GBA1 gene that encodes lysosomalβ-glucocerebrosidase, resulting in accumulation of glucosylceramide(GlcCer), a key lysosomal sphingolipid. GBA1 mutationsalso enhance the risk for Parkinson’s disease, whose hallmarkis the aggregation of α-synuclein (αSyn). However, therole of accumulated GlcCer in αSyn aggregation is not completelyunderstood. Using various biophysical assays, we demonstrate thatGlcCer self-assembles to form amyloid-like fibrillar aggregates in vitro. The GlcCer assemblies are stable in aqueous mediaof different pH and exhibit a twisted ribbon-like structure. Nearlysosomal pH GlcCer aggregates induced αSyn aggregation andstabilized its nascent oligomers. We found that several bonafide inhibitors of proteinaceous amyloids effectively inhibitedaggregation of GlcCer. This study contributes to the growing evidenceof cross-talk between proteinaceous amyloids and amyloid-like aggregatesof metabolites accumulated in diseases and suggests these aggregatesas therapeutic targets.A major risk factor for Gaucher’sdisease is loss of functionmutations in the  The two most frequent GBA1 mutations associatedwith PD (N370S and L444P) are found in 17–31% of all PD patientsin European Ashkenazi Jewish populations and 3% of non-Ashkenazi populations.26 Aggregation of αSyn into amyloid fibrilsis a hallmark PD.1 However, the underliningcause for the correlation between GD and PD remains to be established.27 It has been proposed, for example, that theaccumulation of the GD-substrates alters the homeostasis of αSynin the lysosomes, that the mutant GCase1 causes saturation of theubiquitin-proteasome pathway, thereby reducing its activity of degradationof αSyn, or that loss of GCase1 function impairs activity andstructure of the mitochondria, a process that can aggravate accumulationof αSyn.28 Recent in vitro and in cell experiments revealed that incubationof αSyn with liposomes containing either GlcCer, GlcSph, sphingosine,or sphingosine-1-phosphate leads to aggregation of αSyn, andthe resulting oligomers were capable of cross-seeding endogenous αSynin cultured human neurons.30Interestingly, GD patients were estimatedto have a 20- to 30-foldincreased risk for developing PD.GBA1 gene and restoring the biosynthetic pathway.31 Replacement of the defective GCase1 enzyme by routine infusionof recombinant normal GCase1 has been very successful, especiallyfor type 1 GD, albeit highly costly. Such enzyme replacement lessensthe visceral, hematological, and sometimes skeletal pathologies ofGD; however, since it cannot cross the blood–brain barrier,it does not affect neurological involvement of GD.31 Pharmacological chaperones to mitigate misfolding of mutantGCase1 are also being developed as an alternative treatment approachfor GD, for example, Ambroxol.32 In anotherrecent therapeutic approach for GD, termed ‘substrate reductiontherapy’, rather than enhancing the deficient enzyme levels,the drugs target an earlier step in the metabolic pathway, leadingto inhibition of glucocerebroside production, the lipid that is notbeing efficiently cleaved by the mutant GCase1.33 However, to the best of our knowledge, no therapeutic strategiescurrently aim at directly targeting the accumulated GlcCer. Recentlysome small molecules, including doxycycline, polyphenols, and quinones,which are effective inhibitors of aggregation of proteinaceous amyloids,were found to be efficacious in inhibiting amyloid-like self-assemblyof certain accumulated metabolites.36Current therapeutic approaches for GD focus primarily on overcomingthe metabolic block caused by the mutations in the in vitro studies, includingThioflavin T (ThT) binding, turbidity assay, electron microscopy,and fluorescence microscopy, we demonstrate that the onset of accumulationof GlcCer, characteristic of GD, proceeds through the formation ofamyloid-like aggregates. X-ray scattering analysis of the GlcCer aggregatesenabled us to propose a plausible model that satisfies their twisted-ribbon-likestructure. We observed an inducing effect of GlcCer self-assemblieson the aggregation of αSyn, suggesting a plausible mechanismlinking GD and PD pathologies. Finally, we demonstrate that severalsmall molecules, including allantoin, rosmarinic acid (RA), epigallocatechingallate (EGCG), and naphthoquinone-tryptophan (NQTrp), known to inhibitaggregation of proteinaceous amyloids, efficiently modulate GlcCeraggregation in vitro.Here, using various Figure S1) powder was dissolvedin 100% DMSO to make a stock solution (100 mM), which we refer toas monomers. It was diluted with PBS (pH 7.4) to make various concentrationsand was immediately used. When GlcCer (100 μM) was incubatedin PBS buffer for 10 h, it formed a fibrillary-like morphology whenviewed under transmission electron microscopy (TEM) and atomic forcemicroscopy (AFM) . The morphologyof the GlcCer aggregates looks very similar to that of bonafide proteinogenic amyloid fibrils.37 Thecritical concentration for fibril formation by GlcCer was furtherevaluated using TEM, and we observed that upon dilution from 50 μMto 100 nM, the fibrillary morphology remained the same and no fibrilswere noticed below 100 nM concentration . To examine whether these fibrils exhibit amyloid-likeproperties, we performed several biophysical assays, which are commonlyused to characterize proteinaceous amyloid structures.39GlcCer (39 GlcCer was incubated at different concentrations(10–200 μM) in PBS at pH 7.4 and 37 °C, and itsaggregation was monitored using a ThT fluorescence assay (S4), very similar to reported amyloidogenic proteins.41 At a higher (200 μM) concentration of GlcCer, the solutionbecame turbid faster than at lower concentrations (42 A sharpblue shift (to ∼485 nm) with enhanced intensity was observedwhen ANS bound to different concentrations of GlcCer, whereas ANSalone (control) exhibited emission at ∼515 nm , very similar to fibrils reportedfor proteinaceous amyloids and amyloid-like assemblies of accumulatedmetabolites.43 Thedensity of the fibrillar network was higher as the concentration ofGlcCer increased . We monitored,using TEM, the formation of GlcCer (100 μM) fibrils at differenttime points immediately after dissolution in PBS buffer from the stocksolution (10 mM in DMSO). GlcCer did not show any fibrillary structurein DMSO (which we considered as monomers), and immediately after dissolutionin PBS (at time 0), small aggregates were noticed and clear fibrilswere evident upon further incubation . These fibrils remained very stable even after incubation for 60days . We also examined thethermal stability of the GlcCer fibrils using TEM and observed thatthe fibrillar morphology remained stable from 25 to 80 °C . Scanning electron microscopy . Using fluorescencemicroscopy, we also observed that the GlcCer assemblies were stainedwith ThT , whereas its amyloid-like characteristics describedabove were recorded at pH 7.4 and 37 °C. Therefore, we examinedthe effect of pH on the formation of amyloid-like fibrils and thestructural arrangement of GlcCer assemblies. To that end, ThT assay,turbidity assay, and TEM analysis of GlcCer aggregation were performedat different pHs (4.5 to 7.4), using four different pH media, 100mM acetate buffer , double-distilled or deionized water, and 100 mM phosphatebuffer (pH 6.5 and 7.4).S10). Further, TEM analysis of GlcCer assemblies at differentconcentrations showed a similar amyloid-like fibrillar network atall tested pHs . These resultssuggest that the self-assembly of GlcCer depends mainly on the aqueousenvironment irrespective of the pH. Therefore, the structural characterizationof GlcCer fibrils was further investigated in DDW (pH ∼ 6).As evident by the ThT and turbidityassays, in DDW, AB, or pH 6.5 PBS, GlcCer aggregated similarly tothat in PBS at pH 7.4 (S12). These parameters resemble the previously reported twisted helicalGlcCer fibrils obtained from GD patients’ brain45 and similar to those observed for invitro generated galactosylceramide  fibrils.47A closer examination of the amyloid-like fibrilsof GlcCer underTEM revealed a twisted-ribbon-like structure 2i–l. Cal49 and has been extensively used to understand the molecular interactionbetween lipids, including sphingolipids, within bilayers in aqueousmedia.48 FTIR analysis revealed that themonomeric GlcCer exhibited two sharp peaks at 2850 and 2919 cm–1, indicating characteristic symmetric and asymmetricstretching vibration of the ceramide CH2 groups or with the environmental H2O , powderX-ray diffraction (PXRD), and small- and wide-angle X-ray scattering(SAXS and WAXS) analyses. FTIR spectroscopy provides information onall functional groups present in a molecule2 groups 4a.48 Twof GlcCer 4a. In contal H2O 4b.51 PXRD of the GlcCer monomers exhibited mostly a noisy spectrum  of 0.11 Å–1, correspondingto a lamellar repeat distance (d1), of 5.5 nm  observed in SAXS analysis,which were also evident in PXRD, was further indicative of the presenceof lamellar stacking. The wide-angle region (WAXS) showed differentharmonics (marked with red arrows) and two strong reflections pointingtoward real-space spacing of ∼0.44 nm (d2) and ∼0.41nm (d3)  4f,chara47 In addition, we observed weaker correlation peaks in our scatteringdata with monomeric αSyn (10 μM) in PBS at pH 7.4, and thekinetics of their coaggregation was monitored for 60 h by ThT assay,after which the samples were analyzed by TEM. Untreated monomericαSyn and GlcCer only served as controls. ThT fluorescence ofuntreated αSyn increased gradually with a lag phase of ∼22h and reached a plateau in 50 h  was first aggregated for 10 h toform fibrils as described above 1a,d. Thein vitro at a low pH of 4.5, near the lysosomal condition.At pH 4.5 (acetate buffer), αSyn (10 μM) aggregated muchfaster, with a shorter lag phase of ∼5 h, than at physiologicalpH (lag phase ∼25 h), in accord with previous studies.57 In the presence of different doses of GlcCer PFFs (10–50%v/v), the rate of αSyn aggregation was reduced, with an increasedlag phase of 13–27 h  PFFs of GlcCer PFFs was significantly higher  than in their absence (100%), consideringthe end-point ThT fluorescence of untreated αSyn as 100% . These results suggest that the GlcCerPFFs induce αSyn fibril