This study aimed to compare the diagnostic capability of Pulsar perimetry (Pulsar) in pre-perimetric glaucoma (PPG) and early glaucoma (EG) with that of Flicker perimetry (Flicker) and spectral-domain optical conference tomography (SD-OCT). This prospective cross-sectional study included 25 eyes of 25 PPG patients, 35 eyes of 35 EG patients, and 42 eyes of 42 healthy participants. The diagnostic capability using the area under the curve (AUC) of the best parameter and agreement of detectability between structural and functional measurements were compared. For PPG patients, the AUC of Pulsar, Flicker, OCT-disc, and OCT-macular was 0.733, 0.663, 0.842, and 0.780, respectively. The AUC of Flicker was significantly lower than that of OCT-disc (p = 0.016). For EG patients, the AUC of Pulsar, Flicker, OCT-disc, and OCT-macular were 0.851, 0.869, 0.907, and 0.861, respectively. There was no significant difference in AUC among these methods. The agreement between structural and functional measurements expressed by kappa value ranged from −0.16 to 0.07 for PPG and from 0.01 to 0.25 for EG. Although the diagnostic capability of Pulsar in the PPG and EG groups was equal to that of Flicker and SD-OCT, the agreements between structural and functional measurements for both PPG and EG were poor. Although glaucoma is an irreversible disease, visual field progression can be delayed or arrested by reducing intra-ocular pressure to an optimal level2. Thus, early assessment using structural and functional ophthalmic measurements is crucial to slow the progression of glaucoma.Glaucoma is a chronic and progressive optic neuropathy associated with retinal ganglion cell death or dysfunction and with visual field disorder10, the retinal nerve fibre layer (RNFL)10 and the macular retinal ganglion cell13, generally occur prior to visual field damage. However, structural damage from glaucoma does not always precede functional damage, and both structural and functional measurements are needed to reliably detect early glaucoma15. Although conventional SAP is the clinically accepted method for the diagnosis and assessment of glaucoma, SAP detectability of early glaucomatous visual field damage has been reported to be slightly inferior to the detectability of structural measurements by optical coherence tomography (OCT)17 and to functional measurements from non-conventional perimetry, such as short wave length automaed perimetry19, flicker perimetry (Flicker)21, Moorfields motion displacement test23, flicker defined form perimetry25, and frequency doubling technology (FDT)27.Previous studies using conventional standard automated perimetry (SAP) have suggested that structural changes in early glaucoma, including changes to the optic disc29 and SAP30. Pulsar stimulus was invented in 2000 by Gonzalez-Hernandez and coworkers28. Although a prototype device known as the Pulsar T30W test was used in a pilot study, Pulsar was not commercially available until the Octopus 600 perimeter was released in 2013. Pulsar is a flicker stimulus, displaying a ring pattern with different contrast levels in counter phase. Pulsar has been shown to have high detectability for early glaucoma34. Detectability of early glaucoma using Pulsar has been compared with FDT, rarebit perimetry, the Heidelberg Retina Tomograph II (HRT II), and scanning laser polarimetry (GDx VCC)34, but there has been no report comparing it to other non-conventional perimetric methods or to spectral-domain OCT (SD-OCT). Nomoto and coworkers20 reported that Flicker demonstrates a higher AUC compared to FDT or to SWAP. Thus, there is a need to compare the detectability of Pulsar, Flicker, and SD-OCT. The aim of this study was therefore to assess the diagnostic capability of Pulsar for PPG and EG and to compare this capability with those of Flicker and SD-OCT.The Octopus 600 perimeter  is based on a thin film transistor LCD and was designed to perform both Pulsar perimetry (Pulsar)Three eyes of 3 glaucoma patients and 2 eyes of 2 normal participants were excluded due to failure to meet the inclusion criteria. Thus, 60 eyes of 60 glaucoma patients and 42 eyes of 42 normal participants were analysed. Of the 60 eyes of the 60 glaucoma patients, 25 eyes were classified as pre-perimetric glaucoma , and 35 eyes were classified as early glaucoma . Table The best parameters for discriminating the control group from the PPG group by Pulsar, Flicker, and SD-OCT (OCT-disc and OCT-macular) were mean defect AUC = 0.733), number of abnormal points with a p < 0.01 (AUC = 0.663), vertical cup-to-disc ratio AUC = 0.842), and superior macular RNFL (mRNFL) thickness (AUC = 0.830), respectively. The AUC from the Flicker results was significantly lower than that from the OCT-disc results (p = 0.016), but there was no other significant difference among the three methods. The best parameters for discriminating the control group from the EG group by Pulsar, Flicker, OCT-disc, and OCT-macular were mean defect (AUC = 0.851), square loss variance (AUC = 0.869), inferior circumpapillary RNFL (cpRNFL) thickness (AUC = 0.907), and inferior mRNFL thickness (AUC = 0.861), respectively. There was no significant difference in AUC among the four methods. ROC curves are shown in Fig. 3, number2, and suComparison of sensitivity and specificity values at best cut-off and sensitivity values at 80% and 90% specificity values are shown in Table Figure Table In the current study, we found that the diagnostic capability of Pulsar was equal to Flicker and SD-OCT for both PPG and EG patients. However, the agreement of detectability of structural and functional measurements was poor, and structural measurements appeared to be more sensitive than functional measurements in PPG patients. In contrast, functional measurements using Pulsar were equal to structural measurement in EG patients.37, Flicker (AUC = 0.800)20, SWAP (AUC = 0.660 to 0.704)37, and Pulsar (AUC = 0.733)31. Multiple studies have also investigated the diagnostic capability for PPG using structural measurements with methods such as OCT (AUC = 0.527 to 0.938)40, HRT (AUC = 0.740 to 0.914)40, and GDx (AUC = 0.688 to 0.894)39. Although it is difficult to make a direct statistical comparison between the AUC results of the current study and those of previous studies because of differences in sample size and characteristics, we report an AUC for PPG of 0.733 using Pulsar, similar to the results of previous studies on specific functional measurements37. Although the AUC from Pulsar did not differ from other devices in the current study, the AUCs from specific functional measurements were slightly lower than those that have been reported by previous studies. When sensitivity values at the best cut-off or at fixed specificity were compared for each device, the structural measurements from OCT appeared more sensitive than the specific functional measurements reported by the current study and by a previous study31. This might be because PPG was determined by clinical structural assessment of the optic disc shape based on the general mechanism of pathogenesis42. The best parameter for discriminating between the control group from the PPG patients was cup shape, not cpRNFL thickness. Indeed, the diagnostic capability of structural measurements of the optic nerve head is better than cpRNFL thickness for classifying PPG44.To date, many studies have reported on the diagnostic capability for PPG using specific functional measurements with methods such as FDT (AUC = 0.666 to 0.802)31. Functional measurements appear to be more sensitive for EG diagnosis than structural measurements in both a previous study31 and the current study; however, we report the opposite for PPG diagnosis. This might be due to the fact that all EG patients had abnormal SAP results corresponding with Anderson-Patella criteria45 in addition to a glaucomatous optic nerve head. Additionally, as glaucoma progresses from PPG, superior or inferior RNFL thinning occurs along with changes to the optic nerve head. Thus, it could be reasonably expected that functional measurements are more sensitive than structural measurements for EG diagnosis.For EG, the diagnostic capabilities using AUC of both Pulsar and Flicker were equal to those determined from SD-OCT. This is in agreement with a previous study reporting that the diagnostic capability of Pulsar using AUC was equal to FDT, HRT II, and GDx VCCAlthough the sensitivity for PPG at best cut-off of Pulsar was lower than the sensitivity of OCT, specificity at best cut-off was higher than OCT. However, both sensitivity and specificity for EG at best cut-off with Pulsar was equal to OCT. The agreement between structural and specific functional measurements in both PPG and EG was poor. However, PPG and EG were detected with either structural or specific functional measurements with 100% sensitivity Fig. . There w46.Reliability indices of FP and FN for Flicker were worse than Pulsar, and an especially high FP rate was demonstrated for Flicker in both PPG and EG. Flicker fusion frequency was measured at each test point with a fixed high contrast stimulus of 0 dB, while contrast sensitivity was measured with a fixed temporal frequency of 10 Hz for Pulsar. Eyes with PPG or EG, such as those investigated in this study, can still respond to Flicker stimuli, although with decreased sensitivity. Flicker is difficult for PPG or EG patients to accurately respond to, as the flickering target is close to threshold at slightly decreased sensitivity regions. It was reported that threshold variability increases even the SAP measurements at slightly decreased sensitivity regionThe test duration of Pulsar was shorter than that of Flicker despite the use of the same tendency oriented perimetry (TOP) algorithm. This may be due to the difference in number of test points between Pulsar and Flicker. The 32 P test point of Pulsar is similar to the original Octopus 32 test point program with a 6-degree interval, but the 4 points at the superior and inferior were each removed. In contrast, Flicker was measured with the original Octopus 32 test point program. Another reason for the difference in stimulus presentation time might be that a presentation time of 500 msec was applied for Pulsar, but only a 1 sec presentation time was applied for Flicker.47, but that Flicker is48. However, it was reported that Pulsar was affected with intraocular straylight as well as SAP49. In contrast, Flicker