Here we screened small molecules showing anti-TNF-α activity in the compound library of indole derivatives. 11 out of 41 indole derivatives inhibited the TNF-α effect. Among them, Mitochonic Acid 35 (MA-35), 5--3-indoleacetic acid, showed the potent effect. The anti-TNF-α activity was mediated by inhibiting IκB kinase phosphorylation, which attenuated the LPS/GaIN-induced hepatic inflammation in the mice. Additionally, MA-35 concurrently showed an anti-TGF-β1 effect by inhibiting Smad3 phosphorylation, resulting in the downregulation of TGF-β1-induced fibrotic gene expression. In unilateral ureter obstructed mouse kidney, which is a renal fibrosis model, MA-35 attenuated renal inflammation and fibrosis with the downregulation of inflammatory cytokines and fibrotic gene expressions. Furthermore, MA-35 inhibited TGF-β1-induced H3K4me1 histone modification of the fibrotic gene promoter, leading to a decrease in the fibrotic gene expression. MA-35 affects multiple signaling pathways involved in the fibrosis and may recover epigenetic modification; therefore, it could possibly be a novel therapeutic drug for fibrosis.Renal fibrosis is closely related to chronic inflammation and is under the control of epigenetic regulations. Because the signaling of transforming growth factor-β In fibrotic diseases, the signaling of transforming growth factor-β1 (TGF-β1) plays a key role in its disease progression. TGF-β1 activates resident fibroblasts to turn into myofibroblasts, which produce excessive extracellular matrix (ECM)1. In addition to the TGF-β1 signaling, tumor necrosis factor-α (TNF-α) is an important pro-inflammatory mediator that aggravates renal fibrosis2, because renal fibrosis is preceded by chronic inflammation accompanied by complex inflammatory processes.Fibrosis leads to the irreversible exacerbation of compromised function in various organs such as the liver, lung, heart and kidney3, although several attempts to prevent fibrosis using anti-TNF-α agents have been undertaken. Anti-TNF-α biologics such as infliximab, adalimumab or etanercept have not been shown to be successful in the treatment of renal fibrosis. As in the case of such anti-TNF-α therapy, anti-TGF-β1 agents such as pirfenidone have not been shown to be successful in the treatment of renal fibrosis. Because TGF-β1 is one of the anti-inflammatory cytokines that suppresses inflammatory response, anti-TGF-β1 therapy alone may actually worsen the inflammatory response and thereby promote fibrosis4. Thus, inhibiting both the TNF-α and TGF-β1 signaling pathways may be a potential approach to anti-fibrotic treatment.There is as yet no definitive drug for the treatment or prevention of renal fibrosis1 are also controlled by epigenetic mechanisms6. TGF-β1–induced SET domain-containing lysine methyltransferase 7/9 (SET7/9) promotes the methylation of histone H3 lysine 4 (H3K4) and increases pro-fibrotic gene expression, as we described previously. Thus, inhibition of SET7/9 could reverse histone modifications and it may prevent the progression of renal fibrosis.The pro-fibrotic effects of TGF-β1 activities. From the small molecule library of indole derivatives that we recently synthesized, we found out a novel indole derivative compound MA-35 that showed both anti-TNF-α and anti-TGF-β1 effect. MA-35 treatment attenuated the inflammatory response and renal fibrosis with epigenetic modification of SET7/9 in vitro and also in the animal model.In the present study, we focused on the development of a novel anti-fibrotic agent having both anti-TNF-α and anti-TGF-β7. Recently, we constructed an in-house indole compound library and reported that some of compounds exerted a stimulatory effect on Epo production and increased the cellular ATP level in Epo-producing Hep3B cells9. Then, we utilized this chemical library to screen compounds which have anti-TNF-α activity. As shown in Fig. in vitro analysis, the intravenous injection of MA-35 into mice did not affect the serum Epo level  production in liver-derived Hep3B cells. Epo production by the Hep3B cells increases under the hypoxic condition, and the increase is significantly suppressed by the exposure to TNF-αin vitro system: a cytotoxicity assay using a TNF-α-sensitive L929 cells10. The cell viability of L929 cells was reduced by TNF-α in a dose-dependent manner and the apparent IC 50 was 0.03 ng/dL  pathways13. Since TNF-α signaling pathway consists of phosphorylation of IκB kinase (IKK), p38, ERK and JNK, we examined the each phosphorylation level. The phosphorylation of IKK and p38 was increased 15 min after TNF-α stimulation, and the phosphorylation of ERK and JNK peaked at 30 min  line LX-2, which is a representative model for examining TNF-α−14. In addition, the induced-TNF-α further activates the IKK/NF-κB pathway and enhances the TNF-α expression, forming a positive feedback loop15. To examine whether MA-35 inhibits the downstream signaling of phosphorylated IKK and the positive feedback loop of TNF-α, we investigated the effect of MA-35 on the LPS/ Toll-like receptors 4 (TLR4) signaling pathway  induced-fulminant hepatitis mice model, in which TNF-α plays a main pathway in the pathogenic condition16. As in Fig. in vivo, which ameliorated the hepatic injury in the mice model.To examine the anti-TNF-α effect of MA-35 1 mRNA level is reported to be increased16. Our analysis also showed that the TGF-β1 mRNA expression in the liver was elevated by the LPS/D-GalN. This elevation of hepatic TGF-β1 was significantly reduced by MA-35 treatment  was also increased by the LPS/D-GalN, and this increase was significantly suppressed by MA-35 17. Because many previous reports evaluated fibrosis in the early stage of UUO19, we also examined the anti-fibrotic effect of MA-35 5 days after the UUO. MA-35 was administered by osmotic pump starting 3 days before the unilateral ureter ligation and the mice were sacrificed 5 days after the ligation , fibronectin (Fn1), collagen I (Col1a1) and plasminogen activator inhibitor-1 (PAI-1) (Pai1) were increased in the UUO kidney, and this increase was significantly suppressed by MA-35 20 20 Fig. . PreviouSetd7) was increased and this increase was significantly suppressed by MA-35  and PAI-1 (PAI-1) promoters was upregulated, and this upregulation was significantly inhibited by MA-35  was added to the culture for indicated periods of time. Samples were subjected to quantitative PCR or Western blot (n = 3).Human hepatoma Hep3B cells  were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin. Human hepatic stellate cell line LX-2 cells were kindly provided by Prof. Friedman of the Mount Sinai Medical School, New York. LX-2 cells and Murine TNF-α sensitive L929 fibrosarcoma cells  were cultured in DMEM containing 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. Normal rat kidney interstitial fibroblasts (NRK-49F)  and normal rat kidney tubular epithelial cells (NRK-52E)  were cultured in DMEM containing 5% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. Each cell type was incubated in serum-free medium for 24 h prior to each stimulation. After pretreatment with MA-35 at a dose of 10 μM or 30 μM for 60 min, recombinant human TNF-α , LPS  or recombinant human TGF-βLX-2 cells were treated with indicated concentrations of MA-35 in serum-free DMEM for 24 h. Cell viability was measured using Cell Count Reagent SF  (n = 6). L929 cells were treated with TNF-α (0.03 ng/mL) and actinomycin D  for 20 h in the presence or absence of MA-35 at a dose of 10 μM. Cell viability was measured using a CellTiter 96 Aqueous non-radioactive cell proliferation kit (Promega) (n = 8).All animal experiments were approved by the Tohoku University Animal Care Committee. The experimental protocols and animal care were performed according to the guidelines for animal experiments of Tohoku University. C57BL/6 male mice aged 8–10 weeks were purchased from CLEA Japan, Inc. Acute hepatitis was induced by intraperitoneal injection of LPS (5 μg/kg body weight) and D-GaIN  dissolved in phosphate-buffered saline (PBS). Mice were randomly divided into four groups: calboxymethylcellulose (CMC) (n = 8), MA-35 (n = 8), LPS/D-GaIN (n = 10) and LPS/D-GaIN+MA-35 (n = 10). MA-35 or CMC was orally administered 13 h and 1 h prior to LPS/D-GaIN. Mice were sacrificed to assess the liver damage at 6 h after the LPS/D-GaIN injection.UUO was performed by ligating the left ureter with 4–0 silk at two points and cutting between the ligatures. Mice were randomly divided into four groups: Sham operation+DMSO (n = 4), Sham operation+MA-35 (n = 4), UUO+DMSO (n = 5) and UUO+MA-35 (n = 6). MA-35 at a dose of 80 mg/kg or DMSO was injected by an intraperitoneally-implanted micro-osmotic pump (Alzet) from 3 days before UUO until they were sacrificed at 5 days after UUO.The livers and kidneys were fixed in 10% buffered formalin and embedded in paraffin. Liver sections were stained with hematoxylin and eosin (HE). Kidney sections were stained with Elastica Masson, Sirius red, collagen I and p-Smad3 antibody. The fibrotic area and p-Smad3-positive area were quantified by measurement at ×200 and ×400 magnification, respectively in 10 randomly non-overlapping fields using the National Institutes of Health Image J software.2) for 48 h. Human erythropoietin protein was measured using a Human Erythropoietin Platinum ELISA kit (eBioscience). Mice were injected intravenously with MA-35 at a dose of 40 mg/kg or DMSO (n = 3). The murine Epo levels in the plasma were measured at 4 h after injection using a Quantikine ELISA kit (R&D Systems).Hep3B cells were incubated under normoxic condition for 24 h. Cells were further treated with recombinant human TNF-α (220 U/mL) and our 41 chemical compounds (MA-1 to MA-41) in the culture medium under hypoxic conditions (1% OThe serum ALT level was measured with Fuji DRI-CHEM SLIDE GPT/ALT-PIII (Fujifilm). Murine TNF-α level was measured using a TNF-α ELISA kit (R&D system). All were performed according to the manufacturer’s instructions.TM Real Time PCR System (Applied Biosystems). The primers used are listed in Supplementary Table Total RNA was extracted using TriPure Isolation Reagent (Roche) according to the manufacturer’s instructions. cDNA was synthesized using a transcriptor first-strand cDNA synthesis kit (Roche). Quantitative real-time PCR was performed using the Taqman Gene Expression Assay (Applied Biosystems), according to the manufacturer’s instructions using a StepOnePlusEqual amounts of protein were separated by SDS-PAGE on 10% gel. Proteins were transferred onto a polyvinylidene difluoride membrane. The membrane was incubated overnight with primary antibodies against p-IKK, IKK, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-Smad3, Smad3  and, fibronectin (Santa Cruz Biotechnology), incubated for 1 h with HRP-conjugated secondary antibodies (PIERCE). Protein bands were detected using the enhanced chemiluminescent plus system. Bands intensities were analyzed by NIH Image J software.Col1a1 and PAI-1 was performed using a ChIP Assay Kit (EMD Millipore) according to the manufacturer’s protocol. DNA was purified using the QIAquick PCR Purification Kit (Qiagen). Antibody used is anti-H3K4me1 . Purified DNA samples were subjected to qRT-PCR. Results were normalized to input DNA (n = 5). The primers used in this study are listed in Supplementary Table ChIP assay of P < 0.05 was considered to be statistically significant.All data were expressed as the mean ± SEM. Statistical analyses were performed with one-way ANOVA, Tukey’s test and Kruskal-Wallis test using JMP version 11 (SAS Institute Inc). Supplementary Information