formation. The formation of fibrilsat the end point of the ThT assay, analyzed by TEM .GlcCeraccumulates in the lysosome whose milieu is acidic. We nextexamined the effect of the GlcCer PFFs on αSyn aggregation  13–27 h 5d; howev 13–27 h 5d at ide as 100% S13. ThesFigure S15). As controls, we also treated cells with 50% (v/v) PFFs of GlcCeronly and with untreated αSyn fibrils. The viability of controluntreated cells was set as 100%. In this XTT assay, we observed thatthe untreated αSyn fibrils reduced viability to 70 ± 3%,whereas the PFFs of GlcCer only exhibited 77 ± 5% of cell viability.The αSyn aggregates formed upon incubation in the presence of50% (v/v) PFFs, for 2 h, 5 h, 15 h, 25 h, 30 h, and 60 h caused cellviabilities of 68 ± 2%, 37 ± 3%, 34 ± 5%, 43 ±4%, 54 ± 3%, and 59 ± 3%, respectively. The viability ofcells incubated with αSyn aggregates that formed in the presenceof 50% (v/v) GlcCer PFFs was much lower than that of cells incubatedwith αSyn aggregates that formed in the absence of GlcCer PFFs,indicating that the former were more toxic than the latter. Interestingly,αSyn incubated with GlcCer PFFs for 2–25 h showed a muchhigher toxicity (evident in lower cell viability) in comparison tothe samples incubated for 30–60 h as well as untreated αSynfibrils. Since the concentration of PFFs was same in all of the treatedsamples, the contribution of cytotoxicity by these GlcCer PFFs themselveswould be same. Therefore, the higher cytotoxicity observed for the2–25 h incubated samples is likely due to the increased relativeconcentration of αSyn oligomers vs mature fibrils,which was decreased upon further incubation (30–60 h) concomitantlywith the increase in the relative concentration of mature αSynfibrils. Indeed, the samples incubated with PFFs for 30–60h contained mainly mature αSyn fibrils  GlcCer PFFs, we incubated αSynwith GlcCer PFFs at pH 4.5 and 37 °C for 60 h. At different timepoints, samples were collected and applied to SH-SY5Y cells for 12h, after which cell viability was compared to that of cells incubatedwith untreated αSyn fibrils S15. As c58 Upon 5 h of coincubation, αSyn displayedtwo negative CD peaks at ∼200 nm and ∼ 220 nm and apositive peak at ∼195 nm, reflecting a mixture of β-sheetand random coil conformations, as previously reported.58 Upon further incubation up to 15 h or longer,the control sample presented complete β-sheet conformation asonly one negative maximum was observed at ∼220 nm and a positivepeak at ∼195 nm, suggesting fibril formation by αSyn.In the presence of GlcCer PFFs (50% v/v), at time 0, αSyn exhibiteda negative peak at 200 nm, suggesting random coil conformation andpossibly the presence of monomeric protein conformation .To gain insight into the natureof the species formed upon incubationof αSyn (10 μM) with GlcCer PFFs (50% v/v) at near lysosomalpH (4.5), we employed circular dichroism (CD) spectroscopy and dynamiclight scattering (DLS) at various time points during the 0–25h of coincubation. At time 0, the control sample of αSyn aloneexhibited a negative CD peak at 200 nm, suggesting random coil conformation6a.58 Upoormation 6b. Upon g,and the soluble portion of the reaction mixtures was examined. Thesample of untreated αSyn at 2 h of incubation exhibited a hydrodynamicdiameter of ∼8 nm. With incubation time, the average diameterwas increased to ∼15 nm, ∼55 nm, ∼110 nm, ∼320nm, ∼720 nm, and ∼540 nm at 5 h, 10 h, 15 h, 25 h, 30h, and 60 h, respectively .In contrast, the hydrodynamic diameter of the sample of αSyncoincubated with 50% (v/v) GlcCer PFFs was smaller ∼7 nm, ∼35nm, 40 nm, 45 nm, 122 nm, 255 nm, and 905 nm at 2 h, 5 h, 10 h, 15h, 25 h, 30 h, and 60 h, respectively .For DLS analysis, samples werecollected from the CD experimentat different time points, centrifuged at 50,000ectively S7c.In cectively S7c.In cFigure S17). In the absence of GlcCer PFFs, theαSyn aggregates quickly as evident from the rapid change ininitial slope of ACF, which reflects the presence of small size oligomers,61 from 2 h to 5 h and the slope of the ACF remainsstable after 10 h . Likewise,the ACF curves at 10–60 h exhibited higher values in the timedomain at the midpoint of the transition, indicating larger aggregatescompared to the values of 2 h and 5 h samples. These results indicatethe short life (5 h) of soluble oligomers and rapid formation of largeaggregates (fibrils) by the untreated αSyn. In contrast, αSynsamples treated with GlcCer PFFs were found to aggregate slowly, asthe initial slope of the ACF curve changes gradually from 2 h to 25h and thereafter remains stable . In addition, the ACF curves from 30 h to 60 h exhibited highervalues in the time domain at the midpoint of the transition than thecurves at 2–25 h, suggesting slower formation of larger aggregates(fibrils) by αSyn in the presence of GlcCer PFFs. These resultssuggest that GlcCer PFFs stabilize the oligomers of αSyn. Collectively,all of these in vitro results suggest that the preformedGlcCer fibrils promoted aggregation of αSyn.The size distribution results obtainedfrom DLS were supportedby the ACFs of the scattered light, extracted from DLS analysis , which were reported as effectiveinhibitors of aggregation of various amyloidogenic proteins,63 were examined for their effect on self-assembly of GlcCer at pH4.5 .Accumulation of GlcCer in the lysosome is a hallmarkof GD, and we observed that accumulated GlcCer forms amyloid-likefibrils The kinetics of GlcCer aggregationin the absence or presence of different molar ratios of the inhibitors were monitored by ThT bindingassay 7a–e.In S18). Thedensity of the fibrils declined in a dose-dependent manner in thepresence of EGCG or NQTrp, but not when incubated with RA or allantoin,in agreement with the ThT and ANS results. These observations indicatethat certain bona fide inhibitors of aggregationof amyloidogenic proteins can mitigate the formation of amyloid-likeaggregates of GlcCer.To corroborate the ThT and ANS results, TEM analysis was performedon the samples at the end of the kinetics experiments. Control GlcCeralone could form a densely fibrillar network . We observed that neither of thetested doses (1-fold or 5-fold molar excess) can reduce the ThT signal,suggesting the inefficiency of the tested inhibitors to disaggregatethe PFFs of GlcCer even at higher dose (5-fold molar excess). TEManalysis  was performed tosupport the ThT results. Collectively, these results indicate thatthe candidate inhibitor molecules were unable to disaggregate thePFFs of GlcCer.We further examined the effect of thesetested inhibitors to disaggregatethe PFFs of GlcCer in pH 4.5 at 37 °C. GlcCer alone was firstallowed to aggregate for 10 h to form fibrils, then different dosesof the inhibitors (GlcCer:inhibitors = 1:1 and 1:5) were added tothe PFFs and incubated for additional 10 h. Thereafter, the ThT fluorescencesignal was recorded and the % amyloid was calculated, consideringthe ThT fluorescence of untreated GlcCer PFFs as 100% . In contrast, the change in ThT fluorescencewith time was much lower in the solution of Cer, which is almost comparableto the background ThT fluorescence . The end-point ThT fluorescence intensity was calculated, and wenoticed that the intensity increased up to 10 times in the GlcCersolution in comparison to the control ThT (background fluorescence). Whereas the ThT fluorescenceintensity of Cer showed almost equal or 1.5 times increased fluorescencethan the background fluorescence . These results indicate higher binding affinity and aggregate formationby GlcCer than Cer.To verify whether the ability to form amyloid-liketwisted fibrilsis exclusive to GlcCer or any lipid can exhibit such property, weseparately incubated GlcCer and ceramide (Cer) in AB pH 4.5 at 37°C for 10 h and monitored their aggregation propensity usingThT kinetics, ANS binding, and TEM study. We observed that the ThTintensity increased rapidly with time in GlcCer solutions in all thetested concentrations (10–200 μM) . In contrast, Cer did not show any blue shift, yet exhibited aslight increase in intensity. These results indicate that GlcCer stronglybinds with ANS dye than Cer. We also examined the morphology of theselipid assemblies using TEM, and we observed a clear fibrillar speciesin GlcCer sample, but no such fibrillar structure was noticed in Cersamples . Collectively, theseresults indicate that Cer did not aggregate and form fibrils, butGlcCer aggregates and forms amyloid-like fibrils.ANS binding assay was further performed,and it was observed thatthe samples containing GlcCer exhibited a blue shift with higher intensityin comparison to the ANS alone  of ∼184 nm. Thesestructural values resemble the previously reported twisted helicalGlcCer fibrils obtained from GD patients’ brains .45 Thesestructural parameters of GlcCer are also very similar to those reportedfor in vitro generated GalCer fibrils, where 20:1Δ11GalCer aggregates formed helical ribbons that havea right-handed twist with an average width of 30–35 nm andhelical pitches of 80–90 nm.64Our results indicate that molecules of free GlcCer self-assemblespontaneously 66 Such ribbon-likestructures not only have been shown for synthetic lipids but havealso been described for various biological lipids that form bilayertubules, notably cerebrosides such as GlcCer and GalCer.47Our combined FTIR and X-ray scattering resultsindicate that thefree GlcCer spontaneously forms a lamellar phase in which the long-chainnonpolar tails of GlcCer monomers interact with each other to organizepairwise in a tail-to-tail configuration, and the polar glycan headgroups remain exposed to the aqueous medium. The AFM analysis showedthat the average width of the GlcCer fibrils was 29 ± 8 nm. InSAXS analysis, the lamellar repeat distance was observed as 5.5 nm,indicating that the GlcCer forms a typical lipid bilayer arrangementwith 4–6 lipid bilayers stacked together and extended towardthe ribbon. It appears that the chirality in GlcCer does not allowits monomers to stabilize and pack parallel to their nearest neighbors,but rather at a nonzero twist angle to them, leading the entire lamellarphase to twist into a helical ribbon.et al. have observed that in vitro incubation of a mixture of