didn’t affect ocular media opacity50. Thus, Pulsar may have the disadvantage of robustness to media opacities compared with Flicker.Pulsar and Flicker each have several advantages and disadvantages. Pulsar may have the advantage of ease of use and less fatigue compared with Flicker because it demonstrated good reliability indices and a shorter test duration in the current study, and a previous study reported that Pulsar is not associated with a learning effectThe current study’s main limitation was that the rate of glaucoma type was different between PPG and EG, and PPG patients in particular had almost normal tension glaucoma. Further studies will therefore be required to confirm our results.In conclusion, the diagnostic capability of Pulsar for PPG and EG was equal to that of Flicker and OCT. However, the agreement between structural and functional measurements for PPG and EG was poor. The structural measurements from OCT were more sensitive than the specific functional measurements from Pulsar for PPG, while specific functional measurements by Pulsar were more sensitive than the structural measurement by OCT for EG. Therefore, a combination of structural and functional measurements is recommended to reliably diagnose early glaucoma.http://www.umin.ac.jp/) under the unique trial number UMIN000016055.This prospective cross-sectional study was reviewed and approved by the Kitasato University Hospital Ethics Committee (no. B14-129). All study conduct adhered to the tenets of the Declaration of Helsinki, and all study subjects provided written informed consent. This study was registered in the UMIN Clinical Trials Registry  24–2 or 30–2 Swedish Interactive Threshold Algorithm (SITA) Standard better than a mean deviation of −3 dB. Additionally, the control group consisted of 45 eyes of 45 normal volunteers from a population of Kitasato University Hospital medical staff and Kitasato University staff members who were recruited between January and May 2016 and who had previous SAP measurements from a HFA 24–2 or 30–2 SITA Standard at least 2 times within one year. The diagnosis of glaucoma was determined via a fundus examination using slit-lamp indirect ophthalmoscopy and 90-diopter lens by one of three glaucoma specialists  and based on previous SAP results. All glaucoma patients and normal participants underwent a comprehensive ophthalmic examination, including noncycloplegic refraction testing, visual acuity testing at 5 meters using a Landolt ring chart, intraocular pressure measurement, ocular axial length measurement, and slit-lamp and fundus examination by a glaucoma specialist . Glaucoma patients were included in this study if they had a corrected visual acuity of 20/20 or better, cylindrical power of −1.50 diopter or less, and spherical equivalent of −8.00 to +5.00 diopter. These criteria were also applied to normal participants, with the added criteria of an intraocular pressure of 21 mmHg or less, a normal optic disc appearance, and no ophthalmic diseases in the absence of refractive error.After comprehensive ophthalmic examination, all glaucoma patients and normal participants underwent an initial SAP measurement. This SAP measurement was performed with an HFA  24–2 or 30–2 SITA Standard. SAP results were considered reliable if the fixation loss was <20%, the false positive rate was <15%, and the false negative rate was <33%.45.After SAP measurement, glaucoma patients were classified as EG if they showed structural glaucoma changes such as rim thinning, notching, and nerve fibre layer thinning or defects, and if they showed abnormal SAP results corresponding with Anderson-Patella criteriaAfter SAP measurement, patients were classified as PPG if they showed the abovementioned structural glaucoma changes in the absence of abnormal SAP results. After SAP measurements, all patients and normal participants underwent Pulsar, Flicker and SD-OCT in random order.Pulsar perimetry was performed using the Octopus 600 perimeter 32 P TOP algorithm. The 32 P test point is similar to the original Octopus 32 test point program with a 6 degree interval, but the 4 points at the superior and inferior were each removed because of limitations of the angle of field of the monitor. The stimulus consisted of a circular, sinusoidal, 5-degree diameter grating pattern that was presented for 500 msec. The stimulus underwent a counter phase pulse motion at 10 Hz, in which both spatial resolution  and contrast  were simultaneously modified. Threshold sensitivity is expressed in spatial resolution contrast units (src). Refractive error was corrected to distance by inserting trial lenses with the spherical equivalent correction into the eye piece. The presentation ratios of FP and FN catch trials were configured to 10% of the total number of stimuli presented for Octopus 600 testing reliability, which corresponds with those of the HFA SAP performed with the SITA protocol.2 (4800 apostilbs) that was presented for 1 sec. The flicker targets were presented under a supra-threshold condition with a background luminance of 10 cd/m2 (31.5 apostilbs), and critical flicker frequency values were evaluated at each test point. Threshold sensitivity is expressed in critical flicker frequency (Hz). The presentation ratios of FP and FN catch trials were configured to 10% of the total number of stimuli presented for Octopus 311 testing reliability, which corresponds with those of HFA SAP performed with the SITA protocol.Flicker perimetry was performed using the Octopus 311 perimeter  32 TOP algorithm. The stimulus consisted of a Goldmann size III (0.43 degree) target with a luminance of 1527 cd/m51.SD-OCT imaging was performed using 3D OCT-2000 version 8.1.1  in the 3D optic disc horizontal raster scan mode (OCT-disc) with a 512 × 128 scan resolution and 6 × 6 mm scan area and in the 3D macular vertical raster scan mode (OCT-macular) with a 512 × 128 scan resolution and 7 × 7 mm scan area. This device operates at a speed of 50,000 A-scans per second and has a depth and lateral resolution of 6 and 20 μm or less, respectively. It requires a pupil size of 2.5 mm or larger for imaging. Ocular magnification was corrected based on Littmann’s formulaThe main outcome measures were the diagnostic capability of each device using the best cut-off parameter for discriminating between healthy and glaucomatous eyes and the agreement of detectability between structural and functional measurements. The secondary outcome measures were the comparison of reliability indices, including FP and FN, and the test duration between Pulsar and Flicker.All examinations were performed within a three-month period. The results of the first examination were excluded to avoid learning effects. Right eye results were converted to left eye format for analysis. The exclusion criteria were as follows: fixation loss >20% and false positive rate >15% in HFA measurement; reliability factor >15%, which is the average of the FP and FN rates in Flicker and Pulsar; and image quality index <30 in SD-OCT.Normality of the data distribution was evaluated using the Shapiro-Wilk test. Test results were compared using either paired t-tests or Wilcoxon signed-rank tests. A Bonferroni test was used to correct for multiple testing. The best cut-off parameter for each device for discrimination between healthy and glaucomatous eyes was decided by the highest AUC based on receiver operating characteristic analysis. The detectability of each device was assessed using the AUC of the best cut-off parameter by the DeLong method. Kappa statistics were calculated to evaluate agreement of detectability between structural and functional measurements. All data were analysed using commercially available SPSS version 22.0  and MedCalc version 16.1 .


Chardonnay wines from Burgundy, obtained from musts with three levels of clarification  during two consecutive vintages (2009 and 2010) and for two kinds of closures (screw caps and synthetic coextruded closures) were analyzed chemically and sensorially. Three bottles per turbidity level were opened in 2015 in order to assess the intensity of the reductive and/or oxidative aromas (REDOX sensory scores) by a trained sensory panel. The chemical analyses consisted in polyphenols and colloids quantification, followed by a proteomic characterization. For the two vintages, the REDOX sensory scores appeared to be driven both by the type of closure and to a lesser extent by the level of must clarification. Vintages and must racking prefermentative operations were also distinguished by chemical analyses. All white wines from the lowest must turbidity had the lowest REDOX sensory scores. Such wines exhibited lower concentrations in tyrosol and grape reaction product and higher concentrations in colloids with relatively low molecular weights. Among these macromolecules, grape proteins were also quantified, two of them exhibiting concentrations in bottled wines, which were statistically correlated to oxidative evolution in white wines. Winemaking pre-fermentative operations influence the progress of fermentations and the final quality of wine , elaborated at three different levels of must turbidity, and bottled with two kinds of stoppers, revealed different oxidative evolution trends upon time. We looked here for chemical markers that should be potentially linked to reductive/oxidative perceptions.−1 SO2. Cold racking at 12°C from 12 to 24 h was carried in order to obtain three levels of must clarification, denominated for the present study as “Low,” “Medium,” and “High” corresponding to 300, 600, and 800 NTU, respectively. Musts were directly placed in oak barrels for the alcoholic and malolactic fermentations, and aged for 8 months. Wines were then bottled and 40 mg L−1 of SO2 was added. Two different closures, including screw caps and synthetic coextruded stoppers, were used to study the further impact of oxygenation during bottling. A total of 12 bottles for each vintage  were stored in a cellar with constant temperature and humidity and opened in 2015 for chemical and sensory analysis.Chardonnay dry white wines were elaborated identically during vintages 2009 and 2010. Chardonnay grapes were hand harvested and pneumatically pressed. Must was protected with a 4 g hLThe water used was purified in ultrapure Millipore Milli-Q , methanol, ethanol, acetonitrile, formic acid, tartaric acid and trifluoroacetic acid were purchased from Interchim  at the highest grade of purity. A caustic soda solution at 50% (w/w) was supplied by Sigma-Aldrich . Pure standards of gallic acid, protocatechuic acid, hydroxybenzoic acid, hydroxytyrosol, tyrosol, catechin, (2)-epicatechin, dimer B1 and B2, caftaric acid, gentisic acid, caffeic acid, coumaric acid, chlorogenic acid and ferulic acid were obtained from Extrasynthèse .