Accordingly, there exists net tangential forces for monomers of vesicle, and the rotating movement for vesicle occurs. Meanwhile, rotational angular velocity of vesicle depends mainly on the angular velocity (ω) and area fraction (ρ) of chiral active particles, and there exists an optimal parameter for ω at which the rotational angular velocity of vesicle takes its maximal value. Our results highlight that asymmetric environments can produce a spontaneous and directional rotation of vesicles filled with chiral active particles. Moreover, the shape of vesicle also relies on the area fraction (ρ) and angular velocity (ω) of chiral active particles as well as the vesicle perimeter (L). At low concentration the continuity of curvature is destroyed seriously for chiral active particles with large ω, and at high concentration the chiral active particles cover the vesicle almost uniformly, resulting in fairly homogeneous curvature and nearly circular vesicle shape. The center-of-mass mean square displacement for vesicle is accompanied by oscillations at short timescales, and the oscillation period of diffusion is consistent with the rotation period of chiral active particles. Meanwhile, the diffusion slows down significantly with the additional angular velocity for chiral active particles by about two orders, especially for high concentration. Our investigation can provide a few designs for nanofabricated devices with asymmetric environments that can be driven in a unidirectional rotation by chiral active particles.We have investigated numerically the dynamical behaviors of vesicles filled with chiral active particles using 2D Langevin dynamics simulations. In fact, the behavior of vesicles filled with chiral active particles bears some resemblance with the directed migration of Eukaryotic cells, as observed in would healing assays or in the presence of chemotactic cuesSupplementary Information: Rotational Diffusion of Soft Vesicles Filled by Chiral Active ParticlesVideo S1. Diffusion behavior of a soft vesicle filled by active particles with ω=0.Video S2. Diffusion behavior of a soft vesicle filled by chiral active particles with ω=0.7.Video S3. Diffusion behavior of a soft vesicle filled by chiral active particles with ω=0.04.


The antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been shown to affect maturation, function, and subsequent DC-mediated T cell responses of murine and human DCs. In experimental autoimmune encephalomyelitis (EAE), as prototype animal model for a T helper cell-mediated autoimmune disease, antigen presentation, cytokine production, and costimulation by DCs play a major role. We explore the role of Nrf2 in DC function, and DC-mediated T cell responses during T cell-mediated autoimmunity of the central nervous system using genetic ablation and pharmacological activation in mice and men to corroborate our data in a translational setting. In murine and human DCs, monomethyl fumarate induced Nrf2 signaling inhibits DC maturation and DC-mediated T cell proliferation by reducing inflammatory cytokine production and expression of costimulatory molecules. In contrast, Nrf2-deficient DCs generate more activated T helper cells (Th1/Th17) but fewer regulatory T cells and foster T cell proliferation. Transfer of DCs with Nrf2 activation during active EAE reduces disease severity and T cell infiltration. Our data demonstrate that Nrf2 signaling modulates autoimmunity in murine and human systems  In the periphery, immature dendritic cells (DCs) survey their environment and capture soluble antigens, like microbes and cell debris. Since they are barely immunogenic, immature DC express only low levels of major histocompatibility complex (MHC) II and costimulatory molecules but display marked phagocytic capacity which renders them critical for maintaining peripheral T cell tolerance , 2. Activia proteasomal degradation by Kelch-like ECH-associated protein 1 (Keap1) -like 2 (Nrf2) was described as critical regulator responsible for maintenance of DC redox homeostasis, differentiation, and maturation by modulating the expression of cytoprotective genes, such as heme oxygenase 1 (Hmox1), NAD(P)H: quinone oxidoreductase 1 (Nqo1), and glutathione –12. In g (Keap1) , 21. App (Keap1) , 23, inc (Keap1) . In cont (Keap1) .via NF-κB signaling pathways in vitro  and its primary active metabolite monomethyl fumarate (MMF), have been shown to activate the Nrf2-ARE pathway