two different lipids,purified GlcCerand brain phosphatidylcholines (PCs), at PC:GlcCer 9:1 to 1:3 molarratios, under acidic conditions influenced the aggregation of αSyn.30 However, as the proportion of GlcCer was increased(from 25 to 75%), while keeping the total lipid amount constant , the kinetic profile of αSyn fibril formationwas altered by delaying the formation of insoluble ThT-positive αSynfibrils, extending the lag time from 2 to 16 h. Along these lines,our results indicate that at physiological conditions (pH 7.4), GlcCerassemblies induced αSyn fibril formation with a comparable delayof 4–19 h. However, at acidic conditions (pH 4.5), the delayin the aggregation process was more profound, 13–27h. SEC andSDS-PAGE analyses by Mazulli et al. indicate thatduring the lag phase (between 1 and 5 h), the induced αSyn solubleoligomeric species appeared to become more abundant. In contrast,in the control samples (αSyn only), the proportion of oligomersand monomers was reduced, consistent with their recruitment into insolublefibrils. They suggest that GlcCer has stabilized the formation ofa soluble assembly competent species during the observed lag phase.We observed a similar effect by the GlcCer amyloid-like aggregates.Using immune-EM with syn505 antibody that preferentially detects misfoldedαSyn, they detected αSyn species colocalized with GlcCertubular structures, suggesting a direct interaction between GlcCerand αSyn that facilitates its fibril formation.Mazulli in vivo. GSL accumulationin human induced pluripotent stem neurons promotedαSyn aggregation into toxic conformations.29 Immunofluorescence and immuno-EM showed that GlcCer andαSyn colocalized in these cells, suggesting that a direct interactionmay facilitate αSyn conformational conversion inside neurons.Toxic aggregates of αSyn could be produced in these cells inthe absence of GBA1 mutations, yet with reduced GCase1activity and accumulation of GSL, suggesting that GSLs alone can induceαSyn pathology. GSLs could induce structural conversions ofαSyn also in a mouse model.29Similarresults were subsequently reported The free GlcCer aggregates used in the present study, similar tothe previously reported lipid mixture, possibly stabilized the oligomerizationof αSyn and reduced the process of secondary nucleation, whichmight delay the onset of its aggregation. Subsequently, the solubleoligomeric species started assembling for maturation, leading to theformation of higher-ordered aggregates and exhibited a stronger ThTsignal than the signal observed when αSyn was alone. These findingscan provide a mechanistic explanation for the apparent prevalenceof PD among GD patients.63 The observation that, among the tested molecules, EGCG and NQTrpinhibited amyloid-like aggregation of GlcCer should be helpful forcomparative studies in GD and GD-PD in vivo. We speculatethat the hydroxyl groups of the glycan head of GlcCer may form hydrogenbonds with the tested inhibitors more strongly than its self-assemblyprocess, thus leading to the inhibition of GlcCer aggregation. Inaddition, the aromatic ring of the tested molecules, for example,NQTrp or EGCG, may strongly interact with the cluster of carbon atomsof the hexose ring through the energetically favorable CH−πstacking.68Certain small molecules, includingEGCG, RA, NQTrp, and allantoin,effectively inhibited the aggregation of proteinaceous amyloids  and ameliorated pathology and symptomsof the related diseases in animal models.in vitro and increased the stabilizationof the oligomers known to be a major cause of PD pathogenesis. Finally,we showed that certain inhibitors of protein aggregation could inhibitalso the aggregation of GlcCer. Our findings provide a mechanisticinsight of GlcCer aggregation and its linkage with αSyn pathology,suggesting a target for therapy and drug development toward GD andPD.In conclusion, the present study describesthe characterizationof amyloid-like aggregates of GlcCer, which exhibit a twisted ribbon-likestructure. The GlcCer aggregates were found to delay the aggregationof αSyn MW 726.08; catalog no. 860548P) and ceramide); MW 563.94; catalogno. 860519P) were purchased from Avanti Polar lipids, USA. α-Synucleinwas purchased from rPeptide, USA . Molecularbiology grade ultrapure water and all the solvents and reagents usedfor the XTT assay were purchased from Biological Industries (Israel)and Biological Industries, Israel. All other chemicals were purchasedfrom Sigma-Aldrich .Allof the chemicals used were of the highestpurity grade. Black and transparent, flat-bottomed polystyrene nonbinding96-well plates were purchased from Corning, USA. Glucosylceramide ceramide (d18:1/18:1(9Z)); 280 of 5960 M–1 cm–1) and adjusted to 50 μM concentration as a stock solution.Stock solutions of Thioflavin T  were prepared in DDW.Stock solutions of the tested inhibitor molecules (10 mM) were preparedseparately in DMSO and diluted with PBS/AB before use. The stock solutionsof the GlcCer and protein were used immediately after preparation,and for each assay, a fresh stock was prepared separately.GlcCer powder was dissolved in 100%DMSO to prepare 100 mM stock which was further diluted to desiredconcentrations with different buffer solutions  of pH 6.5 or 7.4, acetate buffer (AB) of pH 4.5, and doubledisstilled or deionized water (DDW)) as required. Commercially availableαSyn was monomerized by a 10 min pretreatment with HFIP, andthe solvent was evaporated using a Speed Vac. The resulted thin filmwas dissolved in PBS/AB and sonicated for 5 min. The strength of theworking buffer system for all assays was 100 mM. Concentration ofthe protein was determined using Nano drop . For the aggregation kinetic of αSyn,the final concentration was used as 10 μM from the stock of50 μM in 100 mM PBS (or in AB). For the αSyn aggregationkinetics, continuous agitation was used. For the inhibition of GlcCeraggregation, the tested inhibitor molecules  were added separately to the designated wells, and kineticsof aggregation was monitored by ThT fluorescence at 37 °C. Tomonitor the effect of GlcCer PFFs on αSyn aggregation, GlcCerwas first allowed to aggregate alone for 10 h to obtain PFFs, andthen different doses of the preaggregated GlcCer fibrils were addedto the designated well and coincubated with αSyn for 60–70h. The data were collected (in triplicates) using Infinite M200 microplatereader , with measurements taken at 15 min intervals.Excitation and emission wavelengths of ThT were 440 and 485 nm, respectively.The plate was incubated at 37 °C. Measurements were performedas independent triplicates. Three recorded values were averaged, andbackground measurements for buffer containing only ThT were subtracted.ThT fluorescence assaywas carried out in a similar manner as described previously to monitorthe protein aggregation.T0)) was subtracted in order to eliminate background absorbanceof the samples.GlcCer was dissolved in 100% DMSO toprepare a stock concentration of 100 mM. The stock GlcCer was dilutedto a desired concentration with PBS (or AB) and incubated at 37 °C.For the turbidity measurement, we examined the absorbance spectraat 350 nm, using an Infinite M200 microplate reader and polypropylene 96-well plates (Corning) in 100 μL volume.Plates were shaken gently for 5 s prior to each measurement cycleand incubated at 37 °C. All optical densities were measured at350 nm wavelength, and their first measurement  in the desired buffer (PBSor acetate buffer). ANS fluorescence intensities were measured withexcitation at 380 nm and emission between 400 and 700 nm using InfiniteM200 microplate fluorescence reader .Samples (10 μL)were placed for 2 min over 400 mesh copper grids covered with carbon-stabilizedFormvar film . Excessfluid was removed, and the grids were negatively stained with 2% uranylacetate solution (10 μL) for 2 min. Then, the excess fluid wasremoved and allowed to dry for 5 min. The samples were viewed usinga JEM-1400 TEM (JEOL), operated at 80 kV.Samples (10 μL)were placed on glass slides and left to air-dry under ambient conditionsfor 12 h. Samples were then coated with Au for conductance and viewedusing a benchtop scanning electron microscope operating at 20 kV.AFM imagingwas performed bydepositing 10 μL solutions onto freshly cleaved V1 grade mica. The samples were allowed to dry underambient conditions for 12 h. The samples were imaged using AFM (JPKInstruments AG) performed with Nano Wizard 3 with 5 N/m spring constanttips and a resonance frequency of ∼150 kHz in soft tappingmode. AFM analysis was performed on different areas for each sample,and the GlcCer fibrils parameters  weremeasured using the Gwyddoin 2.56 software.Congo red powder was dissolvedin 80% aqueous ethanol to prepare a saturated stock solution. Thesolution of aggregated GlcCer (5 μL) was mixed with 5 μLof saturated Congo red solution and incubated for 10 min. The suspension(10 μL) was then drop casted over a glass side, and the sampleswere dried in air and kept in a desiccator before birefringence analysis.The samples were viewed at 20× magnification with a Nikon EclipseTI polarizing microscope. Digitized images were obtained using a NikonDS Ri1 digital camera.Aggregate GlcCersamples (10 μL), obtained from turbidity assay, were mixed with10 μL of ThT dye (20 μM), and the mixture was drop castedover glass slide followed by drying under ambient conditions for 12h. Then the dried samples were viewed under fluorescence microscope(Nikon Eclipse TI polarizing microscope) using a GFP filter.5 cells/mL) was cultured in 96-welltissue microplates (100 μL/well) and allowed to adhere overnightat 37 °C. The stock of GlcCer (in DMSO) was dissolved in DMEM:Nutrientmixture F12 (Ham’s) (1:1)  atdifferent concentrations (1–200 μM) for 6 h to generateGlcCer assemblies (based on ThT results). 1% DMSO was used as thevehicle. The negative control was prepared as a medium without GlcCeror DMSO and treated in the same manner. 