−1 flow rate. Injection volume was 5 μL. Wine polyphenol concentrations were determined following a calibration made from a polyphenol standards mixture with concentrations ranging from 0.1 to 10 mgL−1. For coutaric acid and grape reaction product, concentrations were expressed in mg L−1 caftaric acid equivalent. The area of eluted compounds were measured by the Waters Empower software. The chromatographic methodology was improved from a previous study  from 0 to 6 min and then increasing linearly over 56 min to 100% B  where it was held isocratic until 60 min, with a 0.3 mL min−1 of tartaric acid and a pH adjusted to 3.4, with a caustic soda solution at 50% (w/w). The flow rate was constant at 0.5 mL min−1. Fluorescence detection was realized at an emission of 320 nm with an excitation of 280 nm. Injection volume was 20 μL. The system was calibrated with protein standard markers (Sigma Aldrich) to analyse wine colloids with molecular weight ranging from 5 to 300 kDa, and adapted from a recent comparative study  equipped with a Phenomenex Yarra SEC-2000 column with a length of 30 cm, diameter of 7.8 mm, granulometry of 3 μm and pore sizes of 145 Å . The chromatographic system consists of a L-2130 pump equipped with low-pressure gradient accessory and in-line degasser, a L-2200 autosampler with a sample cooling system, a L-2300 column oven, a L-2455 diode array detector and a L-2485 fluorescence detector  The eluting phase was a hydroalcoholic solution of ethanol/water 12/88, (v/v) with 6 g LL = 15 cm, d = 0,75 cm, granulometry of 3 μm) and optimized from a previous study . A total of eight white wines from vintage 2010, coming from this same experimentation, elaborated from low and high must turbidity and bottled with the two kinds of stoppers, were analyzed in triplicate. The precipitated extracts were reduced, alkylated and digested with trypsin. A 2 pM solution of rat serum albumin was used as a digestion quality control. Peptides were separated on a nanoLC/ESI-Trap  equipped with a LC Thermo Scientific™ Acclaim™ PepMap™ 100 C18 (www.matrixscience.com) within the NCBI, Tandem and Comet databases. Validation of proteins was carried via Protein and PeptideProphet (tools.proteomecenter.org/TPP.php). Peptides were filtered in order to keep peptides that were found in two of the three technical replicates for one white wine sample. Peptides retention times were aligned following the alignment method from MassChroQ software  to (α = 5%). These data were subjected to three-way ANOVAs with wine closures (synthetic stopper or screw cap) and turbidity  as within subject factors. The turbidity x closure interaction was also tested. When a significant effect was reached, pairwise comparisons were carried using Newman-Keuls' test (α = 5%). All statistical analyses concerning sensory analysis were carried out using XLStat 2015 .The effect of must turbidity on wine polyphenolic and colloidal content and on sensory ratings was analyzed by a one-way ANOVA and a Tukey multiple comparison test taking must turbidity and vintage as factors with a confidence interval of 95%. Prediction of REDOX sensory scores (Y dependent variable) from wine chemical characteristics (predictors) was carried out using partial least-squares regression (PLS) performed with Origin Pro 8.0 software .Table For a given vintage, polyphenol concentrations of GRP and tyrosol, appeared to be affected by must racking. Both were significantly more present in bottled white wines (ANOVA with a significance level of 5%) elaborated with less clarified musts (high turbidity), in particular for the 2009 vintage (tyrosol) and the 2010 vintage (GRP). Such differences could be explained by the higher must fermentiscibility  and presented two matched peptides SSLAVDDVDQR and TFFCTDLSR, with molecular weight of 60,279,412 and 57,376,632 Da, respectively. These two matched peptides were found for the seven analyzed wines and seemed to be still present after 5 years of bottling, regardless of the level of must clarification.As presented in Figure P06169) and presenting two matched peptides NATFPGVQMK and NPVILADACCSR, with molecular weight of 54,677,923 and 68,832,675 Da, respectively. Such protein, which is known to be involved during the Ehrlich pathway  turbidity level  but also a significant closure type x turbidity level . The ANOVA of the 2010 data showed only a very significant effect for closure type .The ANOVA on the 2009 data showed significant effect for closure type  but not than the high level (average sensory REDOX score 0.89). High and medium levels were not significantly different.The next step of this experimentation was to establish correlations existing between sensory attributes and the chemical composition. To that purpose, Figure The first two factors of the PLS-DA explained 79.7% of the variance, where the first factor F1 clearly distinguished vintages in terms of chemical composition and REDOX sensory score. Vintage 2009 was: richer in polyphenols, particularly more cinnamic acids; poorer in colloidal matter; and presented lower values of oxidation ratings compared to vintage 2010. The second factor F2 discriminated nicely the must turbidity parameter for both vintages and particularly wines made from low turbidity musts, which were correlated to high contents of wine colloidal matter and low REDOX sensory score. The PLS-DA model also took in account our closure effect observation in terms of oxidative perception, for a given vintage and level of turbidity, where non oxidized screw capped wines presented highest F1 scores and lowest F2 scores compared to the oxidized wines that were closed synthetically.We therefore focused our study in the vintage 2010 which presented the highest amount of colloidal matter and particularly wine proteins for the two bottle closures (screw cap and synthetic coextruded stoppers) in order to cover a large window of wine REDOX sensory scores. The aim was to statistically investigate the possible associations existing between wine proteins content and the level of oxidation reached by each white wines. In that context, a partial least square analysis was carried in order to discriminate among the 123 wine proteins, those that best discriminated white wines in function of their REDOX sensory scores. As shown in Table Eight of them presented on average more peptides for low REDOX wines compared to high REDOX wines. The low-temperature-induced-proteinase-like protein was the most representative for low REDOX white wines, containing in average two matched peptides. Four others proteins were significantly more present in low REDOX wines, but with only one matched peptide: unnamed protein product, basic leucine zipper and W2 domain-containing protein, structural maintenance of chromosomes protein 4 isoform X2 and elongation factor 1-alpha.Saccharomyces cerevisiae showed its functional role in the regulation of the oxidative stress in the yeast metabolism (Farah and Amberg, Two of them: actin-1 and hypothetical protein VITISV_024840 presented on average more peptides for high REDOX wines compared to low REDOX wines: 1.75 and 1, respectively. This result suggests that these two grape proteins could be involved in oxidation mechanisms. A study carried on actin from We reported here the first combined molecular/macromolecular characterization of bottle aged white wines and its correlation with their oxidative state. To that purpose, wines from the 2009 and 2010 vintages, made from the same must but with three levels of clarification, were analyzed for their polyphenolic and chromophoric macromolecular contents. If polyphenol contents appeared to be mostly driven by the vintage, proteins, contributing mostly to the colloidal fraction studied, were directly correlated to the level of clarification, even after several years of bottle aging, and regardless of the vintage. REDOX sensory scores ranging from −5 to +5, made by a trained sensory panel, further enabled to distinguish wines according to their oxidative perception. The type of bottle closure thus appeared to be responsible for the most significant REDOX sensory scores differences, with screw caps leading to low REDOX sensory scores and thus less oxidized wines, while synthetic coextruded closures led to more oxidized ones. A quantitative proteomic analysis of wines from the 2010 vintage possible correlations existing between levels of must clarification and the oxidative perception of white wines. Two sulfur-containing grape proteins: actin and hypothetical protein VITISV_024840 were thus shown to be potential markers of oxidation evolution. These two grape proteins could be in the future quantitatively analyzed to validate their involvement in oxidation deviations in white wines.CC and JB conceived the study. CC analyzed the data and wrote the manuscript. CC, LN, MM, and JB performed the experiments. PJ and JB analyzed the sensory results. MN and RG took part to scientific discussions. All authors contributed to the final form of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Malignant mesothelioma (MM) is an aggressive malignancy of the serosal membranes. Early diagnosis and accurate prognostication remain problematic. BAP1 is a tumour suppressor gene commonly mutated in MM. Germline BAP1 mutation has been associated with early onset and less aggressive disease compared with sporadic MM. Sporadic BAP1 mutations are common and are associated with improved survival in MM, contrary to other malignancies. This study investigated the prognostic role of BAP1 in matched cytology and surgical specimens and aimed to investigate the association between BAP1 and the established prognostic marker VEGFA from a cohort of 81 patients. BAP1 mutation was found in 58% of histology and 59% of cytology specimens. Loss of BAP1 expression in both surgical and cytology specimens was significantly associated with poorer survival in a multivariate analysis when controlling for known prognostic indicators. Increased levels of VEGFA in pleural effusions were associated with poor survival. We conclude that the prognostic