by altering the ability of Keap1 to interact with Nrf2  and reduin vitro –31. In Ein vitro , 32. Simin vitro , 34. Thuin vitro as well as in T cell-mediated autoimmunity of the central nervous system in vivo. We show that Nrf2 deficiency fosters a proinflammatory phenotype in murine and human DC, while Nrf2 activation promotes rather tolerogenic functions by significantly modulating cytokine production and expression of costimulatory molecules.Here, we focus on the effect of Nrf2 signaling on DC maturation, function and subsequent T cell responses Nuclear factor (erythroid-derived 2)-like 2-fl/fl mice were a kind gift from Prof. Pi, China Medical University, Shenyang, China. CD11c-Cre mice were obtained from Prof. Steinkasserer, University of Erlangen-Nürnberg, Erlangen, Germany. Mice harboring a transgenic MOG-specific T cell receptor [2D2 mice ]; were ain vivo studies but MMF for in vitro stimulation experiments.Mice received 15 mg/kg body weight DMF  in 0.8 mg/ml hydroxypropyl methylcellulose (Sigma-Aldrich) twice daily by oral gavage, while control mice obtained 0.8 mg/ml hydroxypropyl methylcellulose. Mice were adapted to DMF treatment 2 weeks before EAE induction. Since the majority of DMF is rapidly metabolized to MMF by esterases in the intestine and only MMF can be detected in the systemic circulation , 37, we 5 DC, cultured in vitro for 10 days with or without 200 µM MMF (Sigma-Aldrich) and stimulated with 1 µg/ml lipopolysaccharide (LPS) (Sigma-Aldrich) for 24 h, were injected intraperitoneally at d2 and d7 after EAE induction together with 200 ng pertussis toxin .Induction of active EAE was performed as previously described , 39. Forl-glutamine) containing granulocyte macrophage colony-stimulating factor (GM-CSF)  and interleukin (IL)-4  for 10 days with/without 200 µM MMF (Sigma-Aldrich). Cells were matured on day 8 with 1 µg/ml LPS (Sigma-Aldrich). For flow cytometry, cells were stained with αCD11c , αCD80 , αCD86 , and αMHCII .Bone marrow cells were obtained from tibias and femurs and maintained in R10 medium . After 20 h of incubation, cells were subjected to flow cytometry and phagocytic cells were detected in the FITC channel.Phagocytosis was assessed by stimulating DC with 1 µg/ml LPS (Sigma-Aldrich) for 48 h before adding opsonized FluoresbriteThe migratory capacity of DC was investigated using a modified Boyden chamber assay and transwell inserts with a 5 µm porous bottom . Mature DC were seeded into the migration chamber in medium containing 0.5% FCS. The lower chamber was supplemented with 10% FCS medium. After 20 h of migration, cells residing in the lower chamber (transmigrated fraction) were stained for CD11c and quantified by flow cytometry .35–55 peptide (20 µg/ml) for 1 h and seeded with naive transgenic T cells harboring a MOG specific TCR at a ratio of 1:6. Naive MOG-specific transgenic T cells were isolated form the spleen of 2D2 mice by magnetic activated cell sorting using the “Naive CD4+ T Cell Isolation Kit, mouse” according to the manufacturer’s instructions . For Th17 differentiations, cocultured cells were additionally stimulated with IL-6  and transforming growth factor beta (TGFβ1) . For Th1 differentiations, cells were cultured in the presence of IL-12p70  and anti-IL-4 . For Treg differentiations, TGFβ1  was added to culture media. T cell frequencies were analyzed by flow cytometry  using the antibodies listed in the section “Mature DC were loaded with MOG+CD62L+CD44lowCD25−) were stimulated by plate-bound anti-CD3  and soluble anti-CD28  and cultured for 4 days in the presence of IL-6  and TGFβ1  for Th17, IL-12p70  and anti-IL-4  for Th1 or TGFβ1  alone for Treg differentiation. T cell frequencies were analyzed by flow cytometry  using the antibodies listed in the section “Differentiation of murine T cells was performed as previously described . SplenicSpinal cords were disrupted with a 5 ml glass homogenizer followed by Percoll  density gradient centrifugation. Cell frequencies were analyzed by flow cytometry  using the antibodies listed in the section “®780 . Nonspecific Fc-mediated interactions were blocked by addition of 0.5 µl αCD16/32 . For surface staining, cells were treated with the respective fluorochrome-conjugated antibodies: αCD4-BV510 , αCD11c-FITC , αCD25-APC , αCD80-APC , αCD86-PerCP/Cy5.5 , and αMHCII-BV510 .As previously described , dead ceFor intracellular cytokine staining, cells were stimulated for 4 h with