100 μL of medium withor without GlcCer aggregates was added to each well. Following incubationfor 24 h at 37 °C, cell viability was evaluated using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT) cell proliferation assay kit according to the manufacturer’s instructions. Briefly, 100μL of the activation reagent was added to 5 mL of the XTT reagent,followed by the addition of 50 μL of activated-XTT solutionto each well. After 2 h of incubation at 37 °C, color intensitywas measured using an ELISA microplate reader at 450 and 630 nm. Resultsare presented as mean and the standard error of the mean. Each experimentwas repeated at least three times.The SH-SY5Y human neuroblastomacell line (2 × 10–1 resolution.FTIR spectroscopywas performed with 30 μL of GlcCer samples (monomers or aggregates)of 200 μM concentration. Monomers were obtained by dissolvingGlcCer powder in chloroform/methanol (2:1) solvent. Aggregates ofGlcCer were prepared in the same manner as described above for theturbidity assay. The samples were deposited onto disposable KBr IRsample cards (Sigma-Aldrich), which were then allowed to dry undervacuum. Transmission infrared spectra were collected using a Nexus470 FTIR spectrometer (Nicolet) with a deuterated triglycine sulfatedetector. Measurements were performed using the atmospheric suppressionmode by averaging 64 scans in 2 cmα radiation source (wavelengthof 1.54 Å) on a BrukerD8Discover X-ray diffractometer equippedwith a one-dimensional LynxEye detector based on compound siliconstrip technology.Powder GlcCer samples (monomersand aggregates) were poured inside a glass capillary of 0.5 mm indiameter. Monomers were obtained by dissolving GlcCer powder in chloroform/methanol(2:1) solvent and evaporated under argon to get solid powder. Theaggregated samples were prepared by incubating 200 μM of GlcCerin DDW for 12 h, and the assembled fibers were lyophilized to obtainfluffy powder. XRD was collected in symmetric Bragg–Brentanogeometry with Cu Kα radiation source (wavelengthof 1.54 Å) and a scatterless slits setup.70 Two-dimensional scattering data with a wave vector amplitude(Q) range of 0.06–2 Å–1 at a sample-to-detector distance of about 230 mm were collectedon a Pilatus 300 K detector (Dectris), and radially integrated usingMATLAB (MathWorks) based procedures (SAXSi). Background scatteringdata were collected from buffer solution alone. The background-subtractedscattering correlation peaks were fitted using a Gaussian with a linearlysloped baseline. For each sample, time-resolved correlation peaksposition, intensity, and width were extracted.The aggregatedGlcCer samples at 30 mg/mL concentration were measured in 1.5 mm diametersealed quartz capillaries. WAXS/SAXS measurements were performed usingan in-house solution X-ray scattering system, with a GeniX (Xenocs)low divergence Cu KRecombinant αSyn  was dissolved in PBS (pH 7.4) or acerate buffer (pH 4.5) to aconcentration of 50 μM. The monomeric protein was mixed withthe either nonaggregated GlcCer (100 μM and 200 μM) orpreformed GlcCer assemblies , takenfrom 500 μM stock solution, to a final concentration of 10 μMαSyn. The samples were then incubated at 37 °C, and thekinetics of αSyn aggregation in the absence or presence of differentGlcCer samples was monitored by ThT and TEM following a similar processas described above.To analyze the secondarystructure of the αSyn aggreagtes in the absence or presenceof different doses of preaggregated GlcCer, 300 μL of the sampleswere taken in a cuvette (path length 1 mm), and CD spectra were thenrecorded on a Chirascan spectrometer between the range of 190 and260 nm, and the background was subtracted from the CD spectra. SinceDMSO absorbs at far UV range, stocks of GlcCer was prepared in methanolfor this assay.72 measurements were performed with the different αSyn samples(in the absence or presence or GlcCer assemblies) on a Malvern nanozetasizer  using a laser source with λ = 633 nmand a detector at a scattering angle of θ = 173°. Priorto DLS measurement, different αSyn samples, with or withoutGlcCer assemblies, were ultracentrifuged for 30 min  at 50,000g to removethe larger αSyn aggregates and GlcCer assemblies. The supernatantsolution was further ultracentrifuged two more times to completelyremove the larger aggregates, and the final supernatant samples containingαSyn soluble aggregates were used for DLS measurement. Priorto the analysis, samples were filtered through a 0.2 μm PVDFmembrane. The samples were placed in a disposable cuvette and heldat the corresponding temperature during the analysis. For each sample,the analyses were recorded three times with 11 subruns using the multimodalmode. The Z-average diameter was calculated fromthe correlation function using Malvern technology software. Representativeautocorrelation functions (ACFs) were directly adapted from the DLSdata obtained for each sample.DLS


Insulin resistance contributes to several disorders including type 2 diabetes and cardiovascular diseases. Carpachromene is a natural active compound that inhibits α-glucosidase enzyme. The aim of the present study is to investigate the potential activity of carpachromene on glucose consumption, metabolism and insulin signalling in a HepG2 cells insulin resistant model. A HepG2 insulin resistant cell model (HepG2/IRM) was established. Cell viability assay of HepG2/IRM cells was performed after carpachromene/metformin treatment. Glucose concentration and glycogen content were determined. Western blot analysis of insulin receptor, IRS1, IRS2, PI3k, Akt, GSK3, FoxO1 proteins after carpachromene treatment was performed. Phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase (HK) enzymes activity was also estimated. Viability of HepG2/IRM cells was over 90% after carpachromene treatment at concentrations 6.3, 10, and 20 µg/mL. Treatment of HepG2/IRM cells with carpachromene decreased glucose concentration in a concentration- and time-dependant manner. In addition, carpachromene increased glycogen content of HepG2/IRM cells. Moreover, carpachromene treatment of HepG2/IRM cells significantly increased the expression of phosphorylated/total ratios of IR, IRS1, PI3K, Akt, GSK3, and FoxO1 proteins. Furthermore, PEPCK enzyme activity was significantly decreased, and HK enzyme activity was significantly increased after carpachromene treatment. The present study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular basis. It increased the expression ratio of insulin receptor and IRS1 which further phosphorylated/activated PI3K/Akt pathway and phosphorylated/inhibited GSK3 and FoxO1 proteins. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway. Insulin resistance is a condition that arises from the defect in insulin-mediated actions on glucose and lipid metabolism, which occurs mainly in liver, muscles, and adipose tissues. This contributes to numerous human diseases including type 2 diabetes mellitus and cardiovascular diseases ,2. The cApproximately 30% of patients taking metformin develop nausea and bloating with abdominal pain, and 5–10% of patients are unable to continue on the medication . Thus, sFicus benghalensis  . After HepG2/IRM cells were obtained, 10,000 cells were seeded per well in triplicates in 96-well plates. Then, the cells were treated with different concentrations  of carpachromene or metformin. After incubation for 48 h, 10 µL of MTT (5 µg/mL) was added per well and incubated in the dark for 3 h at 37 °C. To dissolve the Formazan crystals that were formed, 100 µL of DMSO were used and absorbance was measured using an ELISA reader  µg/mL for different durations. Metformin was used as a positive control. Glucose concentration was assayed in 10 µL of the medium after 12, 24, 36, and 48 h of treatment using glucose assay kit  according to the manufacturer’s instructions .5 cells/well. Cells were treated with 20 µg/mL carpachromene for 24 h. Metformin was used as a positive control. Then, cells were washed three times with PBS. Glycogen assay kit  was utilized according to the manufacturer’s instructions. Microplate reader  was used to measure absorbance at 570 nm. Additionally, the protein content of HepG2/IRM cells was quantified by Bradford method [To examine the effect of carpachromene on intracellular glycogen, HepG2/IRM cells were seeded into a 6-well plate at a density of 3 × 10d method , and theThe present study investigated the protein expression levels of phosphorylated insulin receptor (ph-IR) , total insulin receptor (IR) #ab137747, Abcam), phosphorylated IRS1 , total IRS1 , phosphorylated IRS2 , total IRS2 (RS2) , phosphorylated PI3K (ph-PI3K) , total PI3K (#ab154598), phosphorylated Akt , total Akt (Akt) , phosphorylated GSK3 , total GSK3 , phosphorylated FoxO1 , and total FoxO1 . Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed. HepG2/IRM cells were treated with 20 µg/mL of carpachromene for 24 h. After washing with PBS, the protein extraction was performed in RIPA lysis buffer, containing 50 mM Tris–Cl, pH 7.5; 0.1% SDS, 150 mM NaCl, 0.5% Sodium deoxycholate, 1 mM PMSF, and 1% Nonidet P-40, supplemented with the complete protease inhibitor cocktail . The Bradford method was used to determine the protein concentration , Abcam, . Accordi5 cells per well. Cells were treated with 20 µg/mL carpachromene for 24 h. Metformin was used as a positive control. Then, cells were washed three times with PBS. PEPCK assay kit  and Hexokinase activity assay kit  were utilized according to the manufacturer’s instructions. A standard curve was obtained, and the absorbance was measured at 570 nm and 450 nm for PEPCK and HK, respectively, using an ELISA plate reader. The activities of PEPCK and HK were calculated from the standard curves. The experiments were performed in triplicates. To examine the effect of carpachromene on PEPCK and HK hepatic enzymes activity, HepG2/IRM cells were seeded into a 6-well plate at a density of 3 × 10At least three independent experiments were used to obtain the results. Data were expressed as mean ± standard deviation. One or two-way analysis of variance (ANOVA) followed by post hoc Dunnett test were used to analyse the differences of multiple comparison using GraphPad Prism 9 statistical software (GraphPad) and Excel software . Differences were considered significant when the probability values (P) were less than 0.05.This study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular level. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway.