We evaluated threshold saccadic vector optokinetic perimetry (SVOP) and compared results to standard automated perimetry (SAP).A cross-sectional study was done including 162 subjects  recruited at a university hospital. All subjects underwent SAP and threshold SVOP. SVOP uses an eye tracker to monitor eye movement responses to stimuli and determines if stimuli have been perceived based on the vector of the gaze response. The test pattern used was equivalent to SAP 24-2 and stimuli were presented at Goldmann III. Average and pointwise sensitivity values obtained from both tests were compared using Pearson's correlation coefficient. Two versions of SVOP were evaluated.r = 0.95, P < 0.001). Excluding the blind spot, correlation between SVOP and SAP individual test point sensitivity ranged from 0.61 to 0.90, with 48 of 54 (89%) test points > 0.70. Overall SVOP showed good repeatability with a Pearson correlation of 0.88. The repeatability on a point-by-point basis ranged from 0.66 to 0.98, with 45 of 54 points (83%) > 0.80. Repeatability of SAP was 0.87, ranging from 0.69 to 0.96, with 47 of 54 (87%) points > 0.80.A total of 124 tests were performed with SAP and SVOP version 2. There was excellent agreement between mean threshold values obtained using SVOP and SAP (Eye-tracking perimetry is repeatable and compares well with the current gold standard of SAP. The technique has advantages over conventional perimetry and could be useful for evaluating glaucomatous visual field loss, particularly in patients who may struggle with conventional perimetry.Suprathreshold SVOP already is in the field. To our knowledge, this is the first report of threshold SVOP and provides a benchmark for future iterations. Although newer testing strategies, such as the Swedish Interactive Threshold Algorithm (SITA),4 have improved efficiency, many patients still find SAP difficult.6 The requirement to maintain fixation on a central target, and then signal perception of a peripheral stimulus, without altering fixation, can be challenging. In addition, patients are required to maintain a fixed head position, which may be problematic. All of these factors can lead to longer test times and contribute to test–retest variability and low test reliability.8The use of a static white stimulus on a white background (white-on-white) has become the accepted standard for investigation of visual function in patients with glaucoma. White-on-white automated suprathreshold tests often are used for screening purposes,9 SVOP uses eye tracking to assess the visual field by automated real-time assessment of natural eye movement responses to stimuli, and has no postural constraints.10 Different iterations of the technique have now been evaluated in children and adults.12Saccadic vector optokinetic perimetry (SVOP) is a technique originally developed to enable suprathreshold visual field assessment in children unable to perform conventional forms of perimetry.We have developed a version of SVOP for threshold testing. The purpose of this study was to introduce threshold SVOP and compare SVOP threshold values to threshold values obtained from SAP in patients with glaucoma and normal subjects. A second part to this study is continued in a companion paper, which compares visual field patterns obtained by threshold SVOP and SAP, and evaluates patients' perceptions of threshold SVOP.This cross-sectional study included 103 patients with glaucoma  and 59 healthy subjects . Patients with glaucoma were recruited from a nonconsecutive series of patients attending the glaucoma clinic at the Princess Alexandra Eye Pavilion, Edinburgh. Healthy subjects were required to have no previous history of glaucoma or visual field defect and no neurological conditions that might affect the visual field. All subjects provided written informed consent. The study adhered to the tenets of the Declaration of Helsinki and was approved by the South-East Scotland Research Ethics Committee, NHS Lothian.All patients attending the glaucoma clinic had undergone best-corrected visual acuity assessment, slit-lamp biomicroscopy, intraocular pressure measurement, pachymetry, gonioscopy, and dilated funduscopy. Glaucoma was diagnosed by a glaucoma specialist, based on the presence of typical glaucomatous changes in optic disc morphology and the presence of a glaucomatous visual field defect on SAP, using the Humphrey visual field analyzer  SITA Fast 24-2 test. Patients with strabismus or a history of eye movement disorders were excluded. All patients had a best corrected visual acuity of better than 0.3 logMAR. In the healthy group, eyes with a visual acuity worse than 0.15 logMAR were excluded.Each subject underwent SAP (24-2 SITA fast) and threshold SVOP on both eyes, with the order of testing randomized. A smaller cohort of subjects underwent repeat testing in one eye to evaluate repeatability. For glaucoma patients, repeat testing was performed on the eye with the more advanced SAP visual field loss. For healthy subjects, one eye was selected randomly for repeat testing. Testing was performed during a single session on the same day and breaks offered between tests should they be required. The SVOP and SAP tests used are described in more detail in the following sections.10 Briefly, the SVOP device consists of a personal computer, two display screens (one for the patient and another for the examiner), and an eye tracker. The eye tracker assesses patient eye gaze responses to visual field stimuli presented on the display screen and a software algorithm determines if the stimuli have been perceived based on the direction and amplitude of a subject's eye gaze responses rather than a patient response button. The only task required of the patient is to refixate on a peripheral stimulus, if they have seen it, which then becomes the fixation target for the next peripheral test point. A peripheral stimulus will be presented only if the system determines that the patient is fixating correctly on the fixation target. The eye tracker also provides “real time” data on eye location allowing screen coordinates of visual field stimuli to be calculated based on the position of the patient, meaning that there is no requirement for a chin rest. In other words, the size and position of the stimuli can be adjusted automatically and continually to compensate for changes in the patient's position during testing. The suprathreshold version of SVOP has been previously described.Threshold SVOP uses the same underlying principles as suprathreshold SVOP, with the exception that the fixation target remains in place until a test stimulus is seen and an appropriate eye gaze is made. In the suprathreshold test, the fixation target disappears when a peripheral stimulus is shown so as to incite an eye movement reaction. In a threshold test where many stimuli will not be seen, this approach would lead to lengthy test durations because refixation time would be required for every presented stimulus.13 This method of calibration was performed once at the beginning of the study. A second, simpler screen calibration using an x-rite i1 Display Pro Colourimeter  was used twice during the study to maintain the display color consistency.The eye tracker used for threshold SVOP was an IS-1 model . The patient display screen was a 24″ ColorEdge CG243W LCD display  controlled by a FirePro 2270 graphics card  providing native 10-bit color depth support. The display was calibrated to generate accurate representation of the varying stimulus luminance levels required for threshold testing. Briefly, the screen calibration technique uses an accurate Look-Up Table (LUT) pairing the grey-levels of each pixel to the corresponding required luminance levels. This enabled the accurate reproduction of the background and stimulus luminance levels required for threshold testing.2. Stimuli luminance levels replicated the luminance values corresponding to 14-40 dB on SAP. The SVOP threshold sensitivity values were matched in luminance to those of the SAP to allow direct comparison. Thresholds were obtained using a 4-2 bracketing strategy and began by testing four “seed” locations (one in each quadrant), which then were used to set the starting stimulus luminance levels for neighboring locations, which in turn were used to calculate the remaining starting luminance levels.The test pattern used was equivalent to the SAP 24-2 test pattern. Peripheral stimuli were presented at size Goldmann III for 200 ms each on a background of 10 cd/mThe room lights were off for SAP and SVOP testing. Participants were seated in front of the patient screen with their eyes aligned with the center of the screen at a testing distance of approximately 55 cm, measured using an eye tracking–based on-screen tool. Monocular testing was made possible using custom made test spectacles allowing transferable, full aperture prescription lenses (55 mm diameter) if required, while also occluding the nontest eye with an infrared bandpass filter which enabled the eye tracker to detect the position of the occluded eye. A calibration sequence, in which the subject was required to follow a stimulus to nine different screen locations, was performed before each test to produce accurate eye gaze data during testing. Before testing commenced, a 20-second demonstration of the test was provided for all subjects. During testing the patient was instructed to follow their natural reaction to fixate towards any peripheral stimulus perceived.Throughout the study, the testing clinicians (AMcT and LC) liaised with the research engineers  to suggest improvements. When sufficient improvements were developed, version 1 (v1) of the SVOP software was replaced with version 2 (v2). Key changes between software v1 and v2 were: (1) an indicator for correct patient height and position (before and throughout the test); (2) an improved interactive demonstration test; (3) an increased time period for determining fixation and new fixation target with central pulsating crosshairs to improve fixation; (4) ensuring that starting luminance levels  and subsequent neighboring threshold levels were never below 18 dB; this ensured that threshold point results would never be determined from a single stimulus decision; and (5) two bug fixes .SAP was performed using a HFA 750i (Carl Zeiss Meditec). The SITA FAST algorithm was used with the 24-2 test pattern. The eye being tested was aligned to the central fixation point with the aid of the HFA on-screen camera. Monocular testing was performed using an eye patch and, if required, a near prescription was provided using full aperture trial lenses placed in the HFA lens holder. During testing, participants were instructed to fixate on the light in the center of the perimetry bowl throughout the test and to respond to peripheral stimuli by pressing a patient response button. Individuals who were unfamiliar with visual field testing were shown a short demonstration of the test.14), and those identified as having eyelid or lens rim artefact. The number of excluded tests is reported.SVOP tests excluded from analysis were those that were incomplete. SAP tests excluded from analysis were those with a false-positive response rate exceeding 15%  was used. All tests were 2-sided and a P value less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS version 21 .For group comparison tests, normality assumption was assessed by inspection of histograms and using