ionomycin  and PMA  in the presence of monensin , fixed with 1% paraformaldehyde and made permeable by saponin buffer treatment. Intracellular cytokines were stained with the respective fluorochrome-conjugated antibodies: αFoxp3-PE , αIFNγ-APC , and αIL-17A-PE . Samples were measured with a flow cytometer .l-glutamine (Sigma-Aldrich), and 10 mM Hepes (Lonza) as well as 800 IU/ml (day 0) or 400 IU/ml (day 3) recombinant human GM-CSF and 250 IU/ml (days 0 and 3) recombinant IL-4 (both Miltenyi). Maturation of DC was induced by adding a maturation cocktail consisting of 200 U/ml IL-1β, 1,000 U/ml IL-6 , 10 ng/ml tumor necrosis factor alpha (TNF-α) , and 1 µg/ml PGE2  for 48 h. DC were analyzed by flow cytometry for activation marker expression: αCD80-PE , αMHCII-PE/Cy5 , αCD25-FITC , αCD86-PE , αMHCI-FITC , αCCR7-APC , αILT-4-FITC , and αCD83-PE .For the generation of monocyte-derived DCs from leukoreduction system chambers of healthy donors (HDs) the positive vote from the local ethics committee has been obtained (Re.-Nr.: 4556). Generation of human monocyte-derived DC was performed as previously described . Periphe6 cells/ml in MLR medium consisting of RPMI 1640 (Lonza) supplemented with 5% heat-inactivated human serum type AB (Sigma-Aldrich), 1% Penicillin/Streptomycin/l-glutamine (Sigma-Aldrich), and 10 mM HEPES (Lonza). Afterward, triplicates of 2 × 105 allogeneic T cells were cocultured in 96-well flat cell culture plates  with allogeneic DCs at different ratios for 72 h in MLR medium, and were finally pulsed with [3H]-thymidine (1 μC/well) for 8–16 h. Cultures were harvested onto glassfiber filtermates using an ICH-110 harvester  and filters were counted in a 1450 microplate counter .Mature human DC were generated as described above  and wereDetermination of cytokines  secreted by human DC and/or T cells into the supernatant was assessed by Human Inflammation LEGENDplex™  according to the manufacturer’s protocol.Cytokine concentrations in murine cell culture supernatants were analyzed by ELISA for the secretion of IL-6, TNF-α, IL-23, and IL-12 (R&D Systems) according to the manufacturer’s instructions.actb/β-Actin or gapdh as housekeeping gene. The following TaqMan® real-time PCR assays from Thermo Fisher Scientific were used: actb (β-Actin) Mm00607939_s1, nfe2l2 (nrf2) Mm00477784_m1, il6 Mm99999064_m1, nqo1 Mm01253561_m1, hmox1 Mm00516005_m1, il10 Mm01288386_m1, il12a Mm00434169_m1, arg1 Mm00475988_m1, tgfb Mm01178819_m1, nqo1 Hs02512143_s1, akr1c1 Hs04230636_sH, hcar2 Hs02341584_s1, and gapdh Hs02758991_g1.RNA isolation, reverse transcription and PCR reactions were performed as previously described . RelativCultured cells were harvested in 1× Ripa lysis buffer . Protein concentration was determined using the BC Assay Protein Quantitation Kit . Protein was detected using a rabbit polyclonal Nqo1 antibody . Mouse anti-β-Actin  was obtained from Abcam (ab8226). Donkey antirabbit Alexa Fluor 488-conjugated and goat antimouse Alexa Fluor 647-conjugated secondary antibodies  were used. Detection was performed using a Fusion Capt Advance FX7 .in vitro and ex vivo data were analyzed by either one-/two-way ANOVA followed by Tukey’s posttest, unpaired t-test or Mann–Whitney test after checking for normal distribution (unless otherwise indicated). EAE data were analyzed by Mann–Whitney U-test. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, or ***p < 0.001 were considered to be statistically significant.Statistical testing was performed using GraphPad Prism . All in vitro. Yet CD11c+ cell frequencies were not altered in MMF-treated cell cultures  or starting at the day of LPS stimulation  to analyze if the time point of Nrf2 activation is crucial for its effect on DC maturation and surface marker expression. First, we examined whether activation of Nrf2-dependent signaling pathways influences BMDC generation + cells did not differ between bone marrow obtained from wt, Nrf2KO, or CD11c-Cre/Nrf2-fl/fl mice and treatment with 200 µM MMF did not influence the generation of BMDC in the cell culture  or conditional DC specific knockout mice (CD11c-Cre/Nrf2-fl/fl) with MMF. Again, the yield of CD11cin vitro.In conclusion, modulation of the Nrf2 signaling pathway critically affects the activation status of DC in vitro. As shown in Figure Next, we assessed the influence of Nrf2 on different essential BMDC functions in vitro. In turn, Nrf2 activation not only induced its