Triticum aestivum). Auxin plays an important role in the regulation of branch production. PIN-FORMED 1 (PIN1), an auxin efflux carrier, plays a role in the regulation of tiller number in rice (Oryza sativa); however, little is known on the roles of PIN1 in wheat.Tiller number is a factor determining panicle number and grain yield in wheat  transgenic wheat plants, indicating that auxin might mediate the axillary bud production. By contrast, the spikelet number, grain number per panicle, and the 1000-grain weight were decreased in the TaPIN1-RNAi transgenic wheat plants compared with those in the wild type. In summary, a reduction of TaPIN1s expression increased the tiller number and grain yield per plant of wheat.Nine homologs of TaPIN1 genes increased the tiller number of transgenic wheat lines. Our study suggests that TaPIN1s is required for the regulation of grain yield in wheat.Phylogenetic analysis and protein structure of nine TaPIN1 proteins were analyzed, and subcellular localization of TaPIN1-6a was located in the plasma membrane. Knock-down expression of  Triticum aestivum) is an important agronomic trait that contributes to grain production. Plant hormones, environmental signals, and genetic factors are involved in the regulation of tiller number [Pisum sativum) [Arabidopsis [Tiller number of wheat (r number –4. Auxinsativum)  and Arabbidopsis , 7.Arabidopsis, eight PIN proteins are identified, which possess two conserved domains formed by transmembrane helices and a conserved central hydrophilic loop [PIN protein is specific to auxin transport and is a limiting factor of auxin polar transport –10. In Alic loop .Oryza sativa), maize (Zea mays), and switchgrass (Panicum virgatum) [ZmPIN genes and two PIN-like genes in maize [OsPIN genes in rice had been identified [OsPIN1 gene expression display an increase in tiller number and till angle, which provides a new insight into the functions of the PIN1 family in rice [bif2 maize mutant, ZmPIN1a and ZmPIN1b protein expression patterns and localizations are altered in the tassel and ear [bif2 mutants form a needle-like inflorescence structure and lack branches, spikelet pairs, and florets in male tassels and female ears, which is similar to the phenotype of pin1 mutant in Arabidopsis [The initiation and growth of lateral branches  are important factors in determining plant architecture and yield –14. Previrgatum) –17. Auxiirgatum) , 19. Twein maize  and twelentified . Transge in rice . Maize B in rice . In seve and ear . The bifbidopsis .PIN1 genes and their functions in wheat are rarely reported. In this study, we identified nine TaPIN1 genes in wheat and analyzed their expression patterns. Furthermore, we found that down-expression of TaPIN1s in wheat resulted in more tillers and grain yield of each plant. Our results suggest that TaPIN1s play important roles in the regulation of tiller number and grain yield in wheat.Wheat is an important food crop; however, EnsemblPlants database to identify the homologs of AtPIN1 gene in wheat. Sequence alignment revealed that wheat A, B, and D subgenomes have several homologs, including TaPIN1-6a, TaPIN1-6b1, TaPIN1-6b2, TaPIN1-6b3, TaPIN1-6b4, TaPIN1-6d, TaPIN1-7a, TaPIN1-7b, and TaPIN1-7d. Among these sequences, chromosome 6A has one copy and it was located in 543,394,469–543,397,613, chromosome 6B four copies and they were located in 593,711,770–593,714,658, chromosome 6D one copy which was located in 397,082,345–397,084,736, chromosome 7A one copy located in 148,414,887–148,418,041, chromosome 7B one copy located in 109,702,167–109,705,332, and chromosome 7D one copy located in 146,862,702–146,865,831. The sequences encoded putative products of 588, 521, 589, 595, 587, 589, 587, 586, and 586 amino acids, respectively.We used AtPIN1 protein sequence as query to blastp against wheat Triticum dicoccoides, Aegilops tauschii, and Hordeum vulgare, whereas the TaPIN1-7 proteins were closely related to the OsPIN1a and OsPIN1c proteins  analysis, TaPIN1 proteins harbor the transmembrane helices predicted as the trait of auxin transport carrier candidates  was constructed  by Agrobacteria-mediated genetic transformation to identify the function of TaPIN1 genes in wheat development.ted Fig. a and traTaPIN1-RNAi-positive plants were obtained by polymerase chain reaction (PCR)-based identification. Bar (selective marker gene) and TaPIN1 genes were confirmed simultaneously in transgenic lines  showed that the expression level of TaPIN1 genes in axillary bud after gemination 20 days was significantly decreased in L1, L2, and L3 compared with that in the wild type, particularly the expression level of TaPIN1-6a  were sown in field to observe the agronomic characters. Tiller number was analyzed at three developmental stages, that is, tillering stage Fig. a, jointiTaPIN1s increased the productive tiller number and grain yield per plant of wheat.The agronomic traits of the L1, L2, and L3 transgenic lines, such as plant height, productive tiller number, spikelet number per panicle, grain number per panicle, 1000-grain weight, and grain yield per plant, were evaluated after harvest. The plant height of the L1, L2, and L3 transgenic lines was decreased slightly compared with that of the wild type Fig. a. The prPIN1 gene family had four members, whereas the wheat PIN1 family had nine members. Phylogenetic analysis suggested that six TaPIN1-6, TdPIN1, AePIN1, and HvPIN1 proteins and three TaPIN1-7, OsPIN1a, and OsPIN1c proteins formed a group separately .PIN proteins were aligned with MEGA6.06, and a maximum likelihood phylogenetic tree was prepared with Phylip package using the 500-bootstrap method. The transmembrane helices of TaPIN1 proteins were predicted using TMHMM2  (http://35S::TaPIN1-6a-CDS-GFP was inserted into the pMDC83 vector and infected into Arabidopsis by Agrobacterium tumefaciens (strain GV3101)-mediated transformation to determine the subcellular localization of TaPIN1. Then, T1 transgenic plants were placed in hygromycin (15 mg/L) pressure medium, and after germination, 5 to 6-day-old seedling roots were excised for imaging as described [Arabidopsis plants with a Leica TCS SP5II , and the GFP signal was observed at 505 to 530 nm emission under 488 nm excitation. Fluorescent images were captured using an LSM 880 Airyscan  with a 40 × objective. Fluorescence was detected using a 488-nmbandpass filter for GFP. Images were processed using LSM image processing software .escribed . Staininescribed –39. ConfIn situ hybridization and detection of hybridized signals were carried out as described by Meng et al. [TaPIN1-6 genes was amplified by PCR using the upper primer 5′- ACCGGACTACAACGACGCG—3′ and lower primer 5′—GAATTGTAATACGACTCACTATAGGGGCGCCCATCCCATTGTTGTT—3′. The sense RNA probe was amplified by PCR using the upper primer 5′- GAATTGTAATACGACTCACTATAGGGACCGGACTACAACGACGCG—3′ and lower primer 5′—GCGCCCATCCCATTGTTGTT—3′.g et al. . The wheTaPIN1 genes was performed on the expVIP, which was developed by Ricardo H. Ramirez-Gonzalez (github) and Bijan Ghasemi Afshar (github) as part of the Designing Future Wheat Institute Strategic Program within the Uauy lab (http://www.wheat-expression.com/) [TaPIN1 genes in wheat EnsemblPlants database were used as a query for the analysis of the expVIP.Expression pattern analysis of the on.com/) , 42. TheTaPIN1s cDNA was amplified by PCR using the upper primer 5′- CACCATGATCACGGGCACGGACTTCT—3′ and lower primer 5′—ATGAGCTTCTGCAGCGTGTCG—3′. The PCR product was inserted into the RNAi vector PC336 to trigger specific RNAi of TaPIN1s in wheat, which created a construct, namely, TaPIN1-RNAi. Then, the construct was transformed into callus initiated from immature embryo of CB037 via A. tumefaciens (strain C58C1)-mediated transformation [For the RNA-interfering plasmid construct, a 242 bp cDNA fragment between 1 and 242 of the ormation . Strain Genomic DNA was isolated from the putative transgenic wheat plants following the CTAB method . In addiTaPIN1 genes in transgenic wheat lines were analyzed by qRT-PCR . The first-strand cDNAs were synthesized by using Transcript One-Step gDNA Removal and cDNA Synthesis Supermix . qRT-PCR was performed using the gene-specific primers in Table Ultrapure RNA Kit  was used for total RNA extraction according to the manufacturer’s instruction in our experiment, and the transcript levels of 4 transgenic plants were grown at the Experimental Station of Shandong Agricultural University, Tai’an, Shandong Province, China. Wheat grains were sown and covered with plastic sheeting for insulation on November 17, 2018. Plastic sheeting was removed on March 15, 2019 after 5 days for ventilation and harvested on June 12, 2019. Each experimental line was sown with 25 cm in-row spacing and 10 cm plant-to-plant spacing. Common wheat cv. CB037 was kindly provided by Prof. Xiao Chen at the Institute of Crop Science of the Chinese Academy of Agricultural Sciences in Beijing, China. Arabidopsis for subcellular localization was an ecotype of Columbia-0 (Col.-0), which was planted in the growth room in the Science and Technology Innovation Building of Shandong Agricultural University. The growth room was set at 22 °C and 16 h day-time/8 h night-time mode. Moreover, IBM SPSS Statistics was used in our study for statistical analysis of data.Wild type and T


O-glucoside (C3G) is a widespread anthocyanin derivative, which has been reported in vitro to exert potent antioxidant, antiglycation and α-glucosidase inhibition effects. Nevertheless, the physiological relevance of such properties remains uncertain considering its significant instability in gastrointestinal conditions. A simulated digestion procedure was thus instigated to assess the influence of gastric and intestinal media on its chemical integrity and biological activities. HPLC analyses of digested C3G samples confirmed the striking impact of intestinal conditions, as attested by a decomposition ratio of 70%. In contrast, with recovery rates of around 90%, antiglycation, as well as DPPH and ABTS scavenging assays, uniformly revealed a noteworthy persistence of its antiglycoxidant capacities. Remarkably, a prominent increase of its α-glucosidase inhibition activity was even observed after the intestinal phase, suggesting that classical in vitro evaluations might underestimate C3G antidiabetic potential. Consequently, the present data provide novel and specific insights on C3G’s digestive fate, suggesting that the gastrointestinal tract does not profoundly affect its positive action on oxidative and carbonyl stresses. More specifically, it also tends to support its regulating effects on postprandial hyperglycemia and its potential usefulness for diabetes management.Cyanidin-3- O-glucoside (C3G), also known as kuromanin, have been highlighted in several edible and medicinal species [With several hundreds of derivatives described so far, anthocyanins represent a major class of polyphenolic constituents ,2. Moreo species . Indeed, species , black e species ,9,10.Over the last decades, increasing attention has been paid to the health benefits of anthocyanin consumption. This class of constituents, as well as C3G, have been widely studied for their potent antioxidant activity and numerous investigations have demonstrated positive effects on oxidative stress ,11,12. MIndeed, in vitro assessments of the biological activities of natural compounds and plant extracts is a first and fundamental step to endorse their potential physiological effects. However, such kinds of evaluations might not fully guarantee their