significance of BAP1 mutations in MM cannot be determined in isolation of other prognostic factors, which may vary between patients. Pathologists should employ caution when commenting on prognostic implications of BAP1 status of MM patients in diagnostic pathology reports, but it may be useful for early diagnosis. Malignant mesothelioma (MM) is an aggressive malignancy of the serosal membranes and is attributable in most cases to prior asbestos exposure. Prognosis is currently poor, and most deaths occur within 12–18 months of diagnosis, with even shorter survivals on average for pleomorphic, sarcomatoid, and desmoplastic MMs. Most diagnoses occur at a late stage, related to nonspecific symptoms and the long latency period of the disease, and treatment response is limited. Established prognostic indicators such as histological subtype, age, and gender can give some insight into predicting patient survival ; howeverin vitro has resulted in decreased cell proliferation and mediation of apoptosis in MSTO211H, HMeso, and H2373 mesothelioma cell lines, and reintroduction of wild-type BAP1 in BAP1-null cell line NCI-H226 promoted cell growth, yet another study reported that this was counterbalanced by increased apoptosis, indicating that the consequences of in vitro manipulation may be cell type dependent [in vivo evidence in nude mice has shown that injection of NCI-H226 (BAP1 negative) cells confers less tumorigenicity when these cells are infected with lentivirus carrying wild-type BAP1 compared to the mutated BAP1 [The tumour suppressor gene BRCA-associated protein 1 (BAP1) is located at 3p21 and is commonly mutated in MM tissue samples –17. BAP1ted BAP1 –27. Give1/S checkpoint as well as apoptosis regulation; however, the impact of upregulation or downregulation of BAP1 is dependent on cell type [α) and VEGF [α is regulated by VHL, which along with BAP1 is commonly mutated in renal cell carcinoma and located on the short arm of chromosome 3 [The molecular pathways of BAP1 in carcinogenesis are not well understood, and it was previously suggested that BAP1 interacted with BRCA-1; however, recent evidence has contradicted this theory . There iell type , 22. A nand VEGF –31. HIF1mosome 3 . Mutatiomosome 3 , along wmosome 3 .Initially, acquired BAP1 mutation frequency in MM was underestimated due to the use of Sanger sequencing and NGS, which missed larger deletions , 34. ConThis study recruited a cohort of MM patients diagnosed at Flinders Medical Centre and established BAP1 status by immunohistochemistry on matched surgical and cytology specimens . We aimed to evaluate any prognostic significance of BAP1 status within this cohort, independent of established prognostic indicators, in order to compare our findings with previous contradictory data. This may provide further insight into disease pathogenesis and allow for more specific prognostic predictions in pathological analysis.A cohort of 83 patients was recruited, diagnosed at the Department of Anatomical Pathology at Flinders Medical Centre between the years of 2006 to 2015. Archival tissue and cytology blocks were available through SA Pathology. Patients were included on the basis of histological diagnosis of MM, availability of adequate tissue and cytology blocks, and clinical follow-up information. Additionally, 18 cytology specimens from patients presenting with malignant effusions due to metastatic lung adenocarcinoma were also included to compare BAP1 expression between MM and adenocarcinoma, which may be useful for differential diagnoses in selected cases. This work was approved by the Southern Adelaide Clinical Human Research Ethics Committee .g for 5 min, and the supernatant was stored at −80°C immediately until further use. Of this cohort, 18 samples were available for processing.Pleural effusion samples were obtained through SA Pathology after processing for diagnosis. Samples were spun at 500μm thick, then deparaffinised, and rehydrated in graded concentrations of xylene and ethanol. Slides were immersed in 3% H2O2 in 50% ethanol to quench endogenous peroxidases. Heat-induced alkaline retrieval was performed using Dako Target Retrieval Solution. Sections were blocked with 10% normal goat serum in TBS for 30 mins. Sections were coated with 1 : 3000 mouse monoclonal BAP1 antibody  and were incubated at 4°C overnight. The Novolink Max Polymer Detection kit  and DAB+ Chromogen System  were used for detection, before haematoxylin counterstaining. Diagnostic clinical procedures related to diagnosis of the cases were performed in a NATA-approved laboratory using QAP validated tests.Paraffin sections were cut at 0.4 All slides underwent blinded assessment by two RCPA-qualified pathologists, S.K. and D.M. A positive result was defined as positive nuclear labelling in any number of tumour cells, regardless of background cytoplasmic reactivity. Cytology specimens were excluded if cell count was not satisfactory .Levels of VEGFA, an established prognostic factor in MM, were correlated with survival to assess potential interaction of BAP1 and VEGFA. VEGFA levels in pleural effusions were tested on the 17 patients for whom effusion fluids were available. The VEGFA Duoset ELISA  was used to detect VEGFA levels as per kit instructions. Concentration was determined via comparison to standard curves.p value of <0.05 was deemed statistically significant. Age, sex, and histological subtype were entered into a multivariate model as they are universally accepted prognostic factors in MM.Kappa scores were obtained to analyse agreement between pathologist opinions. Survival was calculated as the number of months between diagnosis and death of the patient, or last follow-up in the case of patients still alive. If a patient was still alive at the last follow-up, survival was censored. Kaplan-Meier curves were implemented to analyse survival, and Cox Regression univariate and multivariate analysis were used to evaluate covariates for statistical significance. A p = 0.015). Chemotherapy and conservative therapy  were the most common treatment options but were not significant prognostic factors in univariate or multivariate analysis. Age and sex had no statistical impact on survival.In total, 81 patients were eligible for analysis of surgical sections, and 57 of these patients had accompanying cytology sections for analysis. κ = 0.88) and surgical versus cytology BAP1 status (κ = 0.85) for matched patients was analysed, both of which indicated good agreement. BAP1 expression was lost in 61% of epithelioid, 62% of biphasic, and 36% of sarcomatoid histology specimens ; however, this was not evident in analysis of other subtypes or in the cytology group.BAP1 expression status according to clinical data is summarised in p = 0.005). Median survival times were 13  and 7  months for high and low VEGFA concentrations, respectively. Of the 7 patients with high VEGFA levels, 6 showed negative BAP1 expression.To further investigate prognostic implications within our cohort and investigate the potential role of BAP1 in regulating VEGFA levels, we explored pleural effusion VEGFA protein concentrations . Of thisRegardless of conflicting evidence and theories on prognostication, it is evident that BAP1 is commonly mutated in MM , 14, 22.The inclusion criteria of this study required at least 20 cells to be present in a matched cytology specimen for this analysis. Most cytology preparations suitable for analysis are from patients with the epithelioid subtype, as sarcomatoid subtypes generally exhibit less shedding of cells into the pleural space , 44. In The current study used immunohistochemistry for detection of mutation, in the absence of sequencing and/or qPCR, but this may not detect all BAP1 mutations, as reported by us in a case of MM and uveal melanoma  and anotVEGFA prognostic data was utilised in this study for two reasons: firstly, it is a previously established adverse prognostic marker when elevated in pleural effusion, and secondly, it is used to investigate the possible functional relationship between BAP1 and VEGFA. Pleural effusions were available for 18 of the recruited patients and were tested in order to validate the role of VEGFA in this cohort, to confirm that our cohort conforms to validated prognostic indicators, and to explore any association between VEGFA, BAP1 status, and survival. As expected, increased VEGFA levels were associated with adverse prognosis in this cohort, and 7 of the 8 patients with high VEGFA levels were BAP1 negative. The prognostication of MM may be more meaningful when a number of markers are used in conjunction. While there has been limited evidence of a link between molecular pathways, BAP1 mutation has coincided with VHL mutation in clear cell renal cell carcinoma (ccRCC) . VHL mutOur study differs from others by the higher proportion of biphasic and sarcomatoid subtypes in our cohort. In addition, our cohort has a higher mean age at diagnosis in comparison to many studies, which may indicate acquired BAP1 mutation in comparison to germline mutations. Germline mutations are associated with earlier onset of disease, as opposed to the mean age at diagnosis of approximately 70 years of age, but were also associated with improved prognosis , 25, 56.It is clear that although a number of genes have been suggested as driver mutations in MM, a single-target approach for therapy may not be the answer. Although BAP1 has been described as a common mutation in MM cases, it is unlikely that this will offer any therapeutic target due to the high mutational load with heterogeneity of genetic changes in MM cases, with resulting treatment resistance. We suggest that care must be taken when interpreting BAP1 status in diagnostic pathology reports and relating BAP1 status to prognosis in isolation. A combination of established prognostic markers could be used to assist in more accurate individual prognostication. At this stage, histology analysis is optimal for the clinical diagnosis of MM, but we hypothesise that the use of BAP1 on cytology samples may become useful in detecting malignancy at an earlier stage. The targeted investigation of patients with multiple previous effusions or clinical suspicion before surgical biopsy may permit earlier diagnosis of MM and may provide some further uses for BAP1 as a marker of malignancy. Further investigation of the functional relationship with VEGFA status may provide further insight into pathogenesis.