Shapiro-Wilk tests. For comparison of two groups, Student's r) for each SVOP software version (v1 and v2). This analysis was repeated for each of the 54 visual field test locations. Due to the limitations of the current SVOP LCD screen, it was not possible to generate stimuli with brightness greater than 136.69 cd/m2 (corresponding to 14 dB on SAP). Therefore, visual field sensitivity on SAP tests lower than 14 dB were truncated to 14 dB for comparison. To assess the repeatability of SVOP (v1 and v2) and SAP tests, Pearson correlations were calculated for each pair of repeat test point measurements on the same eye (54 points per test).SVOP and SAP tests (excluding repeat tests) were compared by correlating the average sensitivity for each test to produce a Pearson's correlation coefficient (P < 0.001).A total of 162 subjects took part in the study, including 103 patients with glaucoma  and 59 healthy subjects . Of those with glaucoma, 90 had open angle and 13 primary angle closure glaucoma. Healthy subjects were younger than those with glaucoma .Overall there was good agreement between threshold sensitivity values obtained with SVOP and SAP. For example, Analysis also was performed on individual visual field locations. This is of interest as the expected thresholds across the visual field are expected to differ dependent on the position tested. For example, r) between tests at each location. There was closer agreement between SAP and SVOP v2 than between SAP and SVOP v1.Similar comparisons at each of the 54 visual field point locations were performed. To assess the repeatability of the numerical scores, Pearson correlations were calculated for each pair of repeat measurements on the same eye. For SVOP v1, the average correlation over the 54 points was 0.66, with a range of 0.37 to 0.85, indicating only moderate repeatability. The lowest correlation of 0.37 was an outlier, corresponding to the blind spot. For SVOP v2 and SAP, the corresponding figures were 0.88 (0.66–0.98) and 0.87 (0.69–0.96). Thus, the repeatability of SVOP v2 was much better than that for v1, and comparable to SAP. The individual test location repeatability Pearson correlations, for SVOP v2 and SAP, are shown in P < 0.001), and also was significantly shorter than SVOP v2 . Additionally, the test duration for patients performing SVOP v2 was significantly longer than SVOP v1 .The mean test duration for patients performing the SITA Fast test  was significantly shorter than SVOP v1  and SAP was significantly shorter than SVOP v2 . Additionally, test duration for healthy subjects performing SVOP v2 was significantly longer than SVOP v1 .The mean test duration for healthy subjects performing SAP  was significantly shorter than SVOP v1 .Finally, the mean SAP test duration for patients  compared to healthy subjects  was significantly longer  values being produced by SVOP than SAP in the central 4 test points, which is a finding reported previously by another group who have developed eye gaze perimetry.15Overall average SVOP v2 and SAP thresholds showed close agreement. A pointwise comparison of SAP and SVOP thresholds revealed agreement between SAP and SVOP varied depending on location, with The lower correlation for test points in the superior visual field was attributed to these points sometimes being tested towards the end of the test, meaning that their presentation became more predictable due to repetitive testing in these locations. The reason for these test locations being left until the end stages of a test primarily is because less screen area is available for fixation that allow these points to be tested and, hence, they are less likely to be selected during earlier stages of the test. With knowledge of these limitations and understanding of the data generated by the eye tracker these issues can be resolved, for example by changing the fixation requirements depending on the test stimulus eccentricity of a point to be tested, and by increasing the priority of testing certain locations before the end of the test.Overall SVOP v2 showed good repeatability with a Pearson correlation of 0.88. The repeatability of SVOP v2 on a point-by-point basis ranged from 0.66 to 0.98 , with 45SVOP v2 showed better agreement with SAP and better repeatability compared to SVOP v1. It was felt that improving the participant starting position provided the greatest benefit, but also the small changes and bug fixes ultimately contributed to the overall improvements.16 has suggested that due to a reduction in the asymptotic maximum response probability, SAP may be unreliable in test locations with sensitivity below 15 to 19 dB. This would make detection of progression in areas of the visual field with sensitivity below this level problematic and illustrates the need for alternative testing strategies to detect progression in areas of the visual field with advanced loss. The display screen also requires calibration to produce and maintain accurate luminance presentation. The display calibration processes  could be automated easily.The SVOP threshold test currently has limitations. First, it does not produce stimuli brighter than equivalent to 14 dB on SAP. With a different display screen, this could be improved to 10 dB, but it would not be possible to achieve the brightness equivalent to 0 dB on SAP. Although the inability to generate stimuli with intensity greater than 14 dB is a limitation of SVOP, a recent study by Gardiner et al.17 To further improve SVOP, collaboration with eye tracking manufacturers would be required to better understand the issues and produce an eye tracker designed with the requirements of SVOP in mind. In addition, there also is useful information on eye tracking quality, which can be measured before the test  and during the test, which could be used to ascertain how likely it is the test will complete.A further limitation of threshold SVOP is that some tests were not completed . Incomplete tests largely were due to poor quality eye tracking data, meaning that the test could not proceed as it was unable to determine accurately whether the subject was fixating correctly. We observed a larger proportion of tests on glaucoma patients being incomplete compared to healthy subjects . This is likely because the eye tracker was developed for a normal population and occurrences of ocular abnormalities (which could interfere with the eye tracking) are more likely to occur in the patient population. The Tobii eye tracker uses video identification of corneal and pupil reflexes. Incomplete tests due to poor eye tracking can occur when the quality of the image of the pupil margin and corneal reflex is impaired . The rate of incomplete tests would need to be reduced to make the system more acceptable for use in clinics. However, newer models of eye trackers have introduced proprietary developments to improve eye tracking and we have begun work on a further iteration of the technique using newer eye tracking hardware with promising results when retesting some patients who produced incomplete tests in this study.It also is important to acknowledge that SVOP test duration was significantly longer than SAP, for both subject groups and for both SVOP software versions. This primarily is due to the way in which the system determines the threshold at each visual field test location. The SITA fast algorithm, which was used for SAP testing, uses a model to estimate threshold values for each point based on responses to stimuli presented at that location, as well as information gathered from nearby locations, and tests considerably fewer points than the SVOP test, which uses the 4-2 bracketing technique. Several techniques can be incorporated into SVOP to improve test time. One simple example would be to reduce the amount of time SVOP waits to make an “unseen” decision. Currently, this is set to 1 second, but a dynamic value based on patient response time for “seen” stimuli could be used. Given that near to 50% of stimuli in a threshold test can be unseen, this would represent considerable improvement in test time. SVOP v2 was on average longer to complete than v1. The reason for this primarily was because the number of stimulus presentations was greater with v2 due to the modification, which ensured that starting luminance levels  were never below 18 dB and because of the longer period used to determine fixation. There was no significant difference in the test times between patients and healthy subjects performing SVOP in contrast to SAP, where on average patients had a longer test duration. This simply may be due to the difference in algorithms used to determine threshold. However, there are aspects of SAP testing that SVOP bypasses , which could contribute to this effect and this warrants further investigation.Our study design also was limited by the need to compare SVOP to SAP. Although SAP is the gold standard, and the obvious choice for device validation, SAP itself is subject to variability. Difficulties also were encountered with 5% of initial SAP tests, where the data were deemed unreliable due to false-positive responses exceeding 15% and subsequently excluded from analysis. In all 30 of the “unreliable” SAP tests the corresponding SVOP test was completed successfully. Given the overall results demonstrating the accuracy of threshold SVOP, this indicates that in these subjects SVOP would have provided a useful result where SAP did not, indicating that threshold SVOP, even in its current form, may prove useful in a group of patients who struggle to perform SAP reliably. False-positive responses on SAP occur if the subject responds by pressing the button when no stimulus is present. It is an indicator that the subject may be “trigger happy” and affects the validity of the visual field results. SVOP requires an eye gaze response in the direction of the test stimulus and, therefore, it is very difficult to make a conscious false-positive response.11 which itself was an extension of work performed by Bertil Damato in 1985.18 As would be expected the testing times for threshold SVOP are longer compared to suprathreshold SVOP . Both forms of SVOP testing perform well in healthy subjects and glaucoma patients .Threshold SVOP is an extension of our previously described suprathreshold SVOP,17 Additional future work will be necessary to assess other outputs available from threshold SVOP tests, such as saccadic reaction times, which may provide additional diagnostic information,19 and correlation of threshold SVOP with retinal nerve fiber layer density.In this study, we demonstrated an alternative method of measuring threshold visual fields that is repeatable and compares well with the current gold standard. Although further work is required to achieve a faster test and improve test accuracy in the central locations, we demonstrated how iterative improvements can be made through software. More data are required to create a larger database of normative SVOP thresholds and assess more subtle differences between SVOP and SAP . Further developments and improvements to the SVOP system are possible through advances in eye tracking technology and newer models of eye tracker currently are available that may improve eye tracking in patients who have proved difficult to test thus far. The second part to this study demonstrates how a further iteration of the threshold SVOP system can improve on the rates of incomplete testing, which, coupled with positive patient feedback, make the test a useful and acceptable form of perimetry.