antioxidative target genes nqo1 and hmox1 but also the anti-inflammatory cytokine il10 , nqo1, and hmox1 expression was almost undetectable in Nrf2-deficient BMDC and tgfb expression levels were also significantly lower compared to wt BMDC , a marker for tolerogenic DC  as well as an increased expression of the tolerogenic surface receptor Ig-like transcript (ILT)-4  in human monocyte-derived DC analogously to murine BMDC , a niacin receptor involved in the regulation of inflammation and oxidative stress independently from Nrf2 signaling . For the generation of monocyte-derived DCs from leukoreduction system chambers of healthy donors, the positive vote from the local ethics committee at the University Erlangen-Nürnberg has been obtained (Re.-Nr.: 4556).AnH, AW, IK, EZ, JB, SJ, KK, and CD planned and performed experiments and analyses. RL designed the study and planned as well as supervised the research. AnH, AW, and RAL wrote the manuscript. AS and JP provided genetic engineered mice. AS, RG, AiH, AB, JP, and DL supervised the research and edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Percutaneous transluminal renal angioplasty (PTRA) improves blood pressure (BP) and renal function only in selected patients with atherosclerotic renovascular disease (ARVD). Hyperuricemia is associated with elevated risk for hypertension and chronic renal disease, but its role in renovascular hypertension is unclear. We hypothesized that hyperuricemia negatively impacts renal and BP outcomes among patients with ARVD undergoing PTRA. This retrospective, observational cohort study included 94 patients with ARVD and preserved systolic cardiac function, who underwent PTRA at Mayo Clinic, Rochester, Minnesota. Renal, BP, and mortality outcomes were compared among patients according to their serum uric acid (SUA) levels. Multivariate analysis was used to determine significant predictors of renal, BP, and mortality outcomes after PTRA.β: 26.0; 95% confidence interval, 13.9 to 38.1). Compared to patients with normal basal SUA levels (≤5.7 mg/dl), patients with very high SUA (≥8.7 mg/dl) had lower baseline estimated glomerular filtration rate (eGFR), more extensive use of antihypertensive and diuretic drugs, increased baseline systolic blood pressure (SBP), and elevated left ventricular mass index. After PTRA, multiple logistic regression analysis showed that, compared to normal SUA, very high SUA was associated with decreased odds ratio (OR) of change in eGFR , but not of change in SBP. In multivariate linear analysis SUA independently predicted delta urine protein/creatinine ratio ( Severe hyperuricemia in patients with AVRD may have a negative impact on outcomes of renal revascularization. Atherosclerotic renovascular disease (ARVD) is a common and progressive disease with manifestation of renovascular hypertension. It affects 7% of individuals older than 65 years and accounts for 90% of cases of renal artery stenosis, resulting in a reduction of renal blood flow (RBF) to the affected kidney . In addiIncreasing evidence over the past century has supported a strong association between serum uric acid (SUA) and hypertension, independent of traditional risk factors . NotablyThe mechanisms responsible for essential hypertension differ from those contributing to renovascular hypertension, yet the role of SUA in hypertension and renal injury in renovascular disease has not been elucidated. SUA is elevated in patients with ARVD compared to healthy controls , but wheAfter receiving approval from the Institutional Review Board of the Mayo Clinic, 94 patients above 18 years of age at Mayo Clinic, Rochester, Minnesota, USA, were enrolled in the present study. Informed written consent was obtained. Patients identified with significant ARVD, using entry criteria analogous to enrolment in CORAL , were reBaseline clinical parameters were recorded at the time of PTRA. Follow-up was achieved via the electronic medical records within 3 years and with the censored point at the last observed clinical visit at Mayo Clinic, the end of the study period, or death. All echocardiograms were performed by certified technicians following standard clinical procedures and read by level III certified echocardiographers. EF was measured by the quantitative 2-dimensional biplane volumetric Simpson method. Some of the data were only available for some patients, such as level of urine protein/creatinine ratio (PCR) . Cardiov