Although increased nasality can originate from basal ganglia dysfunction, data regarding hypernasality in Parkinson’s disease (PD) and Huntington’s disease (HD) are very sparse. The aim of the current study was to analyze acoustic and perceptual correlates of velopharyngeal seal closure in 37 PD and 37 HD participants in comparison to 37 healthy control speakers.Acoustical analysis was based on sustained phonation of the vowel /i/ and perceptual analysis was based on monologue. Perceptual analysis was performed by 10 raters using The Great Ormond Street Speech Assessment ’98. Acoustic parameters related to changes in a 1/3-octave band centered on 1 kHz were proposed to reflect nasality level and behavior through utterance.r = 0.51, p < 0.001) as well as the Unified Huntington Disease Rating Scale chorea composite subscore .Perceptual analysis showed the occurrence of mild to moderate hypernasality in 65% of PD, 89% of HD and 22% of control speakers. Based on acoustic analyses, 27% of PD, 54% of HD and 19% of control speakers showed an increased occurrence of hypernasality. In addition, 78% of HD patients demonstrated a high occurrence of intermittent hypernasality. Further results indicated relationships between the acoustic parameter representing fluctuation of nasality and perceptual assessment (In conclusion the acoustic assessment showed that abnormal nasality was not a common feature of PD, whereas patients with HD manifested intermittent hypernasality associated with chorea. Considerable attention has been given to progressive neurodegenerative diseases affecting the basal ganglia such as Parkinson’s disease (PD) and Huntington’s disease (HD). Both PD and HD are terminal neurodegenerative diseases that elicit a variety of motor and non-motor manifestations, which significantly contribute to decreased quality of life . As PD aThe majority of both PD and HD patients manifest the motor speech disorder termed dysarthria , which iInterestingly, although hypokinetic and hyperkinetic dysarthria manifestations are often counteractive, hypernasality has been reported in both hypokinetic and hyperkinetic dysarthria . In partHypernasality is a result of velopharyngeal impairment and may be defined as the presence of inappropriate air leakage through the nasal cavity during phonation . This leStudies examining hypernasality in PD have yielded controversial results. The etiology of hypernasality in PD and HD is unclear. Although the dysarthria is typically attributed to the disrupted motor control, little correspondence between speech and limb manifestations has been found . NeverthIn HD, the relationship between speech and limb manifestations appears to be more prominent. Correlation between speech timing parameters and overall motor disability has been noted previously . FurtherCurrently the most common method for hypernasality estimation is perceptual rating . In partThe 1/3-octave spectra method is a type of spectral analysis focused on the examination of spectral changes caused by resonatory speech pathologies. This method is based upon the linear source–filter theory of speech, which was first described by The vowel /i/ with the first formant frequency (F1) at approximately 240 Hz and the second formant frequency (F2) at approximately 2,400 Hz appear to be the most sensitive to nasal resonance . Being tBased upon these previous findings, the goal of the present study was to employ methods of objective hypernasality assessment and evaluate the presence and character of hypernasality in PD and HD speakers. A further aim was to examine possible relationships between the severity of hypernasality and disease-specific motor manifestations, to provide more insight into the pathophysiology responsible for development of hypernasality in basal ganglia disorders.The participants in the present study were part of a larger investigation examining speech characteristics of patients with PD and HD. Previous reports generally focused on phonatory, articulatory and prosodic abnormalities including medication effects . A totalThe PD group consisted of 23 men and 14 women, mean age 63.1 ± 14.0 standard deviation (SD) (range 41–80) years, mean disease duration 8.0 ± 4.8 (1–24) years. All PD patients fulfilled the diagnostic criteria for PD . All parThe HD group consisted of 19 men and 18 women with genetically confirmed HD with mean age 49.1 ± SD 12.7 (23–67) years, mean disease duration 6.1 ± 3.4 (1–16) years, mean number of CAG triplets 44.7 ± 3.3 (40–53). Most of the patients (32/37) were treated with monotherapy or a combination of benzodiazepines, antipsychotics, amantadine and antidepressants. All HD patients underwent extensive examination by an experienced neurologist and were rated according to the Unified Huntington’s Disease Rating Scale  . In addiThe healthy control (HC) group consisted of 23 men and 14 women, mean age of 63.1 ± 8.7 (41–77) years. None of the HC participants had a history of neurological or speech disorder. None of the HD, PD or HC subjects suffered from chronic obstructive pulmonary disease, respiratory tract infection, allergy, asthma, facial paresis, or other malady that could negatively influence participant speech performance.The study was approved by the Ethics Committee of the General University Hospital in Prague, Czech Republic, and all participants provided written, informed consent.All recordings took place in a quiet room with a low ambient noise level using a head-mounted condenser microphone  positioned approximately 5 cm from each subject’s mouth. The utterances were sampled at 48 kHz with 16 bit quantization. All the voice signals were obtained during single session conducted by a speech specialist, who asked participants to take a deep breath and perform sustained phonation of vowel /i/ at a comfortable loudness and pitch, as constant and long as possible. The measurement of sustained phonation was performed twice. The participants were also asked to provide freely spoken monologue on a given topic including family, work or interests, for at least two minutes. The both sustained phonation and monologue tasks were part of a comprehensive dysarthria test battery. No time limits were imposed during recording. The inclusion criteria were determined as the ability to sustain prolonged phonation for at least three seconds.As connected speech is more demanding for velopharyngeal control, it is considered the most valid task for perceptual nasality estimation . The ratFor the purposes of instrumental analysis, two recording parts equal to 10% of signal length were cut off from both the beginning and end of the vowel /i/ to avoid distortion by initial vocal fold adjustment and fatigue at the end of the utterance. The remaining signal was then resampled to 20 kHz, which lowered the computational complexity and preserved all useful information . The preEfiltered is energy contained in the single band of 1/3-octave and E(i) is the decibel value of energy contained in the ith band.The process of 1/3-octave spectra analysis based on the multirate filter bank presented by EFn parameter, which represented energy in a 1/3-octave band centered around 1 kHz (passband from 890.9 Hz to 1122.5 Hz). This parameter reflected the addition of nasal resonance and additive nasal pole to the transfer function at 1 kHz. The overall level of hypernasality was estimated by the mean value of EFn parameter (EFn mean) across all windows in the entire utterance. The variability of nasality (EFn SD) in speech was evaluated as the standard deviation of each parameter across the entire utterance. Finally, the evolution of hypernasality in the course of the utterance (EFn trend) was described using a linear regression tangent for each parameter.Considering the effect of spectral flattening, nasality in sustained phonation of the vowel /i/ was evaluated using the EFn mean, EFn SD and EFn trend for each participant were used for all consecutive analyses.As the vowel /i/ was recorded twice for all speakers, average values of estimated acoustic parameters The Kolmogorov–Smirnov test for independent samples was used to evaluate normality. Analysis of variance (ANOVA) with post-hoc Bonferroni adjustment was used for the estimation of group differences between PD, HD and HC groups across acoustic variables.EFn mean, EFn SD, EFn trend, and perceptual assessment) and the level of significance was set at p < 0.0125.Relationships between variables were evaluated using Pearson’s correlation and Spearman’s correlation. Pearson’s correlation was applied to normally distributed data (acoustic speech metrics and disease severity scores), whereas Spearman’s correlation was used for non-normally distributed data . The Bonferroni adjustment for multiple comparisons was performed according to the four measures investigated , by an indecisive class . Use of According to UPDRS III speech item 18, 10 PD patients (27%) demonstrated no speech impairment (score of 0), 32 PD patients (62%) mildly affected speech (score of 1) and 4 PD patients (11%) moderately affected speech (score of 2). According to the UHDRS speech item, eight HD patients (22%) showed normal speech (score of 0) and 29 HD patients (78%) dysarthria without the necessity of repeating speech to be intelligible (score of 1). In summary, the speech of all PD and HD patients was still fully understandable as indicated by UPDRS speech item 18 (ranging between 0 and 2) as well as the UHDRS speech item (ranging between 0 and 1).p < 0.001) across all raters and the intra-rater reliability ranged between 0.77 (p < 0.05) and 0.85 (p < 0.001) between individual raters.The distribution of participants across four perceptual rating grades  are presented in EFn showed strong correlation for mean  and SD  parameters, whereas trend analyses showed only moderate correlation . EFn mean and EFn SD (p < 0.001), particularly due to differences between HD and HC groups (p < 0.001).Analysis of test–retest reliability of the proposed parameter EFn mean, we found increased nasality in 27% of PD, 54% of HD and 19% of HC speakers. In addition, based upon a cutoff value of 3 dB for EFn SD, we observed abnormal nasality variability in 27 % of PD, 78% of HD and 11 % of HC participants.EFn SD parameter  but not EFn mean parameter  or EFn trend parameter .EFn SD  and between UHDRS speech item and EFn SD . We did not detect correlation between perceptual assessment and clinical manifestations in either PD or HD groups.EFn to evaluate the presence and character of hypernasality in prolonged vowels. Using acoustic analysis, we revealed an occurrence of hypernasality in 27% of PD, 54% of HD and 19% of HC speakers. In addition, our results showed a high occurrence of intermittent hypernasality in 78% of HD patients. Perceptual analysis showed the occurrence of mild to moderate hypernasality in 65% of PD, 89% HD and 22% HC speakers. Significant correlation between the acoustic parameter representing nasality fluctuation and perceptual assessment was observed. Furthermore, we revealed significant correlation between acoustic metric representing nasality fluctuation and chorea in HD patients.In the present study, we analyzed hypernasality in PD, HD and HC utterances using objective acoustic analyses as well as perceptual assessment, which represents current gold standard for hypernasality evaluation. Based upon the 1/3-octave spectra analysis presented by Although using acoustic analysis we detected hypernasality in 27% of PD speakers, the non-significant difference between PD and HC groups suggests that hypernasality is a non-prominent speech manifestation. Previous studies focused on hypernasality in PD have provided rather inconsistent conclusions. Based on perceptual evaluation, The presence of hypernasality was observed both perceptually and acoustically in the majority of our HD speakers, which was mainly associated with the occurrence of abnormal nasality variability. Indeed, we observed correlation between acoustic nasality variability and the chorea UHDRS subscore, demonstrating the significant impact of chorea on velopharyngeal mechanism. Although our findings seem to be in accordance with Previous studies have reported perceptual assessment of hypernasality in dysarthria as rather unreliable as hypernasality is less apparent to the listener due to the presence of more dominant dysarthria manifestations . NeverthThere is a little evidence for correlation between perceptual and instrumental measurements of hypernasality in dysarthrias . In our In agreement with our findings, previous studies have perceptually rated the majority of PD participants as mildly hypernasal . HoweverEFn parameter to describe the presence of nasal resonance in speech due to properties of the nasal cavity present in the 1 kHz 1/3-octave band (EFn mean showed significantly increased energy in HD patients, suggesting an abnormal presence of hypernasality in HD patients. Furthermore, using the parameter EFn SD, we revealed significant differences in fluctuations of nasality between HD and control speakers, suggesting intermittent hypernasality in HD patients. The parameter EFn trend was found to be unreliable, as it demonstrated no significant differences between groups and low test–retest reliability.In the present study, we applied an acoustic method designed for the objective evaluation of velopharyngeal insufficiency, to determine the presence and nature of velopharyngeal incompetence in PD and HD. This methodology has been previously found to be superior to other acoustic measures of hypernasality , and wasave band . This asave band . CompareWe did not perform aerodynamic measurements, which would provide direct information about nasal airflow. Nevertheless, a previous study by It is noteworthy that the choice of the vowel /i/ may serve to maximize the impact of nasality or at least the likelihood of an acoustic model finding nasality. Thus, previous research on nasality in children used not only the optimal /i/ but a greater variety of speech material . TherefoOne limitation is that we used different speech tasks for the perceptual and acoustic evaluation of hypernasality, as accurate perceptual evaluation of hypernasality from sustained vowel phonation is not feasible. Indeed, the different speech tasks used likely make correlation analyses between perceptual and acoustic variables problematic. In future studies, it may therefore be beneficial to include rating for consistency, as with the Consensus Auditory Perceptual Evaluation of Voice .We did not test the consistency and reliability of UPDRS and UHDRS metrics. Nevertheless, relationships between nasality and motor abnormalities were found only for the UHDRS chorea subscore, which showed high inter-rater reliability with an ICC of 0.82 .EFn SD is also, to a certain extent, influenced by other manifestations of chorea, particularly laryngeal chorea (As the presence of chorea in HD is unlikely to be limited only to specific parts of the vocal tract such as the soft palate, we cannot exclude that l chorea .As HD generally has an earlier onset than PD, the PD and HD participant groups could not be age-matched. Therefore, we matched the age of the control group to the age of generally older PD group, as nasality is expected to remain stable throughout life or may slightly deteriorate as a consequence of aging . This apPerceptual and acoustic data presented in the current study provide evidence of significantly increased and intermittent hypernasality in HD patients, presumably due to choreatic movements of the velopharyngeal mechanism. Although the presence of hypernasality was also observed in several PD speakers, abnormal nasality is not a prominent feature of hypokinetic dysarthria. However, further research is warranted. The relationships between proposed acoustic metrics and aerodynamic measurements for evaluation of hypernasality in dysarthrias should be explored. Future longitudinal studies are needed to confirm and further elaborate our findings and to show reliability of hypernasality measures as a possible marker of disease progression in basal ganglia disorders. Last but not least, as hypernasality is a prominent sign in several dysarthria subtypes , sensiti10.7717/peerj.2530/supp-1Supplemental Information 1Dataset containing acoustic and perceptual hypernasality scores and clinical informations related to investigated subjects.Click here for additional data file.10.7717/peerj.2530/supp-2Supplemental Information 2Click here for additional data file.