actual benefits on human health since bioaccessibility, bioavailability and metabolization processes are not considered. In particular, the substantial sensitivity of anthocyanin constituents towards gastrointestinal conditions has been reported by several evaluations ,21, highThe present investigation thus aimed at deciphering the digestive fate of C3G by using a simulated gastrointestinal tract model. Chemical and biological evaluations were performed to assess the impact of gastric and intestinal conditions on its structural integrity as well as its bioactivity. Estimations of the Total Phenolic (TPC) and Total Anthocyanin Contents (TAC) were first realized to appraise the intensity of induced degradation at each step of the digestive process. Additionally, HPLC-DAD analyses were implemented to provide more precise data on the engendered chemical degradation. Finally, spectrophotometric and fluorometric assays were achieved to validate the persistence of its radical scavenging, antiglycative and α-glucosidase inhibitory properties in digestive media.w/w) and sodium hydroxide (NaOH) were obtained from VWR Prolabo . α-Glucosidase from Saccharomyces cerevisiae , p-Nitrophenyl-α-d-glucopyranoside (p-NPG), pancreatin from porcine pancreas (8 × USP specification), pepsin from porcine gastric mucosa , bovine serum albumin (BSA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), d-ribose, Folin–Ciocalteu’s reagent, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), sodium chloride, calcium chloride dihydrate, potassium chloride, magnesium chloride hexahydrate, potassium phosphate monobasic, sodium bicarbonate and ammonium carbonate were purchased from Sigma–Aldrich Chemical . DPPH and ABTS solutions were prepared every day and every half-day, respectively, and were stored protected from light at 4 °C.C3G was purchased from Extrasynthese . Ethanol, Methanol (MeOH) and acetonitrile (MeCN) were of chromatographic grade and were bought from Carlo Erba Reagents SAS . All aqueous solutions were prepared with pure water generated by a Milli-Q water (18.2 MΩ) device . Phosphoric acid (85%), hydrochloric acid .The in vitro digestion procedure was achieved following the standardized method published by Minekus et al. . GastricN-Biotek, Bucheon-si, Korea). The resulting gastric mixture was split in half; 5 mL was employed for the intestinal phase and 5 mL was kept back for chemical and biological assessments.A simulation of gastric and intestinal phases was performed as previously reported . For theRegarding the intestinal step, 5 mL of the gastric mixture was mixed with 3 mL of the SIF stock preparation and 1 mL of the pancreatin solution (1000 U/mL in SIF solution). A total volume of 10 mL was reached after the addition of calcium chloride  and water and the pH was adjusted to 7.0 with NaOH (0.1 M). An additional incubation (2 h) was completed with constant shaking (50 rpm) at 37 °C.To normalize C3G concentration in gastric and intestinal samples, a first dilution (1/2 in distilled water) was applied to the gastric solution. All samples were immediately deproteinized by adding four parts of ethanol and were subsequently centrifugated for 15 min at 4300 rpm . Supernatants were split in aliquots of 1 mL, which were kept at −80 °C until further experiments. A control solution of C3G was also performed (1.25 mg/mL in distilled water) to serve as a reference (undigested control). It was submitted to the same deproteinization, centrifugation and conservation processes as the digestive samples.R2 = 0.9979, y = 4.394x + 0.021) and results were expressed as milligrams of gallic acid equivalents (mg GAE) per gram of dry sample. Regarding the TAC, a standard curve of cyanidin 3-O-glucoside (5–200 μg/mL) was obtained  and contents were indicated as milligrams of C3G equivalent per gram of dry sample.The TPC and TAC were evaluated following the previously reported colorimetric methods . AbsorbaR2 = 0.9978, y = 1101.1x + 1.520) and the DPPH scavenging capacity was expressed in micromoles of Trolox equivalent (μmol TE) per gram of sample.The DPPH scavenging activity was assessed following a previously published protocol . Briefly•+ mixture prepared following the protocol of Re et al. [R2 = 0.9992, y = 11,760x + 0.5495) and ABTS scavenging activity was indicated in μmol TE/g of sample.The ABTS scavenging capacity was evaluated as previously reported . After ae et al. . A 10 mid-ribose method as previously described [50 in µg of dry sample/mL. Recovery indexes of the antiglycation capacity were also indicated and 1/IC50 values of digested samples were compared to that of the undigested control.Inhibition of the Advanced Glycation End-products (AGEs) formation was carried out using the BSA/escribed . The AGEp-NPG, 2.5 mM in buffer) was added and a second incubation was performed . Enzymatic hydrolysis led to the formation of colored p-nitrophenol which was recorded at 410 nm using a microplate reader (TECAN infinite F200 PRO). The control was prepared by replacing digested samples with the phosphate buffer. Acarbose was chosen as the reference α-glucosidase inhibitor and was evaluated in the same conditions at concentrations ranging from 0.01 to 1 mmol/L. Results were expressed as IC50 values in μmol/L.Evaluation of α-glucosidase inhibitory activity was performed according to the previously reported method , with sl® Star C8 endcapped column . A gradient elution was settled. The mobile phase was composed of water with 1% phosphoric acid (A) and MeCN (B). The gradient was set as follows: 0–5 min, 5% B; 5–30 min, 5–7% B; 30–45 min, 7–12% B; 45–50 min, 12–40% B. A flow rate of 1 mL/min, an injection volume of 20 μL and a monitoring wavelength of 520 nm were selected.HPLC analyses were performed with a LaChrom Elite system  consisting of two L7100 pumps, a L7200 autosampler, a L2450 diode array detector (DAD) and EZ Chrom Elite software . Retreated samples were diluted two times and analyzed with a reversed phase Purospherp values of 0.05 or less (p ≤ 0.05) were considered statistically significant. All data are expressed as a mean ± the standard error of mean (SEM). All analyses were done in triplicate (n = 3).The statistical significance was evaluated by one-way ANOVA, followed by a Fisher’s Least Significant Difference (LSD) test; C3G and anthocyanin derivatives are reported as particularly unstable constituents and their chemical integrity can be affected by numerous factors such as pH, light or temperature . Given tp < 0.05). The higher pH of that second phase might be considered as an important contributing factor to C3G instability. In fact, anthocyanin pigments are known to change to a blueish-colored quinoidal form in such pH conditions [By contrast, with a quantitative recovery of 37.9 ± 2.7%, a tremendous decrease of the TAC was highlighted in an intestinal medium .The potent antiglycative and radical scavenging capacities of C3G have been highlighted by several in vitro evaluations ,14. Neve50 of 40.52 ± 2.61 µg/mL. In accordance with previous investigations [p < 0.05) than aminoguanidine (IC50 = 140.9 ± 5.8 µg/mL), a commonly employed positive control. As observed for radical scavenging experiments, gastric and intestinal steps did not induce major modifications of the antiglycation capacity of C3G. In fact, with respective values of 95.8 ± 2.6 and 93.8 ± 3.4%, the recovery rates of these two digestive phases were not significantly different from the undigested matrix (p > 0.05).Substantial antiglycation activity was also observed for undigested C3G, as was ascertained by its very low ICigations , C3G wasTaken together, these data indicate that antiglycoxidant properties of C3G might be slightly affected by the gastrointestinal digestion process. Nevertheless, a very good level of persistence was observed during the three performed assays. Such results clearly indicate that the intense structural modifications spotted by chemical analyses are not associated with a pronounced alteration of C3G potential benefits on radical and carbonyl stresses. It strongly suggests that C3G decomposition products also contribute to the activities observed in simulated intestinal conditions. Indeed, owing to their above-confirmed phenolic nature, these components are likely capable of exerting prominent antiglycoxidant activities.Located in the intestinal brush border, α-glucosidases can be regarded as key carbohydrate hydrolysis enzymes which are capable of converting non-absorbable oligosaccharides and disaccharides into absorbable monosaccharides . They th50 of 22.7 ± 7.1 μmol/L, C3G was shown to exert a stronger effect (p < 0.05) than the acarbose positive control (IC50 = 340.2 ± 21.2 μmol/L). As illustrated in p < 0.05), as is attested to by a two-times lower IC50 of 10.2 ± 1.6 μmol/L. These results clearly support that an intestinal-induced chemical transformation exerts a major and positive impact on C3G’s inhibition effect. It tends also to indicate that classical in vitro evaluations of its α-glucosidase inhibitory properties might underestimate the real in vivo potential of C3G. In fact, the obtained data implies that this constituent may partially act as a prodrug whose activity gradually increases during the intestinal passage.By using a common spectrometric evaluation, α-glucosidase inhibition activities of digested samples of C3G were thus assessed to estimate its actual antidiabetic potential in physiological conditions. Considering the intestinal location of such enzymes, evaluations focused on the comparison of undigested and intestinal matrices. In accordance with previous investigations ,36, a suInterestingly, these results can be tied to previous in vivo investigations of C3G antidiabetic potential. Indeed, its marked inhibition potency on α-glucosidase should substantially contribute to its hypoglycemic effect reported in diabetic mice . More geBy using a standardized in vitro digestion procedure, the present investigation ascertains the modest bioaccessibility of C3G and confirms its extensive instability in intestinal conditions. HPLC experiments actually indicated that 70% of the digested C3G suffered chemical modifications. Considering its intense digestive susceptibility, classical in vitro evaluations of C3G biological properties might not be sufficient to fully validate its health benefits. Indeed, confirmation of the persistence of its effects after gastrointestinal-induced transformations is of utmost importance. Interestingly, a remarkable preservation of its antiglycoxidant properties was determined after both the gastric and intestinal steps. In fact, with recovery rates of around 90%, antiglycation, as well as DPPH and ABTS scavenging assays, uniformly revealed a marginal impact of gastrointestinal treatment. This clearly indicates that the digestive environment does not profoundly affect the action of C3G on oxidative and carbonyl stresses and supports its potential preventive action on several non-communicable diseases. More specifically, the antidiabetic potential of C3G might even be underestimated by in vitro experiments, as is attested to by the substantial intensification of its α-glucosidase inhibition activity in a simulated intestinal medium. Consequently, the present data also suggest that the consumption of C3G-rich fruits might help to regulate postprandial glucose levels in T2D patients. Nevertheless, further evaluation of the impact of more complex food matrices on C3G’s digestive fate would be worth investigating to fit more precisely with its actual mode of consumption.