Methylmercury selectively damages the central nervous system (CNS). The tumor necrosis factor (TNF) superfamily includes representative cytokines that participate in the inflammatory response as well as cell survival, and apoptosis. In this study, we found that administration of methylmercury selectively induced TNF-α expression in the brain of mice. Although the accumulated mercury concentration in the liver and kidneys was greater than in the brain, TNF-α expression was induced to a greater extent in brain. Thus, it is possible that there may exist a selective mechanism by which methylmercury induces TNF-α expression in the brain. We also found that TNF-α expression was induced by methylmercury in C17.2 cells  and NF-κB may participate as a transcription factor in that induction. Further, we showed that the addition of TNF-α antagonist (WP9QY) reduced the toxicity of methylmercury to C17.2 cells. In contrast, the addition of recombinant TNF-α to the culture medium decreased the cell viability. We suggest that TNF-α may play a part in the selective damage of the CNS by methylmercury. Furthermore, our results indicate that the higher TNF-α expression induced by methylmercury maybe the cause of cell death, as TNF-α binds to its receptor after being released extracellularly. Methylmercury is an environmental pollutant known as the cause of Minamata disease, and severe central nervous system damage12456Microglia and astrocytes, types of brain cells, regulate the neuroprotective immune response that participates in maintaining brain homeostasis. However, there are cases in which they have instead been shown to be the cause of damage to neurons. In recent years, glial contributions have been noted in the progression and exacerbation of neurodegenerative disorders including Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease8910et al.et al. investigated the effect of adding recombinant CCL2 to culture media on the methylmercury sensitivity of primary rat neurons. Instead of CCL2 reducing methylmercury toxicity, they found it amplified toxicity at high concentrationset al. reported that methylmercury induces IL-6 expression in astrocytes, and that IL-6 exhibits a defensive effect against the neuronal damage caused by methylmercuryActivated microglia and astrocytes produce inflammation mediators , inflammatory cytokines and other products. The production of inflammatory cytokines is known to damage neurons through the invasion of inflammatory cells and cytotoxic T cells1214The TNF superfamily (TNFSF) are representative inflammatory cytokines that participate in processes such as cell survival, induction of apoptosis, the inflammatory response, and cell differentiation. Approximately 20 molecular species have been identified, including cytokines that participate in the induction of apoptosis, such as: TNF-α, lymphotoxin α, the Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL), as well as the RANK ligand (RANKL) and the CD30 ligand (CD30L)1920Male C57BL/6 mice were injected subcutaneously with methylmercuric chloride . Seven days after methylmercury administration, the cerebrum and cerebellum were dissected and any changes in the expression of 18 TNFSF genes were analyzed using quantitative PCR. The expression of TNF-α (TNFSF2) and CD30L (TNFSF8) were found to have increased markedly in both the cerebrum and cerebellum after methylmercury administration . AdditioNext, methylmercury was administered to mice under the same conditions as the experiment depicted in CD30L expression in the cerebrum and cerebellum was significantly elevated on the seventh day; however, by contrast to TNF-α, CD30L expression also rose on the fifth day in the kidneys at approximately the same rate as in the cerebrum and cerebellum . The expWe found a concentration- and time-dependent induction of TNF-α expression resulting from the treatment of C17.2 cells, which are mouse neural stem cells, with methylmercury . The TNF222425As discussed above, TNF-α levels were found to increase significantly in culture medium as a result of methylmercury treatment . TNF-α t27+-dependent transporters39In this study, methylmercury was shown to specifically induce TNF-α expression in brain tissue, and significantly induce TNF-α expression in cultured neural stem cells. TNF-α is an inflammatory cytokine produced by monocytes, macrophages, and fibroblasts. In the central nervous system, astrocytes and microglia are involved in TNF-α production, and in particular, activated microglia produce TNF-α continuously293031333536In addition, although the methylmercury administered in our study to the mice accumulated at a higher concentration in the liver and kidneys than in the brain, the induction of TNF-α expression was found to be most pronounced in brain tissue . This su414322Cytokines are known to regulate the expression of other cytokines, which results in a chain reaction among the cytokines46C57BL/6 male mice (8 weeks old) were purchased from Japan SLC Inc. . Mice were kept in plastic cages  under pathogen-free conditions in a room at 22 ± 2 °C and 55 ± 20% relative humidity, with a light/dark cycle of 12 hr. Mice had free access to commercial food that had been sterilized by radiation , and filtered water ad libitum. The Tohoku University Committee for Animal Experiments approved all animal experiments, and the experiments were performed in accordance with the Regulations for Animal Experiments and Related Activities at Tohoku University and Guidelines for Proper Conduct of Animal Experiments by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. After an adaptation period, mice were randomly divided into control (n = 5) and methylmercury-treated groups (n = 5). Methylmercuric chloride, dissolved in physiological saline, was administered via a single subcutaneous injection at a dose of 25 mg/kg. Control mice received injections of saline only. The cerebrums, cerebellums, kidneys, and livers of the mice were dissected under isoflurane anesthesia. Tissue samples were immediately preserved in Isogen II . Aliquots of the samples were taken for analysis of their total mercury content.® RT reagent kit  according to the manufacturer’s instructions. qPCR was undertaken using SYBR Premix EX Taq (Takara) and a Thermal Cycler Dice® (Takara) with the oligonucleotides shown in Total RNA was isolated from mouse tissue and C17.2 cells using an Isogen II kit (Nippon Gene) according to the manufacturer’s protocol. Reverse transcription was performed using 1 μg of total RNA as the template and a PrimeScript+, Lumex Ltd., Russia) based on the atomic absorption spectrometry. This instrument can real-time detect extremely low mercury concentration in biological samples (detection limit was 1 ug/kg). The accuracy of mercury measurement was ensured by using reference NIES CRM No. 13 .The total mercury concentrations in the various tissues of the mice (approximately 10 mg for each tissue) were measured by Zeeman Mercury Spectrometer (RA-9152 atmosphere at 37 °C. C17.2 cells (4 × 105 cells) were plated in 6-well plates and cultured for 24 hr, followed by treatment with methylmercuric chloride.The C17.2 cell line, which is composed of neural stem cells that were derived from the external germinal layer of mouse cerebellum, were cultured in Dulbecco’s modified eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL of penicillin, and 100 mg/mL of streptomycin in a humidified 5% COTNF-α secreted into culture medium was quantified using a Quantikine ELISA kit  according to the manufacturer’s protocol.2, 1 mM dithiothreitol [DTT] and 10% Nonidet P-40) containing protease inhibitor  for 10 min on ice, and centrifuged at 15,000 × g at 4 °C. The supernatant (post-nuclear fraction) was removed, and the pellet was resuspended in nuclear lysis buffer  containing protease inhibitor (Roche). The resulting suspension was sonicated and centrifuged at 15,000 × g at 4 °C to obtain the nuclear fraction. The protein concentration of each fraction was determined using a DC protein assay kit . Proteins in nuclear and post-nuclear fractions were separated by SDS-polyacrylamide gel electrophoresis, transferred to an Immobilon-P membrane , and visualized using primary antibodies against p65 , lamin A/C , GAPDH (Santa Cruz Biotechnology), and horseradish peroxidase-coupled secondary antibodies .C17.2 cells were washed twice with cold PBS and lysed with a hypotonic buffer .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Norovirus Chiba strain were crystallized and data were collected to 3.2 Å resolution. X-ray analysis revealed that although 38 nm VLPs with T = 3 symmetry had been prepared for crystallization, the crystal contained 23 nm VLPs with T = 1 icosahedral symmetry.Virus-like particles (VLPs) from  Norovirus is a major causative pathogen of nonbacterial acute gastroenteritis. Despite the sequence similarity among various strains, noroviruses of different genotypes show different antigenicities and different binding profiles to histo-blood group antigens (HBGAs). To reveal the relationships between the structure of the capsid and the diversity in antigenicity and the HBGA-binding profile, virus-like particles (VLPs) of the Chiba strain that belongs to genogroup I, genotype 4 were crystallized for X-ray structural analysis. Diffraction data were collected and processed at 3.2 Å resolution. The crystal belonged to space group I222, with unit-cell parameters a = 290.0, b = 310.4 c = 350.4 Å. The possible packing model indicated that the diameter of the particle was 280 Å, which was much smaller than the 38 nm VLPs of Norovirus Norwalk strain (NV) with T = 3 icosahedral symmetry and composed of 180 VP1 proteins. The structure was solved by molecular replacement using the structure of the VP1 pentamer of NV 38 nm VLPs as a search model, revealing that the VLPs were smaller particles: 23 nm VLPs with T = 1 icosahedral symmetry, the structure of which has not yet been analyzed at high resolution. The structure of 23 nm VLPs will enable the two different VLPs of Norovirus to be compared, which will provide important information for understanding the structural basis of capsid formation. It is well known as a causative pathogen of nonbacterial acute gastroenteritis in humans. Based on sequence diversity, human noroviruses are divided into two major genogroups, GI and GII, in which at least nine and 22 genotypes have been identified, respectively  of 38 nm in diameter. Because these VLPs are structurally and antigenically similar to the intact viruses, they have been used as immunogens in vaccine development. There are also smaller VLPs of 23 nm in diameter that consist of 60 copies of VP1 and have T = 1 icosahedral symmetry  was solved by Prasad et al.  was identified as the cause of an oyster-associated outbreak of gastroenteritis in Chiba prefecture, Japan in 1987 . Macromolecule-production information is shown in Table 1VLPs were prepared as described in Someya  al. 2011. The