The appearance of detritus shed from mountain ranges along the northern margin of the Tibetan Plateau heralds the Cenozoic development of high topography. Current estimates of the age of the basal conglomerate in the Qaidam basin place this event in Paleocene-Eocene. Here we present new magnetostratigraphy and mammalian biostratigraphy that refine the onset of basin fill to ∼25.5 Myr and reveal that sediment accumulated continuously until ∼4.8 Myr. Sediment provenance implies a sustained source in the East Kunlun Shan throughout this time period. However, the appearance of detritus from the Qilian Shan at ∼12 Myr suggests emergence of topography north of the Qaidam occurred during the late Miocene. Our results imply that deformation and mountain building significantly post-date Indo-Asian collision and challenge the suggestion that the extent of the plateau has remained constant through time. Rather, our results require expansion of high topography during the past 25 Myr. The timing of mountain building along the Tibetan Plateau remains unclear. Here, the authors present new magnetostratigraphic and mammalian biostratigraphic data from sediments to show that mountain building at the edge of the Tibetan Plateau commenced at 25.5 Ma with a separate emergence in the north at 12 Ma. Time-space patterns of deformation within the Eurasian continent reflect the evolution of high topography associated with the Indo-Asian collision12467894In northeast Tibet, the onset of mountain building is considered to have initiated in early Eocene time (50–45 Myr), based on three sources of proxy data. First, low-temperature thermochronology from the hanging wall blocks of major thrust systems suggest that cooling during erosion marks the development of topography above active faults9687The >10 km thick Cenozoic deposits in the Qaidam basin  play an 1461214612614186In this study, guided by the recognition of a new mammalian fossil assemblage, we reevaluate the age of the Lulehe Fm. in the Honggou section, along the northern margin of the Qaidam basin202161214202122The Honggou section is exposed in a fault-related anticline along the northern margin of the basin . Along tAlong the Honggou section, the Lulehe Fm. is 490 m thick and unconformably overlies lower Cretaceous dark-red beds. This formation is predominantly purple to brick red conglomerate intercalated with red sandy mudstone and siltstone . The con24The uniform purple to brick red colour and poor sorting of conglomerates are the most significant features of the Lulehe Fm., which make it easy to distinguish from underlying and overlying strata. These characteristics are the primary basis for lithostratigraphic correlation to its type Lulehe sectionThe 980 m thick Xia Ganchaigou Fm. contains green to yellow, sandstones, siltstones and red to green mudstones . Sandsto262724The 1,400 m thick Shang Ganchaigou Fm. is composed of variable coloured mudstone, yellowish sandstone and thin limestone . Sandsto30The Xia Youshashan Fm. is 820 m thick and is dominated by light brown mudstones and siltstones . Mudston32The Shang Youshashan Fm. is 1070, m thick and is characterized by interbedded mudstone, gravel sandstone and conglomerate . Clast-s24The Shizigou Fm. is 550 m thick and contains pebble, cobble, clast-supported and matrix-supported conglomerates . Clast-sMioechinus? sp., Monosaulax tungurensis, Plesiodipus sp., Zygolophodon sp., Turcocerus sp., and Rhinocerotidae indet , we recovered abundant mammalian fossils which are referred to as the Honggou fauna. Six taxa can be recognized from the Honggou fauna; these are ae indet . The folMioechinus? sp.: A right p4 is present  were identified . As mentBelow the R25, we correlate two short normal intervals of N25 and N26 to chrons C5Cn . AlthougCollectively, the correlation of the Honggou magnetostratigraphy to the GPTS implies that the onset of sediment accumulation of the Qaidam basin along the Honggou region occurred at ∼25.5 Myr and that sediment accumulation was continuous to ∼4.8 Myr. Our results imply that the Lulehe Fm. was deposited from 25.5 Myr to 23.5 Myr, the Xia Ganchaigou Fm. was deposited from 23.5 Myr to 16.5 Myr, the Shang Ganchaigou Fm. spans from 16.5 Myr to 11 Myr, the Xia Youshashan Fm. spans from 11–9 Myr, the Shang Youshashan Fm. was deposited from 9–6.3 Myr and the Shizigou Fm. ranges from 6.3 to 4.8 Myr.Chilotherium, Cyprideis, and Gomphotherium in the strataOur new chronologic constraints from the Honggou magnetostratigraphy suggest that the onset of Cenozoic sediment accumulation in the northern Qaidam basin is much younger than the previous magnetostratigraphic correlations202122The proximity of the Honggou and Dahonggou sections allow reasonably confident correlation of the strata containing the Honggou fauna within the Shang Ganchaigou Fm.  along the Dahonggou q section . Supportet al.et al.East of the Dahonggou section , the ∼50et al.et al.Finally, our new interpretation of the age of the Cenozoic strata in the northern Qaidam basin is also consistent with a well-constrained magnetostratigraphic section in the Huaitoutala anticlineOverall, all of the previous magnetostratigraphic studies along the northern margin of the Qaidam basin are consistent with our new chronology of the Honggou section. Thus, we suggest that the onset of Cenozoic sediment accumulation in the Qaidam basin occurred in Oligocene time, significantly younger than previously thought612Armed with this new chronostratigraphic framework, we use the ages of detrital mineral systems to determine sediment provenance. Previous study of the Honggou sectionThe binomial peak fitting methodThe sharp increase for the population peak ages at ∼12 Myr require an abrupt change in sediment provenance, because progressive unroofing of a single source region will yield systematically younger ages upsection as exhumation drives cooling of rocks. The mirror-image distribution peak ages in the upper part of the section probably represent deposits in the section containing first-cycle detritus after 7.5 Myr ago.et al.These interpretations are supported by detrital zircon provenance analysis. In addition to the previous work by Bush In our Honggou section, two pre-12.5 Myr samples  exhibit similar detrital-zircon age spectra. Zircon ages in the both sample are distributed between 184 and 2,581 Myr, with a major 250 Myr peak and three minor peaks at 440, 1,750 and 2,350 Myr . Differeet al.Along the northern Qaidam basin margin, granite bodies in the Qilian Shan are dominantly early Paleozoic in age4343Our detrital AFT and the detrital-zircon age distribution results are also supported by paleocurrent data. The north- to northeast-directed paleocurrent indicators in the lower part of the section are generally consistent with the source of the East Kunlun Shan and the appearance of south-directed paleocurrent orientations in the upper part of the magnetostratigraphic section provides an independent evidence of the Qilian Shan derived material entering the Qaidam basin since the late Miocene time .Our new chronology from the Honggou reversal stratigraphy significantly revises the timing of sediment accumulation in Qaidam basin during the Cenozoic. The onset of deposition (Lulehe Fm.) occurred around ∼25.5 Myr and persisted until ∼4.8 Myr. Accumulation rates were relatively steady through the interval between ∼25.5–14 Myr at ∼14.4 cm per kyr . SometimFirst, the new chronology of the Honggou section reveals that the onset of Cenozoic sediment accumulation in the Qaidam basin occurred at ∼25.5 Myr ago, which is significantly younger than the previous interpretations which held that deposition of the coarse detritus in the Lulehe Fm. occurred between 65–50 Myr ago4647Second, our results indicate that no significant Paleogene tectonic deformation occurred in the region north of the East Kunlun Shan. The Honggou magnetostratigraphic section is adjacent the Qilian Shan, which separates the Qaidam basin from the Hexi Corridor basin in the north. The absence of Qilian Shan derived materials in the pre-middle Miocene deposits, as revealed by the detrital AFT ages and detrital zircon ages , require6Third, our results support the conclusion that significant tectonic deformation and uplift of the Qilian Shan began at 14–12 Myr. The key results from the Honggou magnetostratigraphy and provenance analysis are the significant increase in accumulation rates at 14–12 Myr and abrupt shift to a sediment source north of the Qaidam basin at this time. These changes are interpreted to reflect the onset of rapid uplift of the Qilian Shan during the late Miocene. Notably, a similar shift in sediment provenance is observed along the northern margin of the Qilian Shan, in the Hexi Corridor at ca.12 MyrOur refined ages for sediment deposition as a proxy for mountain building in northern Tibet carry important implications for the geodynamics of plateau growth. The onset of deformation along the East Kunlun Shan at ∼25.5 Myr, accompanied by relative tectonic quiescence of the Qilian Shan, suggests that shortening related to the Indo-Asian collision during Eocene time was restricted to the Hoh Xil region, south of the East Kunlun Shan15495055561A gasoline powered drill was used to collect the magnetostratigraphic samples at intervals of 4–6 m throughout the section and at least three cores per were drilled. In total, 2,970 cores were collected from 990 stratigraphic horizons. All the drilled cores were oriented by a magnetic compass corrected ∼1.8° to account for the local magnetic declination anomaly.All 2,970 core samples (990 × 3 pilot samples) were shorten to standard specimens of 2 cm in length in the laboratory. At last one specimen per site was subjected to stepwise thermal demagnetization in a TD-48 thermal demagnetizer with an internal residual field less than 10 nT. The maximum of 20 steps for thermal demagnetization were applied for the samples with the following stepwise heating routine: 20, 150, 200, 250, 300, 350, 400, 450, 500, 525, 550, 585, 610, 620, 630C, 640, 650, 660, 680 and 690 °C. The magnetic remanence of each sample was measured with a 2G Enterprises Model 760, three-axis, cryogenic magnetometer shielded in field-free space (<300 nT), at the Paleomagnetism Laboratory of the Institute of Geology and Geophysics, Chinese Academy of Sciences.