Leishmania, and that NETs stimulated by LMs alone display leishmanicidal activity. The ability of LM to induce NET release may have potential implications for the course of inflammation or infection.Neutrophils are recruited from the blood and transmigrate through the endothelium to reach tissues, where they are prone to respond through different mechanisms, including the release of neutrophil extracellular traps (NETs). These responses occur in close contact with proteins from the basement membrane and extracellular matrix, where laminins are abundant. Thus, we investigated the interactions between neutrophils and different laminin (LM) isoforms and analyzed the induction of NETs. We showed that neutrophils stimulated with LM isoforms 111, 211, 332, 411, 421, and 511 released NETs. The same occurred when neutrophils interacted with polymerized LMs 111, 411, and 511. LM-induced NETs were partially inhibited by pretreatment of neutrophils with an anti-α6 integrin antibody. Furthermore, NETs triggered by laminins were dependent on elastase and peptidylarginine deiminase (PAD)-4, enzymes that participate in chromatin decondensation. We also found that LMs 411 and LM 511 potentiated the NET release promoted by promastigotes of the protozoan parasite  In response to infection, neutrophils are recruited from the blood and transmigrate through the endothelium to reach the tissue. Once in tissues, neutrophils are prone to respond to infection through different mechanisms, including the release of neutrophil extracellular traps (NETs). NETs are web-like structures composed of decondensed chromatin associated with proteins from different neutrophil compartments, and they contain and kill microorganisms . SeveralTo reach the inflammatory site, neutrophils must cross the endothelium and the vascular basement membrane (BM) and NETs have been reported during this transmigration process . AdhesioLaminins are recognized by several integrins that are noncovalently linked αβ heterodimers forming 24 known integrins, 11 of which can bind to LM ,15,16. TSince the effector functions of neutrophils occur in a milieu rich in ECM, where different LM isoforms are present, we decided to study the effect of LM isoforms on NET induction and analyze the LM receptor and signaling pathways involved in this process. Our results showed that the six laminin isoforms tested were able to induce NET release through recognition of the integrin α6 chain. Elastase- and PAD-4-dependent NET release mechanisms were also characterized.Leishmania protozoan infection [Leishmania induces NETs in human neutrophils that trap and kill the parasites [Leishmania and examined the leishmanicidal properties of LM-induced NETs. Our data demonstrated increased NET release after neutrophil stimulation by both stimuli and showed that NETs released by LM-stimulated neutrophils are leishmanicidal, indicating that NETs induced by LM may reduce parasite survival.Moreover, neutrophils are among the first cells recruited during the early stages of nfection , and Leiarasites . Thus, wThe human LM isoforms 111, 211, 332, 411, 421, and 511 were purchased from BioLamina, Sweden, and used at the indicated concentrations.2. The polymers formed in solution were adsorbed onto glass-bottomed 96-well plates to produce the matrices used for the NET release assays. For the control, the unpolymerized 111, 411, and 511 LMs were diluted to 50 µg/mL in neutral buffer  containing 1 mM CaCl2.To produce polymerized LMs (polyLM), isoforms 111 , 411 and 511  were diluted to 50 µg/mL in 20 mM sodium acetate (pH 4) containing 1 mM CaClPeripheral blood collected from healthy donors was used to isolate neutrophils via density gradient centrifugation as described . The pro6) were stimulated with the LM isoforms 111, 211, 332, 411, 421 and 511 with or without Leishmania promastigotes (1 × 103) or stimulated with polyLMs 111, 411 and 511. After 90 min, NET release in culture supernatants was quantified using a PicoGreen Kit  and a SpectraMax Paradigm microplate reader  set at 485/538 nm excitation/emission. PMA , was used as a positive control.Neutrophils (1 × 106) were treated with elastase inhibitor  or chloroamidine  for 30 min at 37 °C and 5% CO2 and then stimulated with the LM isoforms or PMA for 60 min under the same conditions. NETs were quantified in culture supernatants as described above.Neutrophils (1 × 105) adhered to 0.001% poly-L-lysine-treated coverslips were stimulated with soluble LM  or directly adhered to either LM- or polyLM-treated coverslips  and incubated at 37 °C. After 90 min, the neutrophils were fixed with 4% formaldehyde and blocked against nonspecific binding with 100% AB-positive human serum for 60 min. Cultures were stained with antibodies against pan-LM , α1 LM chain , α4 , α5 , anti-human neutrophil elastase  or anti-DNA/histone H1  for 1 h at room temperature. Then, goat anti-rabbit or anti-mouse secondary antibodies labeled with Alexa Fluor 488 or 546  were added. The slides were mounted in ProLong Gold Antifade Mounting with DAPI (ThermoFisher). Images were obtained with a Zeiss DMi8 confocal microscope.Neutrophils (3 × 106) were incubated with anti-α6 integrin antibody  in 20 µL in RPMI for 15 min at 37 °C, 5% CO2, and the expression of α6 integrin was determined by flow cytometry using a FACS Calibur cytometer. The data were analyzed using Summit v.4.3 software. Alternatively, neutrophils were treated with 2 µg/mL of the same anti-α6 integrin antibody and stimulated with LM 111, 411 or 511. An additional 80 µL of RPMI was added, and cultures were incubated for 60 min. NET production was assessed as described above.Neutrophils (1 × 10L. amazonensis promastigotes (MHOM/BR/75/Josefa) were grown at 26 °C in Schneider medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum , and 40 µg/mL gentamicin . Promastigotes were obtained after 5 to 6 days of culture (stationary phase), washed three times in PBS , resuspended in RPMI 1640 medium (Sigma), and used throughout the experiments.6, 200 μL) were incubated with Leishmania at the indicated ratios or with 2 µg/mL LM 511 for 4 h at 35 °C, 5% CO2. Then, NET-rich supernatants were obtained by centrifugation at 400× g for 10 min. Supernatants obtained by stimulation with parasites were again centrifuged at 2760× g for 20 min to remove parasites.Neutrophils (4 × 10Leishmania promastigotes (103) for 60 min. NETs in culture supernatants were quantified using PicoGreen. Neutrophils (105) were seeded in 96-well plates and stimulated with 1 µg/mL LM 511 for 30 min. Subsequently, they were stimulated or not with delipidated E. coli LPS  at 37 °C with 5% CO2. After 4 h, NET was quantified in the supernatants as above.Neutrophils were incubated with or without LM 111, 411, and 511 (1 µg/mL) in solution for 30 min and then further incubated in the presence or absence of 6, 200 μL) were incubated with NET-rich supernatants (0.5 µg/mL) for 2 h at 35 °C, 5% CO2, and cell viability was assessed with 4 μM ethidium homodimer-1  staining solution for 30 min. Promastigotes killed by 4% formaldehyde served as the positive control. The data were collected with a FACS Calibur flow cytometer and analyzed with FlowJo software.Promastigotes  constitutes a barrier for neutrophils to reach the infection site. LMs 411 and 511 are the major isoforms found in vascular BMs and contribute to the neutrophils’ transmigration ,24,25,26Next, we quantified NET release by neutrophils stimulated with different concentrations of LM 111, 411, and 511 E,J,O andIt is well-established that neutrophils express integrin α6 (CD49f), which is a laminin receptor . ConfirmTo evaluate the signaling pathways involved in NETs induced by LM, we evaluated the role of PAD-4 and elastase. Neutrophils were treated with chloroamidine (12 µM) or elastase inhibitor (10 μM) for 30 min and then incubated with LMs 111, 411, or 511 for 60 min; NET release was then measured in culture supernatants. Our results reveal that the elastase inhibitor decreased the release of NETs stimulated by LMs 111, 411, and 511 by 39%, 68%, and 62.5%, respectively A. InhibiWhen entering the tissue, neutrophils will adhere and migrate on the existing LMs within the tissue, both in the BM and the interstitial ECM. Moreover, neutrophils can be stimulated by secreted LMs produced by themselves or other cells. To better understand the initial interactions leading to NET formation induced by LM isoforms, we compared the effect of NET release induced by soluble and plastic-adsorbed LMs. Neutrophils were incubated with the indicated isoforms at 1 μg/mL in suspension for 90 min, and DNA was quantified in culture supernatants. Alternatively, NET release was assayed on 10 μg/mL LM-coated plates, and DNA was quantified in culture supernatants. Our data demonstrate that in either form—suspension A or adsoPrevious studies have shown that acidification induces LM polymerization, giving rise to a hexagonal sheet-like matrix of fractal dimension ,30. ThisLeishmania, can induce NETs [Leishmania, we incubated neutrophils with LMs 111, 411, and 511 for 30 min and then with the parasite for 60 min. Our data showed 42%, 70%, and 54% increases in NET release when neutrophils were costimulated with Leishmania and LMs 111, 411, or 511, respectively, compared with neutrophils stimulated only with Leishmania (E. coli LPS could be modulated by LM 511. Our results showed that NET induction by LPS increased 60% when neutrophils were stimulated by LPS plus LM 511 (It is known that parasites, including uce NETs . To inveishmania A–C. We as LM 511 . Leishmania or LM 511 and evaluated parasite viability (L. amazonensis alone.We further evaluated leishmanicidal activity by incubating parasites for 2 h with NET-rich supernatants obtained after neutrophil stimulation with iability . Our resUpon different stimuli, neutrophils leave the blood vessels, cross the endothelium and the basement membrane, and move to the inflammatory focus through chemotactic, haptotactic, and topotactic stimuli ,8,9. CelYersinia pseudotuberculosis invasin protein to the β1 integrin chain [LM is recognized by different receptors, including several integrins, which mediate a diversity of effects in leukocytes . The rolin chain .The expression of integrin α6β1, the most ubiquitous and specific receptor for LM isoforms, on neutrophils is increased during transendothelial migration ,19,47. HVarious stimuli may induce NET release and activate different signaling pathways . LM-induIt has been reported that acidification induces LM self-polymerization into a three-dimensional polymer presenting the overall appearance of natural matrices ,49. ThisLeishmania infection, promastigote forms of the parasite are inoculated by the insect vector into a blood pool in the host dermis, allowing contact between the parasites, neutrophils, and ECM. Since our group has been investigating NET release during Leishmania infection [Leishmania. Furthermore, an additive effect was demonstrated for another proinflammatory stimulus, as LM 511 potentiated LPS-induced NET formation.During nfection ,50,51, wL. amazonensis promastigotes are killed by NETs [Leishmania with LM may influence the homing of the parasite [Leishmania capacity to induce NETs and are toxic to the parasites.It has been demonstrated that NETs are toxic to several microorganisms such as bacteria and fungi, and our group has demonstrated that  by NETs ,50. Path by NETs . Of partparasite . In thisTaken together, these data demonstrate that LM isoforms may contribute to the effector mechanism of neutrophils, although further studies are needed to elucidate the impact of these interactions in the course of inflammation or infection.