tra al. 2011a. Macro2.2.M MES pH 6.5 and they were concentrated to 4 mg ml−1. Initial screening was performed using PEG/Ion Screen (Hampton Research), which was diluted fivefold and tenfold because the crystallization-condition information in VIPERdb 2HPO4 and the molecular weight of PEG resulted in the growth of large single crystals 2HPO4 or PEG tended to cause the formation of aggregates. The final crystallization condition is shown in Table 2VLPs were crystallized at 293 K in 96-well plates using the sitting-drop vapour-diffusion method. Prior to crystallization, the buffer solution of purified VLPs was replaced with 20 mls Fig. 2. Higher 2.3.v/v) glycerol, 20 mM (NH4)2HPO4, 2%(w/v) PEG 10 000 and the crystals were cooled in liquid nitrogen. All data were collected on BL41XU at SPring-8 2HPO4, 1–3% PEG 10 000. Data processing showed that the crystals belonged to space group I222 or I212121, with unit-cell parameters a = 290.0, b = 310.4, c = 350.4 Å. The self-rotation function in Fig. 4I222 because to the best of our knowledge there are no virus structures in space group I212121. In the plausible VLP packing in this space group, there were eight particles at the corners of an orthorhombic cell and one particle at the centre. However, the largest allowable particle diameter in this packing model was 280 Å, which was much smaller than the size of 38 nm VLPs. To solve the structure, molecular replacement (MR) was performed with Phaser .To obtain the capsid structure of ChV, we crystallized ChV VLPs. Diffraction data were collected and processed at 3.2 Å resolution using multiple crystals grown in 10–30 mon Fig. 1b.et al.  to analyse the crystals after they had been dissolved in the buffer. The results Fig. 5 showed tA/B dimers and ‘flat’ C/C dimers (Prasad et al., 1999A/B or C/C dimers in 38 nm VLPs. The formation of T = 1 VLPs by the digestion of N-terminal residues has also been reported for Hepatitis E virus (HEV; Yamashita et al., 2009et al., 2009et al., 2010Grouper nervous necrosis virus (GNNV; Chen et al., 2015C/C dimer adopt a flat conformation (Xing et al., 2010T = 3 VLPs (Chen et al., 2015et al., 1999T = 1 VLPs.The crystal structure of 38 nm NV VLPs showed that the N-terminal residues tended to form two distinct dimer conformations in VLPs: ‘bent’ et al., 1981et al., 2016It should be noted that particles of 23 nm in diameter have been isolated in stool samples from norovirus-infected patients (Taniguchi 10.1107/S2053230X17013759/nw5058sup1.pdfData-collection condition of 26 data sets.. DOI:


Specifically, PGE2 accumulation in astrocyte activated the p38 and JNK/c-Jun signaling pathways via phosphorylation, resulting in TNF-α expression. In contrast, the administration of PGI2 attenuated the effects of PGE2 in stimulating the production of TNF-α by inhibiting the activity of TNF-α promoter and the binding activity of AP1 on the promoter of TNF-α. Moreover, our data also showed that not only Aβ1-42 oligomers but also Aβ1-42 fibrils have the ability to involve in mediating the antagonistic effects of PGE2 and PGI2 on regulating the expression of TNF-α via a p38- and JNK/c-Jun-dependent, AP1-transactivating mechanism. Reciprocally, the production of TNF-α finally accelerated the deposition of β-amyloid protein (Aβ)1-42 in β-amyloid plaques (APs), which contribute to the cognitive decline of AD.Cyclooxygenase-2 (COX-2) has been found to be induced during the early stage of Alzheimer’s disease (AD). Using mouse-derived astrocyte and APP/PS1 transgenic (Tg) mice as model systems, we firstly elucidated the mechanisms underlying COX-2 metabolic production including prostaglandin (PG)E Genotyping was performed at 3-4 weeks after birth. The mice were housed in a controlled environment under a standard room temperature, relative humidity and 12-h light/dark cycle with free access to food and water. Mice were randomly separated into several groups and each group contains 10 mice for treatment. The general health and body weights of animals were monitored every day. The brains of animals from the different groups were collected under anesthesia and perfusion as previously described , 61.2, PGI2, Aβ, or Aβ antibody or vehicle (PBS) solutions were injected (i.c.v) into WT or APP/PS1 transgenic mice as previously described [NS398, PGEescribed , 61. In escribed , 61.Brain tissues were freshly collected from WT C57BL/6 mice at 6 months of age. Serial sections (400-µm thick) were cut using a chopper without fixation. The tissue sections were immediately cultured in DMEM/high glucose medium with 10% fetal bovine serum (FBS). In a separate set of experiments, the tissues were grown in serum-free medium for an additional 24 h before incubation with Aβ oligomers or fibrils, as previously described , 61. The2, PGE2 or vehicle (PBS) solutions were then injected (i.c.v) into the other side of ventricle. At different time intervals, mice were anesthetized and injected (i.c.v) with luciferin into the cerebral ventricle, which was preseeded with D1A cells. The scan was performed exactly after 5 min of luciferin introduction as previously described [in vivo imaging systems .The D1A cells that were transfected with an TNF-α promoter were pre-seeded in one side of a ventricle. PGIescribed , 61. AllSac 1 and Xho1 restriction sites (underlined) at the 5’ ends and 3’ends, respectively. The amplicon was then inserted upstream of the luciferase reporter gene in the pGL3-basic vector (Promega). All construct sequences were confirmed using DNA sequencing.A 2, 000-base pair (bp) TNF-α promoter construct, corresponding to the sequence from -1774 to +226 relative to the transcription start site of the 5’-flanking region of human TNF-α gene, was generated from human genomic DNA, using specifically designed forward 5’-TATCGAT AGGTACCGAGCTCCACTTCAGCCCCAGCAGTGT-3’ and reverse 5’ GAT CGCAGATCTCGAGTTTTGGGGGAGTGCCTCTTC-3’ primers incorporating Renilla luciferase activities were measured by use of the Dual-Luciferase Report Assay kit (Promega). Firefly luciferase activities were normalized to the Renilla luciferase controls. Data are expressed as ratios of shear to static normalized firefly luciferase activity unless otherwise stated.Firefly and This assay was performed using the EZ ChIP kit following the manufacturer’s instructions (Upstate Biotechnology) as described previously -65. ForwqRT-PCR assays were performed with the MiniOpticon Real-Time PCR detection system (Bio-Rad) using total RNA and the GoTaq one-step Real-Time PCR kit with SYBR green (Promega) and the appropriate primers as previously described . The GenTissues or cells were lysed in radio-immune precipitation assay buffer  that contained a protease inhibitor cocktail . The protein content of the cell lysates was determined using the bicinchoninic acid (BCA) protein assay reagent . The total protein lysates (4 μg) were separated using SDS-PAGE, transferred to a membrane, and probed with a panel of specific antibodies. Each membrane was only probed with one antibody. β-actin was used as a loading control. All western hybridizations were performed at least in triplicate using a different cell preparation each time.The coding sequence of p38 was generated from mouse total mRNA, using specifically designed forward 5’- TTAAACT TAAGCTTGGTACCATGTCGCAGGAGAGGCCCAC-3’ and reverse 5’-GATATCTGCA GAATTCTCAGGACTCCATTTCTTCTTGGTCAAG-3’ primers, which was incorporated to pCDNA 3.1 (+) vectors by Kpn 1 and EcoR 1 restriction sites (underlined) at the 5’ ends and 3’ ends, respectively. Similarly, the coding sequence of c-Jun was inserted to pCDNA 3.1 (+) vectors by Kpn 1 and EcoR 1 restriction sites using specifically designed forward 5’-TTAAACTTAAGC TTGGTACCATGACTGCAAAGATGGAAACGACC-3’ and reverse 5’-GATATC TGCAGAATTCTCAAAACGTTTGCAACTGCTGC-3’ primers. Cells were transfected with 1.6 µg/6 cm dish of p38 or c-Jun cDNA plasmids. In control experiments, the cells were transfected with 100 nM scrambled siRNA. The transfected cells were allowed to recover for at least 12 h in growth medium and then incubated overnight in serum-free medium before extraction. In live animal scanning experiments, the D1A cells were transfected with 1.6 µg/6 cm dish TNF-α promoter plasmid for 48 h before screening the positive clones by puromycin. The positive clones were then injected or preseeded in the ventricles of mice.1-42 protein  was initially monomerized by dissolving it to a final concentration of 1 µg/µl in 100% hexafluoroisopropanal (HFIP) and the solution was aliquoted in sterile eppendorf tubes. HFIP was then evaporated under vacuum and the peptide was stored at -20 °C before reconstituent. For preparing Aβ1-42 oligomers, the peptide was initially resuspended in dimethylsulfoxide (DMSO) to 20 µg/µl with water bath ultrasonication for 10 min and the solution was then diluted to a final concentration of 0.2 mg/ml in phenol red-free F-12 media, and incubated at 4°C for 24 h. To prepare Aβ1-42 fibrils, Aβ1-42 was resuspended in sterile Milli Q water and incubated at 37°C for 1 week before use.The methods for preparing Aβ oligomers or fibrils had been described previously . In brie2 (2 µg/5 µl) or PGE2 (2 µg/5 µl). Serial sections (5-µm thick) were cut using a paraffin microtome . Sections were first rehydrated in a graded series of ethanol and submerged in 3% hydrogen peroxide to eliminate endogenous peroxidase activity. The activity of TNF-α or Aβ was determined by immunostaining TNF-α or Aβ antibody using an immunohistochemical staining kit, following the manufacturer’s instructions .Brain tissues were collected from WT or APP/PS1 transgenic mice. In selected experiments, brain tissues were collected after injection (i.c.v) of PGI2 (2 µg/5 µl) or PGE2 (2 µg/5 µl). Serial sections (10-µm thick) were cut using a cryostat . Slides were stained with GFAPor Iba1 antibody with Alexa Fluor 555 secondary antibodies  before observing under confocal microscopy .Brain tissues were collected from WT or APP/PS1 transgenic mice. In selected experiments, brain tissues were collected after injection (i.c.v) of PGI1-42 levels in the media of both control and pharmacologically treated cells or the brain of mice were determined using TNF-α or Aβ1-42 enzyme immunoassay kits following the manufacturer’s instructions. The total protein used for ELISA was used as a loading control, and the results are expressed as pg of TNF-α or pmol Aβ1-42 per mg of total protein.The TNF-α or AβHuman brain samples were obtained from New York Brain Bank, serial numbers TT4263 , T4308 , T4339 and T4304 .All animals were handled according to the care and use of medical laboratory animals  and all experimental protocols were approved by the Laboratory Ethics Committees of College of Life and Health Sciences of Northeastern University.t-test [All data are represented as the mean ± S.E. of at least three independent experiments. The statistical significance of the differences between the means was determined either using Student’s t-test .


AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live‐attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost‐effective recombinant vaccine. Here, we report the plant‐based production of a virus‐like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens‐mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus‐based HyperTrans (CPMV‐HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell‐based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease.African horse sickness ( AHS is infectious but noncontagious, and is endemic to sub‐Saharan Africa  is a devastating illness of horses, which is frequently fatal in susceptible hosts, and has been part of South Africa's veterinary disease landscape for several centuries. It is widely recognized as one of the most lethal viral diseases of horses worldwide  produced by Onderstepoort Biological Products, based on products developed in the 1930s  and VP7 (37 kD) as well as the VP7 trimer (135 kD) was demonstrated, the proteins being visualized as distinct bands of the correct expected molecular weight in SDS‐PAGE analyses of crude extracts. Apparent differences in gel loading are sometimes observed as a result of natural leaf‐to‐leaf and plant‐to‐plant total soluble protein (TSP) variation. Bands corresponding to VP3 (103 kD) and VP5 (57 kD) were not detected due to a peculiarity of the available antiserum, which has been shown to detect only VP2 and VP7. However, fully formed AHSV‐5 VLPs were imaged by TEM analysis of these crude extracts, indicating that all four capsid proteins were expressed and indeed had self‐assembled into complete particles  were each vaccinated with AHSV VLPs mixed with 5% Pet Gel A adjuvant , while five control animals (C1‐C5) were immunized with PBS plus adjuvant. Prior to the boost inoculation, a further purification was carried out which yielded sufficient AHS VLPs to increase the amount of the next inoculum. As this was a first vaccination study, the optimal dose required was unknown and animals were therefore boosted on day 13 with a greater amount of VLPs than used for the prime inoculation, or PBS. Due to accidental traumatic injury, guinea pig V1 was euthanized on day 15 and, together with control guinea pig C1, was excluded from the main study results. Sera from all other animals were collected on day 41 and final and prebleed sera (1 : 10 000) were used to probe a Western blot of VLPs used in the initial inoculations. Strong signals for VP2, VP5 and VP7 (both monomers and trimers) were detected by final bleed sera but not by the control guinea pig sera (C2–C5) nor the prebleed sera from all VLP‐vaccinated guinea pigs , following recombinant DNA expression of these proteins within transiently transfected plant cells. The VLPs are noninfectious and inherently safe, as they assemble in the absence of any viral genetic material or other viral proteins. The proteins were each expressed from different recombinant‐carrying Agrobacterium strains co‐infiltrated in equal concentrations, and particles formed within 7 days. VLPs seen in crude extracts resulting from small‐scale expression in a single plant were almost all fully formed, whereas larger‐scale expression and subsequent iodixanol gradient purification produced a more heterogeneous mix of particles including assembly intermediates as well as some partially damaged particles. This phenomenon was observed regardless of whether particles contained the wild‐type or mutated VP7 protein, indicating that minor modification of VP7 by seven amino acid substitutions did not impact on the nature of viral particle formation. However, modifying VP7 in this way did appear to increase the number of particles produced relative to use of the wild‐type VP7. The average number of particles counted over 10 fields of view at a magnification of x54 000 was 9.2 for particles containing the wild‐type VP7 protein compared to 30.7 for particles incorporating the mutated VP7. Furthermore, a comparison of Western blot analyses of gradient fractions containing the two VLP species also seemed to indicate greater VLP formation with VP7mu expression  expressing VP2 and VP5 of AHSV‐4 developed serum VNAb titres of 20–40 (1.3–1.6 log10). Alberca et al. (8 pfu/mL of a modified vaccinia Ankara (MVA) virus expressing VP2 of serotype 9 and reported VNAb titres of 1.6–2.4 log10. In this study, guinea pigs were first each vaccinated with 16.5 μg plant‐produced VLPs as this was the amount of highly purified VLPs available at the start of the study. Subsequent to the initial vaccination, further purifications yielded sufficient VLPs to increase this dosage. Because no previous studies using plant‐produced AHSV VLPs have been described and as Theunemann et al. showed that 50 μg plant‐produced BTV VLPs gave good protection against BTV in sheep, we decided to boost inoculate the guinea pigs with 50 μg VLPs instead of a second dose of 16.5 μg. The unfortunate accidental injury and subsequent euthanization of one of the guinea pigs (V1) 2 days after the boost vaccination (day 15) gave us the opportunity to test the immune response prior to the end of the study. Even at this early stage, sera from this guinea pig developed a VNAb titre of 224, twice that of the AHS‐positive control (112)  titres ranging between 640 and 5120 (2.8 and 3.7 log10) against homologous AHSV‐5 VLPs. These results exceed VNAb titres obtained in studies by others using poxvirus vectors expressing the outer capsid proteins VP2 and VP5  Table . It is tOur results thus demonstrate that fully assembled AHSV‐5 VLPs could be produced in plants via a transient expression strategy and that plant‐produced AHSV 5 VLPs elicit a strong serotype‐specific neutralizing antibody response in guinea pigs. Our study also confirms the suitability of the guinea pig as a small animal model for preliminary testing of recombinant AHSV vaccines. The study will now be extended to include safety and immunogenicity testing in horses, the main target animals, with a view to producing a supplemental vaccine to AHSV‐5 to complement the standard live vaccine mixture.N. benthamiana and synthesized by GenScript Biotech Corporation  with flanking AgeI and XhoI restriction enzyme sites. The codon‐optimized VP7 consensus sequence, modified as described by S. Bekker , was also synthesized. Restriction enzyme cloning was used to insert the genes into the pEAQ‐HT expression vector obtained from George Lomonossoff, John Innes Centre, UK  for these genes listed in GenBank, using CLC Mainbench bioinformatics software . Consensus sequences were codon‐optimized for expression in . Bekker  to incluN. benthamiana plants. Agrobacterium transformants each carrying one of the AHSV‐5 capsid protein genes were subcultured and grown overnight with agitation at 27 °C in Luria–Bertani broth (LBB) base supplemented with 50 μg/mL kanamycin, 20 μm acetosyringone and 2 mm MgSO4. The cultures were diluted in resuspension solution  to the desired optical density and incubated for 1 h at 22 °C to allow for expression of the vir genes. For single infiltrations, each AHSV‐5 Agrobacterium recombinant suspension was diluted to OD600 = 0.5 or 1.0, while co‐infiltration suspensions contained all four AHSV‐5 recombinants in a ratio VP2 : VP3 : VP5 : VP7 of 1 : 1 : 1 : 1 or 1 : 1 : 2 : 1. Plants were grown at 22–25 °C under 16‐h/8‐h light/dark cycles. Agrobacterium suspensions were infiltrated into the leaf intercellular spaces using either a blunt‐ended syringe or a vacuum infiltrator, applying a vacuum of 100 kPa. For optimization of the expression, three leaf discs were obtained from each plant, clipped with the lid of a microcentrifuge tube on 3, 5 and 7 days postinfiltration (dpi) and homogenized in three volumes of PI buffer  using a micropestle. The homogenate was incubated on ice for 30 min and then clarified by centrifugation at 13 000 rpm for 15 min in a benchtop microfuge. SDS‐PAGE samples were prepared by adding 50 μL 5× sample application buffer to 200 μL clarified crude extract and heat‐denatured at 95 °C for 5 min before loading 40 μL per gel lane. For large‐scale expression, leaf tissue was harvested 7 dpi, as this time span was shown to be optimal for expression of all four capsid proteins. Harvested leaves were immediately homogenized in three volumes PI buffer using a Moulinex™ juice extractor. The homogenized leaves were re‐incubated with the extracted juice and incubated at 4 °C for 1 h with gentle shaking. Crude plant extracts were filtered through four layers of Miracloth™  and the filtrate was clarified by centrifugation at 13 000 rpm for 15 min at 4 °C.Expression of the AHSV‐5 capsid proteins was achieved by agroinfiltration of 5‐ to 6‐week‐old AHSV‐5 VLPs were purified by iodixanol density gradient ultracentrifugation. Iodixanol  solutions (20%–60%), prepared in PBS, were used to create a 12 mL step gradient (2–3 mL of each gradient in 10% incrementing steps) under 27 mL clarified plant extract and centrifuged at 32 000 rpm for 2 h at 4 °C in an SW 32 Ti rotor . Fractions of 1 mL were collected from the bottom of the tube and 30 μL from fractions representing the 30%–40% region of the gradient was electrophoresed on a 10% SDS‐polyacrylamide gel, followed by Coomassie blue staining. Particle quantification was achieved by visual comparison of the four capsid protein bands to known amounts of bovine serum albumin (BSA) run in separate lanes on the same SDS‐PAGE gel. To further purify and concentrate VLP samples for use in animal studies, VLP‐containing fractions were diluted with PBS to 20% iodixanol and subjected to a second round of ultracentrifugation as per the same protocol described above. Both crude plant extracts and gradient‐purified VLPs were analysed by Western blot: heat‐denatured samples were separated on 10% polyacrylamide gels and then transferred onto HyBond™ C Extra nitrocellulose membranes  using a Trans‐blot® SD semi‐dry transfer cell . Membranes were first probed with a 1 : 1000 dilution of AHSV‐5‐specific horse serum , washed four times with PBS containing 0.05% Tween® 20  (PBS‐T) and then probed with 1 : 5000 dilution of anti‐horse alkaline phosphatase‐conjugated secondary antibody . After washing again, proteins were detected with 5‐broo‐4‐chloro‐3‐indoxyl‐phosphate (BCIP) and nitroblue tetrazolium (NBT) phosphatase substrate .et al. . Gel pieces were washed and fragmented by in‐gel trypsin digestion as per the protocol described by Shevchenko et al. . The pepGlow‐discharged copper grids (mesh size 200) were floated on 20 μL crude plant extract or 20 μL density gradient fractions for 3 min and then washed successively by floating on five drops of sterile water. Particles were negatively stained for 30 seconds with 2% uranyl acetate and then imaged using a Technai G2 transmission electron microscope (TEM).n = 5) were injected subcutaneously with 16.5 μg purified AHSV‐5 VLPs or 30% iodixanol in PBS, both formulated in 5% Montanide PET Gel A adjuvant . Animals were boosted once on day 13 with 50 μg VLPs or PBS, and on day 41, they were euthanized by anaesthesia with ketamine/xylazine and exsanguinated. Serum was tested for antibodies by Western blot analysis, where guinea pig antisera were used at a dilution of 1 : 10 000 as per the protocol described above.Approval for the immunization experiments was obtained from the Faculty of Health Sciences Animal Ethics Committee, University of Cape Town (FHS AEC ref No.: 016/019). Prior to the study, 100 μL of blood was drawn from each of 10 female guinea pigs (Hartley strain). Guinea pigs  as previously described (House Figure S1 Optimization of plant‐based expression of recombinant AHSV‐5 structural proteins.Click here for additional data file.Figure S2 Increased formation of AHSV‐5 VLPs incorporating a mutated version of VP7.Click here for additional data file.Figure S3 Purification of AHSV‐5 VLPs.Click here for additional data file.Figure S4 Mass spectrometry analysis of the 4 protein bands recovered from SDS‐PAGE separation of density gradient fractions from leaves co‐infiltrated with Agrobacterium AGL1 pEAQ recombinants for co‐expression of AHSV capsid proteins VP2, VP3, VP5 and VP7mu.Click here for additional data file.Figure S5 Quantification of AHSV‐5 VLPs by gel densitometry.Click here for additional data file.Table S1 Virus neutralizing antibody titres of serum samples from vaccinated and control guinea pigs V1 and C1.Click here for additional data file.