−2 A/m, with a range of 10−1–10−3 A/m. Progressive thermal demagnetization successfully resolved multiple components of magnetization  of the Honggou section samples was typically on the order of 10tization . Most satization . The lowtization . In the tization .A reversal testThirteen medium- to coarse-grained sandstone samples were collected from the Honggou section to analyse their provenance. More than 5 kg of sandstones for each of the samples were collected from a single outcrop. The apatite and zircon crystals in the samples were extracted using the following methods: (1) the sandstone samples were first crushed to pass a 60 mesh (250 μm) sieve in the laboratory. (2) Manual washing with water and then alcohol was performed to get the denser component of various grain sizes. (3) Electrometer was used to remove magnetic minerals. (4) Different density heavy liquids were employed to separate the apatite and zircon crystals, respectively. (5) The apatite and zircon grains are checked and some non-apatite or non-zircon grains are picked out by hand under a binocular.3 at 20 °C for 20 min. Induced fission tracks in the low-U muscovite external detectors, which covered apatite grain mounts and glass dosimeters during the irradiation were later etched in 40% HF at 20 °C for 20 min. Fission tracks and track length measurements were measured on an OLYMPUS microscope using a magnification of 1,000 under oil immersion objectives for apatite. Generally, the detrital apatite fission track age represents mixed multiple component ages. We therefore employ the binomial peak-fit methodThe external detector method207Pb/206Pb, 207Pb/235U, 206Pb/238U (235U =238U/137.88) ratios. Standard NIST 610 silicate glass was used for concentration information and the Th/U ratio determination. Analyses with ≥20% discordance or ≥5% reverse discordance were excluded. We adopt the 206Pb/238U ages for grains younger than 1,000 Myr and 207Pb/206Pb ages for grains older than 1,000 Myr. The detrital zircon age populations for individual sample are plotted on relative age-probability diagrams derived from the probability density function.Detrital zircon grains were dated by laser ablation inductively coupled plasma mass spectrometry in the Institute of Geology and Geophysics, Chinese Academy of Sciences, following the standard procedurePaleocurrent orientations were primarily determined from clast imbrication and cross stratification along the Honggou section. All the orientation data were measured by a magnetic compass corrected ∼1.8° to account for the local magnetic declination anomaly. For each site, at last 18 imbricated clasts or cross stratifications are measured. The measured data just have been correlated for tilted bedding because the paleomagnetic data13The authors declare that all data supporting the findings of this study are available within the paper and its How to cite this article: Wang, W. et al. Expansion of the Tibetan Plateau during the Neogene. Nat. Commun.8, 15887 doi: 10.1038/ncomms15887 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Astroviruses are recognized as a leading cause of gastroenteritis in humans and animals. They are also associated with extra-intestinal diseases, such as hepatitis in ducklings, nephritis in chickens, and encephalitis in cattle. In February 2017, a fatal infection of goslings characterized by visceral urate deposition was reported in the Shandong province, China. Our systematic investigation led to the isolation of an astrovirus, designated AAstV/Goose/CHN/2017/SD01, and similar disease was reproduced by experimental infection of healthy goslings, fulfilling Koch’s postulates. The isolated astrovirus replicated well and resulted in 100% mortality of goose embryos. Complete genome sequence analysis revealed that the isolate was genetically distinct from known astroviruses and closely related to members of the avastrovirus genogroup II. Experimental infection showed that the isolate was highly pathogenic in goslings, causing clinical signs, growth repression and in many cases mortality. Histopathological examination indicated that lesions occurred mainly in the kidneys of infected birds. However, virus-specific genomic RNA was detected in all representative tissues, and virus shedding was detected up to 12 days after inoculation, suggesting that the isolate was able to spread systemically and replicate efficiently in vivo. Collectively, our study demonstrates, for the first time, the etiological role of a genetically distinct astrovirus in the fatal infection of goslings. Astroviridae family. Currently, two genera: namely Mamastrovirus and Avastrovirus are distinguished within this family. The genus Mamastrovirus includes astrovirus species isolated from humans and a number of mammals. Isolates originated from avian species, such as turkey, chickens, ducks, and other birds are classified into the genus Avastrovirus2. AstVs have been detected in humans and a variety of animal species, including non-human primates, other mammals and avian species5. Their genomes are 6.8–7.9 kb in length, consisting of a 5′-untranslated region (UTR), three open reading frames (ORFs), a 3′-UTR and a poly (A) tail6. The high degree of genetic diversity among AstVs and their recombination potential signify their capacity to cause a broad spectrum of diseases in multiple host species8. Human classical AstVs are a frequent cause of acute gastroenteritis in young children and the elderly, occasionally with encephalitis8.Astroviruses (AstVs) are non-enveloped, positive-sense, single-stranded RNA viruses belonging to the 16. Increasing evidence indicates that there is a high degree of cross species transmission of AstVs between domestic birds, and even the potential to infect humans17. By comparison, fewer AstV infection cases have been described in domestic goose flocks. Bidin et al.18 reported the detection of avian nephritis virus infection in Croatian goose flocks and provided evidence that this AstV was associated with stunting and pre-hatching mortality of goose embryos. Studies to detect AstV genomes from the clinical samples of geese suggested that these viruses might distribute widely among goose flocks, as seen in other poultry flocks20. In February 2017, an outbreak of disease was reported in a goose farm in Weifang, Shandong Province, China. Affected flocks (containing 2000–3000 goslings) experienced continuous mortality rates ranging from 20 to 30% during the first 2 weeks of the outbreak despite antibiotic and supportive treatment. We conducted a systematic investigation to identify the causative agent of this disease and report here the isolation and characterization of a genetically distinct avian AstV. The pathogenicity of this virus was evaluated by experimental infection of goslings.In poultry, AstV infections have been found to be associated with multiple diseases, such as poult enteritis mortality syndrome, runting-stunting syndrome of broilers, white chick syndrome, kidney and visceral gout in broilers and fatal hepatitis of ducklings, leading to substantial economic lossesIn the field, affected goslings displayed signs of depression and were observed sitting alone Fig. . The palRdRp) gene21. Sequence and phylogenetic analysis of the amplified RdRp gene with other known AstV sequences retrieved in the GenBank database showed that the detected virus could be assigned to the subgroup 1.2 within the avastrovius group 1, with the closest relationship to the astrovirus detected from dropping samples of northern shovelers (Anas clypeata) in Hong Kong . The subsequent passage of the isolate caused 60–100% mortality of the embryos by 5 dpi. The dead embryos exhibited severe subcutaneous edema and hemorrhages with necrotic foci in the liver  as proposed by Martella et al.23.Virulent bacteria were not isolated and tissue samples were negative by PCR for goose parvovirus, goose hemorrhagic polyomavirus, reovirus, or Tembusu virus. However, a DNA fragment was amplified from the RNA sample extracted from the pooled spleen, liver and kidney tissues using pan-AstV RT-PCR targeting the AstV RNA-dependent RNA polymerase  in length with similar gene organization to other known avastroviruses, consisting of a 5′-UTR of 10 nt, three sequential ORFs , a 3′-UTR of 236 nt and a poly (A) tail stretching 30 nt Fig. . ORF1a o225 to Q225. The potential effect of the mutation on the virus adaptation need to be further evaluated.To determine the potential genetic mutation(s) that might occur during the goose embryo passage, the initial virus genome was sequenced using the total RNA extracted from the clinical case tissue homogenate. Nucleotide differences between the initial virus genome and that of the fourth embryo-passaged isolate was shown in Table The complete genome sequence of AstV SD01 had the highest similarity to those of turkey AstV 2 (TAstV-2) strains, at the level of 61.6–62.4%  during the experimental period. At necropsy, slight to moderately swollen kidneys were noted for the deceased birds Fig. . HistoloInfected goslings exhibited signs of depression from 3 dpi and this symptom persisted for 3–4 days. One bird died at 5 dpi and severe urate deposition, similar to that seen in the field cases, was evident on the surface of the heart, liver, and kidney Fig. . For theWhen the samples were tested by RT-PCR for virus shedding evaluation, the AAstV specific RNA was sequentially detected from