t-test) and nonparametric  tests were used to compare continuous variables among the groups and paired t-tests within groups. Pearson's chi-squared test was used for categorical data. We defined improvement of systolic BP (SBP) as mean reduction by more than ≥5 mmHg after PTRA compared to baseline, which is clinically and statistically meaningful, as done before [Statistical analysis was performed using JMP version 13.0 . Continuous variables are expressed as mean±SD and skewed variables as median (range). Parametric , in which the change in SBP was greater than in the normal group , p<0.05.Significant falls in eGFR were observed only in very high SUA group , p<0.01,α-blocker had a greater change in urine PCR compared to nonusers (p<0.01). A multivariate linear analysis revealed that SUA independently predicted delta PCR , as did use of α-blockers  . Patients using o -61.0) .Timing of the last follow-up after revascularization was similar among the groups . A univaWe evaluated the associations of serum uric acid levels with outcomes of renal revascularization in AVRD patients. Our study shows that renal function in patients with severe hyperuricemia may benefit less from renal revascularization than those with normal SUA, given that very high SUA was associated with a decreased odds ratio of a rise in eGFR. Our findings in renovascular disease are consistent with the role of hyperuricemia as a risk factor for incident or progression of CKD . On the Renovascular hypertensive patients with severe hyperuricemia had higher BP and left ventricular hypertrophy, indicated by increased LVMI, which may result from activation of the renin-angiotensin system , reducti2) in a subgroup of patients with an initially lower eGFR. In another prospective, single-arm, multicenter clinical study [2). Conversely, in the present study we observed a significant fall in eGFR in patients with very high SUA group, whose baseline eGFR (41.2±15.8 ml/min/1.73m2) was very close to the previous studies. Severe hyperuricemia was associated with greater renal dysfunction following renal artery revascularization even after adjustment for baseline renal function. These findings imply that UA may blunt renal recovery in renovascular hypertensive patients independent of baseline renal function. Renal function in normal SUA group was unchanged, although we cannot rule out that PTRA improves GFR in stenotic and decreases it in contralateral, nonstenotic kidneys [In several studies, renal function in patients with ARVD that was deteriorating before PTRA stabilized thereafter. Ramos et al.  studied al study , and PTR kidneys .α-blocker usage was associated with decreased PCR after PTRA, consistent with previous study in which doxazosin reduced proteinuria by 34% [Furthermore, SUA remained an independent predictor for increased proteinuria after adjustment. Proteinuria not only predicts worse renal outcome but is also associated with an increased risk for cardiovascular disease . Severala by 34%  in patieWe also found that baseline SUA was associated with, but did not independently predict, all-cause mortality after PTRA. A relationship between UA and mortality has been previously shown in stages 3-5 CKD patients , but ourThis study has some limitations. First, this small single-center retrospective study included primarily Caucasian patients, limiting the generalizability of the results. Second, although we observed no significant difference in follow-up time from revascularization to the outcomes studied among groups, the pertinent conclusion would have been more robust had the outcomes been studied with a scheduled timetable. Furthermore, we excluded patients with severe cardiac dysfunction, which may introduce selection bias, because UA is associated with increasing incident heart failure in elderly people  and seveThis study shows that severe hyperuricemia may be associated with greater residual renal dysfunction and increased proteinuria after PTRA in ARVD patients, whereas no significant relationship between hyperuricemia and BP outcome was observed. Thus, severe hyperuricemia in AVRD patients may have a negative impact on the outcomes of renal revascularization, although this preliminary finding requires confirmation in larger clinical trials. Further studies are needed to explore the use of severe hyperuricemia as an exclusion criterion for PTRA, or the utility of a UA lowering drug before intervention.