EUS-guided FNA biopsy has been widely performed to aid in the diagnosis of submucosal tumors (SMTs). However, in cases of small tumors, the diagnostic yield of EUS-FNA is poor. Therefore, it is necessary to develop a new needle for the diagnosis. We developed a device with a new mechanism that we refer to as a drill needle aspiration biopsy (DNAB). The aim of this study was to evaluate the use of DNAB in resected gastric SMT specimens.A drill needle with a sharp tip and wide ditch was inserted into a catheter for angiography. Continuous suction is enabled through the catheter at the tip. DNAB was performed with one pass and one stroke in 13 gastric SMTs resected by operation. Similarly, FNA was performed by one pass and ten strokes. These gastric tumors included nine diagnosed gastrointestinal stromal tumors and four undiagnosed SMTs by preoperative examinations. The tissue quantity between DNAB and FNA was macroscopically and microscopically examined.All 13 drill biopsy specimens were obtained. Additionally, all 13 gastric SMTs, including 4 undiagnosed tumors, could be diagnosed by DNAB. The quantity of each specimen obtained by DNAB was macroscopically and microscopically much greater than that by FNA. In particular, for tumors <25 mm in the longer axis, the ratio of microscopic diagnosable cases was 100 % (7/7) for DNAB and 42.9 % (3/7) for FNA.DNAB is a novel method that can obtain more tissue than FNA for small gastric SMT. Gastrointestinal stromal tumor (GIST) was first defined in 1983 as a tumor in the gastrointestinal tract and mesentery, characterized by a specific histological and immunohistochemical pattern . DiffereWe designed a special drill that was manufactured by TOKO Co., Ltd. . The drill has three main characteristics: a sharp tip, front-like cutter and deep helical ditch Fig. a. The drWe examined a total of 13 resected gastric SMT specimens from consecutive patients  with a mean age of 63.0 years (range 33–78 years) who underwent an operation at the Department of Frontier Surgery, Chiba University Hospital, between March 2013 and July 2015. Among 13 gastric SMTs, 9 were diagnosed GISTs, and 4 were undiagnosed SMTs by preoperative examinations. Thirteen gastric SMTs were fixed on the corkboard, and DNAB was performed under continuous suction. Similarly, FNA with a 22-gauge needle  was performed using one pass and ten strokes at a site different from the insertion site of DNAB in SMT. The obtained biopsy specimens were then placed on filter paper, and the tissue quantity was macroscopically evaluated. Finally, all specimens were stained in hematoxylin and eosin and immunostained and evaluated by a pathologist.P values of <0.05 were considered to be statistically significant.Fisher’s exact test was used to evaluate differences in the proportions between the two groups. All statistical analyses were conducted using the SPSS 15.0 software package . P = 0.047). There were significant differences between DNAB and FNA regarding the ratio of microscopic diagnosable tumors <25 mm in the longer axis . Moreover, all specimens could be pathologically diagnosed according to the specimens obtained by DNAB. In particular, two gastric SMTs undiagnosed by preoperative EUS-FNA were diagnosed as aberrant pancreas and schwannoma by DNAB. Additionally, a substantial amount of epithelial tissue was obtained from specimen no. 6 using DNAB.The characteristics of the specimens are summarized in Table DNAB is a novel method that can obtain more tissue than FNA in small gastric SMTs. The main direction of the movement in DNAB is a turn, whereas that in FNA is a back-and-forth movement . Therefore, DNAB will enable tissue sampling in small tumors having difficulty with tissue sampling in FNA.GIST was first described in 1983 as a tumor in the gastrointestinal tract and mesentery, characterized by a specific histological and immunohistochemical pattern . The EurIn our study, there was a difference in the thickness of the needle between DNAB and FNA; however, each specimen obtained by DNAB was macroscopically larger in quantity than that by FNA. Additionally, FNA required more stroke times than DNAB (10 vs. 1). FNA generally requires 3–5 passes and 10–20 stroke times per one pass within the lesion . For theThere were significant differences in the ratio of microscopic diagnosable tumors <25 mm in the longer axis between DNAB and FNA. The differences might be associated with the mechanisms of DNAB and FNA. The mechanism of FNA is to plane the tissue by to-and-fro movement and then aspirate it. It is difficult to move the needle in the lesion if the target lesion is small. On the other hand, the mechanism of DNAB is to cut the tissue by turning and then aspirate it.DNAB was able to obtain a more substantial amount of useable tissue compared with FNA, except in one case that included epithelial tissue. We were surprised that all the tissue obtained by DNAB was useful for a diagnosis. Two gastric SMTs undiagnosed by preoperative EUS-FNA could be diagnosed as aberrant pancreas and schwannoma by DNAB. Therefore, DNAB, with its cutting and vacuuming mechanism, is useful if the tissue is hard.In one case, we attempted to watch the drill tip during DNAB using ultrasonography. The drill tip and helical ditch were clearly visualized Fig. . We specIn conclusion, we devised a new mechanism called DNAB to obtain more tissue than FNA for small gastric SMTs. However, many challenges remain before it can be used in clinical practice. Future animal experiments to evaluate DNAB under ultrasonography as a pre-stage of EUS-DNAB are currently planned.


Because shedding alters characteristics of cells in a rapid and irreversible manner, it should be strictly regulated. However, the molecular mechanisms determining membrane protein susceptibility to shedding (shedding susceptibility) are largely unknown. Here we report that alternative splicing can give rise to both shedding-susceptible and shedding-resistant CADM1 (cell adhesion molecule 1) variant proteins. We further show that O-glycans adjacent to the shedding cleavage site interfere with CADM1 shedding, and the only 33-bp alternative exon confers shedding susceptibility to CADM1 by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. These results demonstrate that shedding susceptibility of membrane protein can be determined at two different levels of its biosynthesis pathway, alternative splicing and O-glycosylation.Ectodomain shedding (shedding) is a post-translational modification, which liberates the extracellular domain of membrane proteins through juxtamembrane processing executed mainly by the ADAM ( In addition, through the detailed analysis of a newly identified shedding target, CADM1 (cell adhesion molecule 1), we find that the shedding susceptibility of CADM1 depends entirely on the inclusion of the only 33-bp alternative exon, exon 9. We further show that O-glycans adjacent to the shedding cleavage site interfere with shedding of CADM1 and that exon 9 makes CADM1 susceptible to shedding by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. This study provides evidence that shedding susceptibility of membrane protein is regulated at two different levels of its biosynthesis pathway, post-transcriptional alternative splicing and post-translational O-glycosylation.To further elucidate the molecular mechanisms determining the shedding susceptibility of membrane proteins, comprehensive analysis of naturally occurring shedding is indispensable. Thus we previously developed a proteomic screening system for shedding target proteins using 2D-DIGE  or LPS + BB94−  treated cells were quantified and compared using LC-MS/MS  with the geometric mean H/L ratio >1.5 were defined as candidate shedding targets  and TSLC1 (tumor suppressor in lung cancer 1)), a member of the immunoglobulin superfamily cell adhesion molecules1517181921To confirm that the candidate shedding targets identified in this study are shed in LPS-stimulated Raw 264.7 cells, we focused upon CADM1  and v9 CADM1, the former lacking all alternative exons and the latter containing only exon 9 , and evahttp://www.ensembl.org/) and found that SIRPα , a member of the immunoglobulin superfamily inhibitory receptors23We searched for other shedding targets having multiple splice variants using Ensembl database  . These oIn contrast to SIRPα variant proteins, those of CADM1 have almost the same molecular structures, although the insertion of only 11 amino acids encoded by exon 9 completely converts their shedding susceptibility. We assumed that the 11 amino acids contain important factor(s) determining shedding susceptibility of membrane proteins, and thus analyzed in detail.Since both scissile bond sequence and sufficient stalk length are considered to be required for shedding susceptibility of membrane proteins, we first examined whether exon 9 encodes the shedding cleavage site of CADM1. After confirming that v8/9 CADM1 is significantly more sensitive to shedding than v8 CADM1 in HEK 293 cells, the cytoplasmic PA-tagged v8/9 CADM1 was highly expressed in HEK 293 cells, and the ~15 kDa membrane-remaining shedding product was affinity purified . The N-tO-glycosylated by NetOGlyc 4.0 prediction server (http://www.cbs.dtu.dk/services/NetOGlyc/)O-glycosylated residues using an algorithm, the analysis results suggest that a large percentage of threonine residues encoded by exon 8 are O-glycosylated. Thus, we examined whether O-glycans attached to the threonine residues encoded by exon 8 interfere with CADM1 shedding. Alanine substitutions of two threonine residues which reside immediately upstream of the shedding cleavage site conferred shedding susceptibility to v8 CADM1  treatment significantly decreased the molecular weight of endogenous CADM1, indicating that endogenous CADM1 is highly N-glycosylated as reported previouslyN-glycans, both neuraminidase and O-glycosidase treatment further decreased the molecular weight of endogenous CADM1 , v8, and v9. After removal of N-glycans, neither neuraminidase nor O-glycosidase treatment decreased the molecular weight of v(-) CADM1 , suggesting that the change of O-glycosylation does not participate in the activation of CADM1 shedding.We found that the O-glycosylation level of v9 CADM1 mutants described in O-glycosylation and shedding susceptibility. After treatment with both PNGase F and neuraminidase, O-glycosidase treatment did not decrease the molecular weight of shedding-susceptible Ax5 mutant of v9 CADM1, similarly to that of wild-type v9 CADM1  receptor family members2829O-glycosylation is artificially reduced, suggesting that O-glycosylation prevents these proteins from aberrant shedding under physiological conditions, even though it is not clear which proteases are responsible for the shedding. In addition, it has been reported that the shedding of TNFα is potentiated by the deletion of GalNAc-T2 O-glycosylation enzyme both in cell line and in vivoin vivo studies indicate that O-glycosylation generally prevents shedding, the results obtained from in vitro cleavage assays are somewhat confusing. O-glycosylation of synthetic peptides reduces cleavage by recombinant soluble ADAM in many cases, but potentiates it in a few cases3233O-glycans attached to the threonine residues adjacent to the shedding cleavage site of CADM1 significantly prevent its ADAM17-mediated shedding, and provide clear evidence that O-glycosylation of physiological ADAM17 substrate prevents its shedding. We speculate that O-glycosylation of membrane proteins generally prevents their ADAM-mediated shedding on the cell surface, and that in vitro cleavage assay is not suitable to evaluate ADAM-mediated shedding in which both enzyme and substrate are tethered to the same membrane and thus conformationally restrained.First, we show that O-glycans. Among 11 amino acids encoded by exon 9, five C-terminal non-glycosylatable amino acids which reside immediately upstream of the shedding cleavage site are indispensable for conferring shedding susceptibility, and these amino acids can be substituted to alanine residues without affecting the shedding susceptibility of exon 9-containing CADM1 variants. These results indicate that “a stretch of five non-glycosylated amino acids” is sufficient to confer shedding susceptibility, that is, exon 9 confers shedding susceptibility to CADM1 by increasing the distance between interfering O-glycans and the shedding cleavage site  and shedding-resistant (“JM-b”) splice variant proteins generated by the switching of two mutually exclusive exons (75 bp and 45 bp), both of which