the cloacal swabs of infected goslings from 2 to 12 dpi Fig. . Viral ROrally inoculation of goslings with the isolate resulted in depression of 4 birds from 5 to 8 dpi. One gosling died at 7 dpi with evident urate deposition on the surfaces of heart and liver at necropsy. Another severely ill bird was killed humanely at 8 dpi for animal welfare reasons. Viral RNA was detected by RT-PCR in the cloacal swabs from 4 dpi Fig.  and grow19, but the prevalence and pathogenicity of AstV among domestic geese remains poorly understood due to the lack of efficient in vitro culture techniques and diagnostic assays. In this study, AAstV SD01 was successfully isolated by inoculating tissue samples into goose embryos, as confirmed by RNA detection methods similar to those for pan-AstV. We successfully cultured the virus in vitro provided a convenient method for virus propagation and laboratory diagnosis. Isolation of the AAstV from field cases and reproduction of the disease in a gosling infection model with the inoculating virus prepared in embryos fulfils Koch′s postulates.Several studies have reported the existence of AstV in goose flocks10. In this study, the mortality of young goslings caused by subcutaneous or oral inoculation indicated that AAstV SD01 was highly pathogenic. The experiment was fairly a represent of the situation in the field, where susceptible goslings are exposed to AstV soon after they are placed in contaminated houses. Apart from mortality, avian AstV infection can decrease feed intake and alter feed conversion efficiency, leading to growth repression. The decrease in body weight of infected goslings is a major concern as a 29% lighter body weight at 14 dpi has a considerable economic impact.AstV infection occurred within the first days or week of life usually resulted in a worse outcome, as the age-dependent pathogenicity of AAstV has been reported25. Extra-intestinal infection with nephritis has been reported in birds infected with chicken astrovirus and avian nephritis virus26. Viral RNA was found in all of the tissues sampled from the infected goslings killed on 5 dpi, indicating that the goose AstV has a wide tissue tropism and spread systemically after inoculation. Virus shedding was detected by RT-PCR and persisted in the infected goslings for about 12 days, further indicating that the virus replicated efficiently in vivo.Histologic examination revealed the presence of a proteinaceous substance in the renal tubules, indicating that AAstV SD01 infection caused increased permeability of the kidney epithelia barrier. Degeneration and necrosis of tubular epithelial cells found in the deceased goslings provided further evidence of kidney function damage. These results could explain the development of visceral urate deposition in infected goslings. Increased epithelium permeability due to AstV infection has been reported in both human and avian species27. Nonetheless, based on the limited number of goslings infected in present study, it is not likely to get accurate evaluation for the virulence of the isolate.It is interesting that encephalitis lesions were observed in the deceased goslings (data not shown), along with the detection of AAstV SD01 RNA in the brain tissue Fig. . HoweverThe present work describes the isolation of the astrovirus AAstV/Goose/CHN/2017/SD01 from tissue samples of goslings dying from a disease characterized by visceral urate deposition. The successful reproduction of the disease by experimental infection demonstrates the etiological role of this AAstV. Based on the genetic analysis of the complete capsid region at amino acid level, the isolate should be assigned as a member within the genotype consisting of TAstV-2 and DAstV-1 strains. The high variability of the genomic sequence to other known astroviruses suggest more detailed antigenic investigations should be performed.2 for 48 h. Then the spleen, liver, and kidney tissue were pooled and tested for the presence of goose parvovirus28, goose hemorrhagic polyomavirus29, AstV21, reovirus30, and Tembusu virus31, respectively.For bacteriological diagnosis, liver, and kidney samples from dead goslings were first inoculated onto tryptic soy agar plates  containing 2% fetal calf serum, and incubated at 37 °C under an atmosphere with 5% COg, at 4 °C for 10 min. The supernatant was filtered using a syringe-driven filter unit with a pore size of 0.2 μm and the filtrate was inoculated into five 9-day-old goose embryos (0.2 ml/egg) via the chorioallantoic membrane route. Embryos were incubated at 37 °C and candled daily. Embryos that died beyond 24 h and those that survived until 5 day after inoculation were chilled to 4 °C overnight. The allantoic fluids were collected for a hemagglutination (HA) activity test performed by a standard method using 1% chicken red blood cells as an indicator, and then were subjected to additional passage in goose embryos.To isolate AstV, the kidney, spleen, and liver samples were homogenized with sterile phosphate buffered saline  to a 20% suspension (w/v) and centrifuged at 8000×th and 9th passage, the virus suspension was 10-fold serially diluted with PBS and inoculated into 9-day-old goose embryos via the chorioallantoic membrane route. The embryos were incubated for 7 days at 37 °C and the mean embryo lethal dose (ELD50) of infectious virus was calculated using the Reed–Muench method32.To determine the infectious titers of the 4pEASY-Blunt Simple Cloning Vector . The recombinant vector was transformed into competent Escherichia coli Trans-T1 and transformants containing the PCR amplified fragment were selected by PCR following the manufacturer’s instruction. At least two representative transformants were subjected to bidirectional DNA sequencing using Applied Biosystems ABI3730 . The 5′ and 3′ ends of the viral genome were amplified using the 5′/3′ RACE kit  following the guidelines of the manufacturer. The initial complete genome was assembled and manually edited using the Software ContigExpress. Based on the initial genome sequence, additional primer pairs were designed  and the cDNA was synthesized using a Reverse Transcription System  with random primers following the manufacturer’s instructions. Viral genomic fragments were amplified by PCR with primer sets designed against conserved regions of the AstV sequences retrieved from the GenBank database Table . PCR proed Table , and PCRTo evaluate the potential adaptive mutation (s) of the virus that might occur during the process of goose embryo passage, we sequenced the complete genome of initial virus using the total RNA extracted from the clinical case tissue homogenate of kidney, spleen, and liver using the method described above. The genome sequence was compared with that of the isolate of fourth passage.Nucleotide sequences of the virus genome and the deduced amino acids of the ORFs were compared with known AstV ORF sequences retrieved from the GenBank database. Neighbor-joining trees of the complete genome nucleotide sequences, ORF1b and ORF2 amino acid sequences were constructed using MEGA 7.0 software, with bootstrap values calculated from 1000 replicates. The mean amino acid genetic distance (p-dist) of the viral RdRp and capsid were calculated using MEGA 7.0 with a bootstrap test obtained from 100 replicates.Anser anser domesticus) were obtained from a local hatchery. Birds were raised in negative pressured isolators with ad libitum access to feed and water. Three experiments were conducted to investigate the pathogenicity of the isolate. Animal infection experiments were approved by the China Agricultural University Animal Ethics Committee.One-day-old goslings (4 ELD50). Goslings inoculated with sterile PBS (uninfected control) were kept in a separate isolator. Clinical signs and mortality were recorded for 10 days. Dead birds were necropsied immediately, and tissue samples of the heart, liver, spleen, kidney, lung, thymus, bursa, and brain were collected. A portion of these tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and 4–5μm sections were cut and stained with hematoxylin and eosin (H&E). Pieces of liver and kidney tissue were frozen and subjected to virus isolation.The aim of this experiment was to evaluate whether the isolate was pathogenic in goslings. Thirteen 2-day-old goslings were infected by subcutaneous inoculation with 0.5 ml of the virus suspension prepared from the infected goose embryos at the fourth passage , <0.01 (**).This experiment was designed to investigate the in vivo replication of the virus and its impact on the growth of infected birds. Fourteen 5-day-old goslings were infected as in experiment 1. Birds inoculated with sterile PBS were used as the uninfected control. The tissue distribution of the virus was analyzed in three infected goslings 5 dpi. Representative tissues samples were collected and subjected to histopathological examination and virus RNA detection. For virus shedding detection, cloacal swabs were collected from both infected and control birds on 2, 4, 6, 8, 10, 12, and 14 dpi. Swabs were immersed in 1 ml of 1 × Dulbecco’s modified Eagle’s medium  and frozen at −75 °C. Simultaneously, individual birds were weighed. The difference between the mean body weights of infected and uninfected control birds was tested by Student’s n = 6) or intranasally (n = 6) at 5-day old with the astrovirus isolate at the dose as experiment 1. Five goslings were kept as uninfected control. Mortality were checked daily. Cloacal swabs were collected for virus shedding detection as described in experiment 2.To investigate the possible infection route, three groups of 1-day-old goslings were kept at separate isolator. Birds were inoculated orally  in PBS and were homogenized using beads in a high-throughput Tissuelyser . Then, the homogenate was centrifuged at 8000×Table S1Table S2Table S3Figure S1Figure S2Figure S3Figure S4Figure S5