encode its stalk region35O-glycosylated in their stalk regions, ruling out the involvement of O-glycosylation in the determination of their shedding susceptibility.In this study, we also show that alternative splicing of SIRPα gene generates both shedding-susceptible (long) and shedding-resistant (short) variant proteins. Consistent with the notion that the stalk length is a determinant of shedding susceptibility, long SIRPα has a longer stalk region than short SIRPα, however, the long SIRPα mutant containing the stalk region of short SIRPα was still susceptible to shedding (data not shown). Unlike CADM1, SIRPα variant proteins have completely different extracellular domains. We, therefore, speculate that the extracellular domain of membrane proteins is a determinant of their threshold stalk length. SIRPα variant proteins have no serine/threonine residues predicted to be Since v8/9 CADM1 is significantly more sensitive to shedding than v8 CADM1 even in SBC-5, MDCK, and HEK 293 cells, we consider that the shedding susceptibility of CADM1 is determined by exon 9 in many cell types and species. We have previously reported that the relative abundance of exon 9-containing CADM1 variant mRNAs is strictly regulated in a tissue- and disease-specific manner3815O-glycosylation occurring in the Golgi apparatus. These two modification mechanisms enable the simultaneous expression of both shedding-susceptible and shedding-resistant variant proteins with different ratios in a cell-type-specific manner, thereby contributing to high selectivity of naturally occurring shedding. We believe that the very existence of these elaborate mechanisms strongly indicate the physiological importance of ectodomain shedding.Taken together, this study’s results clearly show that shedding susceptibility of cell surface protein can be regulated at two different levels of its biosynthesis pathway, alternative splicing occurring in the nucleus and A murine macrophage cell line, Raw 264.7, was cultured in high glucose DMEM supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, and antibiotics. Transfections were performed with FuGENE HD  in general and with a NEPA21 electroporator  in the siRNA knockdown and glycosidase treatment experiments. For Western blotting, cell extracts and proteins of conditioned culture media were prepared as described previouslyO-glycosidase were purchased from New England Biolabs .Antibodies were purchased from Promega , MBL , BioLegend , ProSci , Abcam , Santa Cruz , and WAKO . The N-terminal-tagged expression vectors were constructed as described previously12C6 and L-Arginine-12C6,14N4 (Sigma-Aldrich) in the light medium, or L-Lysine-13C6 and L-Arginine-13C6,15N4  in the heavy medium, respectively. Raw 264.7 cells were grown in light or heavy medium for 1 week to metabolically label all expressing proteins. Twenty million cells were washed twice with serum-free medium and seeded into a single 10-cm culture dish filled with serum-free light or heavy medium in the day before sampling. Cells were cultured in fresh serum-free light or heavy medium containing 1 μg/ml LPS with or without 20 μM BB94 for 60 min, and conditioned media were collected, centrifuged, and mixed. To enrich peptides derived from membrane proteins, N-glycosylated peptides were concentrated from the media according to the method described previously41DMEM medium for SILAC (Thermo Fisher Scientific) was supplemented with 10% dialyzed fetal bovine serum (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol, and 100 mg/l L-Lysine-4 at room temperature for 30 min. Proteins were precipitated with methanol/chloroform solution and suspended in a buffer containing 8 M urea and 400 mM NH4HCO3. Approximately 15 μg of protein was obtained from 2 × 107 cells, and 100 μg was reduced with 10 mM DTT at room temperature for 30 min, alkylated with 20 mM iodoacetamide for 30 min in the dark, diluted 4-fold with distilled water, and digested by trypsin (Promega) at an enzyme:substrate ratio of 1:50 (w/w) at 37 °C overnight. The digested peptides were incubated with Affi-Gel Hz hydrazide gel in Affi-Gel Hz coupling buffer  at room temperature overnight. After sequential washing with 1.5 M NaCl, 80% acetonitrile, CH3OH, distilled water, and 50 mM NH4HCO3, the resin was incubated with 1000 U PNGase F in 50 mM NH4HCO3 at 37 °C overnight. The beads were centrifuged and the supernatant was divided into three parts and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) in triplicate.The media were acidified by 50 mM acetic acid buffer (pH 5) and oxidized by 10 mM NaION-glycosylation and having H/L ratio ranging from 0.1 to 10 were selected, and the geometrical mean of H/L ratio was calculated for each protein.Peptides were analyzed by nano-LC-ESI-MS/MS using an UltiMate 3000 nano-LC system (Thermo Fisher Scientific) linked to a QSTAR Elite hybrid LC-MS/MS system . Samples were loaded onto an L-column 2 ODS 0.075 mm × 100 mm, 3 μm  and separated at 300 nl/min with 33 min linear gradients from 6 to 34% acetonitrile in 0.1% formic acid. Identification and quantification of peptides were performed using ProteinPilot software version 4.0.8085 (AB SCIEX). The following search parameters were selected: sample type ), cys alkylation (iodoacetamide), digestion (trypsin), instrument (QSTAR Elite ESI), special factors (default), species (mouse), ID focus , databases (UniProt_Sprot_contaminants + iso_08092010 database), and search effort (thorough). The accuracy tolerance for both peptides and peptide fragments was set to 0.10 Da with a 95% confidence threshold. The false discovery rate (FDR) analysis was done using the integrated tools in ProteinPilot. Peptides containing deamidation(s) of asparagine residue(s) indicating former Raw 264.7 cells expressing both N-terminally Halo-tagged CADM1 variant proteins and BFP  were stained with cell-impermeable HaloTag Alexa Fluor 488 Ligand (Promega) according to the manufacturers’ instructions. Fluorescence images were taken under a FLUOVIEW FV10i-DOC confocal fluorescence microscope .O-glycosidase according to the manufacturers’ instructions. To detect subtle decrease of molecular weights, 12-amino-acid PA tag was used instead of ~33 kDa Halo-tag in the experiments of glycosidase treatment.Cell extracts were digested by PNGase F, neuraminidase, and/or How to cite this article: Shirakabe, K. et al. Mechanistic insights into ectodomain shedding: susceptibility of CADM1 adhesion molecule is determined by alternative splicing and O-glycosylation. Sci. Rep.7, 46174; doi: 10.1038/srep46174 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Objectives. To evaluate methotrexate effect on tumor necrosis factor (TNF) alpha bioactivity during infliximab (IFX) therapy in rheumatoid arthritis (RA) patients and to correlate TNF bioactivity with antibody towards IFX (ATI) development and RA clinical response. Materials and Methods. Thirty-nine active women RA patients despite conventional synthetic disease modifying antirheumatic drugs (csDMARDs) requiring IFX therapy were enrolled, and clinical data and blood samples were recorded at baseline (W0) and at 6 weeks (W6), W22, and W54 of IFX treatment. TNF bioactivity as well as IFX trough and ATI concentrations were assessed on blood samples. Results. TNF bioactivity decreased from W0 to W54 with a large range from W22 at the time of ATI detection. From W22, TNF bioactivity was lower in presence of methotrexate as csDMARD compared to other csDMARDs. IFX trough concentration increased from W0 to W54 with a large range from W22, similarly to TNF bioactivity. Methotrexate therapy prevented ATI presence at W22 and reduced TNF bioactivity compared to other csDMARDs (p = 0.002). Conclusion. This suggests that methotrexate plays a key role in TNF bioactivity and against ATI development. Since last two decades, management of rheumatoid arthritis (RA) improved strongly RA prognosis due to tight control management and large availability of biological disease modifying antirheumatic drugs (bDMARDs). Infliximab  is a human-murine chimerical monoclonal IgG antibody targeting tumor necrosis factor (TNF) alpha. IFX was approved to treat RA  and otheThirty-nine women RA patients with active disease despite csDMARDs and naïve to bDMARDs were enrolled as previously described . All patAnti-CCP2 and rheumatoid factor (RF) were measured by ELIA method on ImmunoCap 250 . Anti-CCP2 was considered to be positive at a cut-off value of 10 U/mL and RF IgM at 3.5 IU/mL as recommended by the manufacturer.μg/mL. ATI positivity was defined by a concentration higher than 20 ng/mL  or number (%) and nonparametric tests . As expected, TNF bioactivity was heterogeneous during IFX therapy . High TNF bioactivity was observed at W0 (8.20 ng/mL [6.35–9.46]) and was strongly reduced at W6 , further to the IFX loading doses . So TNF bioactivity could explain heterogeneity of clinical response to IFX.TNF bioactivity strongly correlated with DAS28 when pooling all time points  to W54 . Furthermore, ATI concentration was lower in case of methotrexate as csDMARDs compared to leflunomide or hydroxychloroquine . Median IFX trough concentration increased from 0.04 μg/mL [0.03–0.07] at W0 to 0.88 μg/mL [0.08–3.45] at W54 . Then IFX trough concentration decreased with a higher range at W22  and at W54 . At W22 and W54, ATI were detected only in RA patients with low IFX trough concentration .TNF bioactivity at W22 was not explained by baseline clinical parameters including body mass index, smoking status, or baseline corticosteroid doses. However, in RA patients with methotrexate therapy, TNF bioactivity at W22 was lower than that in patients treated with other csDMARDs (Wilcoxon rank sum test p < 0.0001). High TNF bioactivity was observed only in the group “Low IFX trough concentration with ATI”  with highest level in the “Low IFX trough concentration with ATI” group compared to the “High IFX trough concentration without ATI” group . In the group “Low IFX trough concentration without ATI,” IFX trough concentration was higher (1.19 ng/mL [0.59–1.54]) than in the group “Low IFX trough concentration with ATI”  explaining absence of TNF bioactivity in the “Low IFX trough concentration without ATI” group.Due to negative correlation between IFX trough concentration and ATI concentration, we explored TNF bioactivity in 3 groups according to IFX trough concentration and ATI positivity at W22: (1) Low IFX trough concentration without ATI, (2) Low IFX trough concentration with ATI, and (3) High IFX trough concentration without ATI. TNF bioactivity was heterogeneous in the 3 groups  and ATI concentration . At W22, DAS28 correlated negatively with IFX trough concentration and positively with ATI concentration .IFX trough concentration (At the time of personalised medicine , only feWe then explored the heterogeneity of TNF bioactivity at W22. At baseline, the only factor influencing TNF bioactivity at W22 was presence of methotrexate as csDMARDs. By preventing ATI development, RA patients with methotrexate therapy obtained higher IFX trough concentration with a lower TNF bioactivity than other RA patients. This heterogeneity of TNF bioactivity as for IFX trough concentration could explain only some part of clinical response heterogeneity as previously described in RA , 16. DesPreventing effect of methotrexate on ATI development was not extensively analyzed. In ATTRACT study , all RA Interestingly, in the group “Low IFX trough concentration without ATI” no TNF bioactivity was observed, whereas TNF bioactivity was observed in the group “Low IFX trough concentration with ATI.” Explanation of this putative discrepancy between TNF bioactivity in both groups was due to higher IFX trough concentration in absence of ATI. This also explained heterogeneity of DAS28 at W22 in these three groups. The association between ATI and low TNF bioactivity demonstrates that the presence of ATI is preventing endogenous TNF neutralization by infliximab .Our data have some weakness with 39 patients included at baseline. We failed to observe correlation between low IFX trough concentration and development of ATI probably due to absence of blood samples around 3 months of IFX therapy. The paradox results between TNF bioactivity and low IFX trough concentration in presence or not of ATI will require further investigations.To conclude, we described a major role of methotrexate to reduce ATI development, to reduce TNF bioactivity, and to improve DAS28 compared to other csDMARDs. Our result confirmed mechanisms of lack of IFX efficacy by ATI development with impact on TNF bioactivity.