Glaesserella parasuis strains were characterized by serotyping PCR, vtaA virulence marker Leader Sequence (LS)-PCR, clinical significance, and geographic region. Overall, the serovars 4, 5/12, 7, 1, and 13 were the most commonly detected. Serovars of greatest clinical relevance were systemic isolates that had a higher probability of being serovar 5/12, 13, or 7. In comparison, pulmonary isolates had a higher likelihood of being serovars 2, 4, 7, or 14. Serovars 5/12 and 13 have previously been considered disease-associated, but this study agrees with other recent studies showing that serovar 7 is indeed associated with systemic G. parasuis disease. Serovar 4 strains illustrated how isolates can have varying degrees of virulence and be obtained from pulmonary, systemic, or nasal sites. Serovars 8, 9, 15, and 10 were predominantly obtained from nasal samples, which indicates a limited clinical significance of these serovars. Additionally, most internal G. parasuis isolates were classified as virulent by LS-PCR and were disease-associated isolates, including serovars 1, 2, 4, 5/12, 7, 13, and 14. Isolates from the nasal cavity, including serovars 6, 9, 10, 11, and 15, were classified as non-virulent by LS-PCR. In conclusion, the distribution of G. parasuis serovars remains constant, with few serovars representing most of the strains isolated from affected pigs. Moreover, it was confirmed that the LS-PCR can be used for G. parasuis virulence prediction of field strains worldwide.The online version contains supplementary material available at 10.1186/s13567-021-00935-9. Glaesserella parasuis is an important pathogen of swine and remains one of the most important bacterial causes of mortality in swine production worldwide. Clinical signs of infected pigs range from fever, respiratory signs, and swollen joints with lameness, to central nervous signs, and sudden death. Chronic infections are also associated with reduced growth rates and food conversion efficiencies. The main pathological findings include fibrinopurulent polyserositis, polyarthritis, and meningitis, commonly known as Glässer’s disease [ disease .G. parasuis is a highly diverse pathogen, with still poorly known cross-protection against disease-causing field strains [Glässer’s disease is considered one of the most prevalent swine bacterial infections worldwide . Relianc strains , 8. In mG. parasuis is also a commensal of the upper respiratory tract (URT) [G. parasuis strains into an immunologically naïve pig population, typically through the mixing of pigs from different sources. Other predisposing factors include poor management, high stocking density, inadequate ventilation, and coinfections with other respiratory pathogens [ct (URT) . Diseaseathogens , 11.G. parasuis-associated disease, and preventive health management, relies on identifying specific G. parasuis serovars colonizing and or causing illness. Fifteen G. parasuis serovars that differ in virulence have been described [Epidemiological investigation of escribed , 13. Howescribed , 15.G. parasuis species in the field requires surveillance tools to detect disease-relevant strains. Whole-genome sequencing enabled detailed analysis of the capsule loci of G. parasuis [G. parasuis serovars but not serovars 5 and 12. A new G. parasuis serovar-specific PCR scheme containing primers that differentiate serovars 5 and 12 was subsequently described [This broad diversity within the parasuis  and the parasuis , 18. Thiescribed . HoweverG. parasuis isolates. A group of specific virulence-associated genes, named virulence-associated trimeric autotransporters (vtaA) group 1-translocator, were associated with virulent G. parasuis strains [vtaA genes. A test for predicting the virulence potential of G. parasuis strains was developed based on the leader sequence of the vtaA genes, called LS-PCR [In parallel to molecular serotyping, novel PCR methods were developed to track virulence markers for  strains . Additiod LS-PCR .G. parasuis from top pork-producing countries in terms of the relative geographic prevalence of molecular serovars and their relationship with vtaA markers.Such epidemiological investigations are essential for designing preventive health programs, supporting vaccine candidate selection, improving pig flow management, and limiting the reliance on prophylactic antimicrobials. This study’s objective was to characterize strains of G. parasuis isolates in this collection originated from pigs from different geographical regions; USA (n = 223 from 15 different states), Europe (n = 436 from 13 different Member States), China (n = 167 from 12 different provinces), Vietnam , and Canada   were obtained from the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL), where isolates were confirmed as G. parasuis using Bruker matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI_TOF MS). Data for Canadian strains (n = 66) were obtained from the Diagnostic Laboratory of the Faculty of Veterinary Medicine of the University of Montreal (DLFVMUM), where isolates were also firstly identified by MALDI_TOF MS and confirmed by PCR [n = 275) and Germany (IVD Innovative Veterinary Diagnostics (IVD GmbH), (n = 165) and represented 13 different member states. Confirmation of G. parasuis by both laboratories was achieved by PCR [n = 167) and Vietnamese isolates (n = 56) were obtained from Huazhong Agricultural University Veterinary Diagnostic Laboratory. The Chinese and Vietnamese isolates were firstly identified with biochemical tests [Data for US isolates n = 3 were obal tests .G. parasuis strains isolated from internal organs other than the lungs, such as pleura, pericardium, peritoneum, joint, and meninges, were considered systemic strains. All URT strains in this study were isolated from nasal cavities.Metadata for a subset of these isolates included isolation site, year of isolation, and country of origin  without calcium and magnesium  and vortexed. Bacterial DNA was extracted with the MagMAX™ Viral DNA/RNA Isolation Kit  using extraction protocol per manufacturer instructions.All participating laboratories conducted the serotyping PCR as previously described  with som–1 of primer mix 1, 2 or 3, 8 µL of nuclease-free water, and 2 µL of extracted DNA. The reaction was run using the following conditions: first cycle at 95 °C for 15 min, followed by 40 cycles of 94 °C for 30 s, 58 °C for 90 s, and 72 °C for 90 s. This was followed by one cycle at 72 °C for 10 min. Electrophoresis and visualization of the PCR products were performed using QIAxcel Capillary Electrophoresis with screen gel software (Qiagen).Briefly, each PCR reaction (one for each of the primer mixtures) consisted of 12.5 µL of 2 × Multiplex PCR Master Mix , 2.5 µL of the 100 nmol 1For isolates obtained by IRTA-CReSA, PCR conditions were amended using the same primers as Howell et al. , but thevtaA genes domain as a diagnostic tool to predict G. parasuis virulence [–1 of each primer, 4.5 µL of nuclease-free water, and 2 µL of extracted DNA in a final volume of 25 μL. Cycling conditions were 15 min at 95 °C, followed by 30 cycles of 45 s at 94 °C, 45 s at 52 °C and 1 min at 72 °C, then a final incubation at 72 °C for 10 min.Isolates from North America and Europe were tested using a PCR to detect the leader sequence (LS) of the irulence . Brieflyp-value of lower than 0.05 was defined as indicating a significant difference. All analyses were completed using SAS version 9.4.Associations between serovars, sample site, country of isolation, and LS-PCR results were calculated using Chi-square and Fisher’s exact tests. A multinomial logistic regression model was fit to compare the sample site and serovar classification to the reference (untypeable or NT) serovar. All tests were 2-sided, and a G. parasuis isolates , were typeable by serotyping PCR, and the remaining 64 (9.7%) were identified as non-typeable (NT). All 15 serovars were identified. Serovars 4 (17.4%), 5/12 (16.8%), 7 (11.4%), 1 (10.7%), and 13 (9.1%) were the most commonly detected serovars across the participating regions  were obtained from pulmonary sites, while 40.8% (212/519) were of systemic origin  was found among Chinese isolates, while only 1.4% 3/210) of USA internal isolates were untypable  of all isolates for which an attributable sample site was recorded and belonged to a variety of serovars, especially serovars 8 12%), 1 (12%), 4 (11.4%), 9 (11.4%), 15 (10.8%) and 10 (9.5%)  Table , 4, 5, 6G. parasuis nasal isolates were from Asian countries, including 25 from Vietnam and ten from China. Most of the nasal isolates from China were either from serovar 5/12 or 1, while most of the nasal isolates from Vietnam were from serovars 2, 9, 4, and 10 . A multinomial logistic regression model was further used to predict serovar classification based on sample type. Given the sample type , samples collected from the nasal cavity had a higher probability of being of serovars 8, 9, 10, or 15 (p < 0.05), but no association was found between serovar and internal samples, during this initial analysis.Results of the chi-square analysis showed an association between sample type  and serovar (p < 0.01). In comparison, pulmonary isolates had a higher likelihood of being serovars 2, 4, 7, or 14 (p < 0.01). No statistical significance was observed when comparing systemic and pulmonary isolates. These results agree with Olvera et al. [Using the same model, internal isolates were classified as either pulmonary or systemic and compared against nasal isolates for the subsequent analysis. Given the sample type, systemic isolates had a higher probability of being of serovar 5/12, 13, or 7 compared to the nasal sample collection  were classified as non-virulent by LS-PCR, and 223 isolates were not tested (Table p < 0.001). Further analysis was performed within each serovar to evaluate the association of virulent and non-virulent strains with the site of isolation. A significant association was found for serovars 5/12 and 7, which had a higher chance of being virulent and from internal sites (systemic or pulmonary). Contrarily, serovar 10 had a higher chance of being non-virulent and from a nasal site   and China (167/223) were isolated more than ten years ago or have an unknown date of isolation. Thus, the actual distribution  of G. parasuis serovars is still unknown. Even though our data do not represent prevalence, most of these isolates originate from field cases and reflect strains frequently found in disease-associated cases, and thus useful information for swine practitioners and researchers.Serotyping has been a necessary means of characterizing R method , 18, we The serovars’ overall distribution varied slightly among countries, and four serovars  accounted for half of all isolates. Serovars 4 and 5/12, in particular, were consistently represented across all regions in this study and previous studies , 22–28.The serovars of utmost clinical significance were identified in samples from internal sites  in this study. These serovars have been previously considered disease-associated –30. VariG. parasuis. In this study, serovar 7 was assigned to 10.9% of all internal samples  and 15.6% of all systemic isolates . Serovar 7 was initially found non-virulent by intraperitoneal challenge [An important finding in this present study and other recent studies –30 was thallenge , and it hallenge . Convershallenge . Additiohallenge , 30, demhallenge .Based on their detection in fewer than 1% of all internal samples, the least clinical significance serovars were serovars 3, 11, and 15. Serovars 3 and 11 were previously considered non-virulent . Still, G. parasuis strains in their URT, and potentially pathogenic serovars can be isolated from the URT of healthy pigs [Nevertheless, pigs harbor various thy pigs , 14. In G. parasuis serovar 4 strains. The reference isolate for serovar 4 was initially isolated from the nose of a healthy pig and considered mildly virulent [G. parasuis serovars’ virulence may have been wrongly assigned by the Kielstein and Rapp-Gabrielson scheme [Different pathogenic properties have also been described for virulent . In our virulent . When twvirulent . These rvirulent –33. Thesn scheme .vtaA genes’ role in G. parasuis virulence [G. parasuis strains, a new PCR, based on the leader sequence of the vtaA genes, was developed [Glasser’s disease is a complex multifactorial disease, and several different putative virulence genes have been identified –37. Stilirulence , 38, 39.eveloped .vtaAs in that strain, which may represent a strain with an intermediate phenotype [In the present study, most internal isolates were identified as virulent by LS-PCR, while most nasal isolates were non-virulent. Our results agree with previous reports , 39–41 ahenotype .vtaA genes are positively correlated with G. parasuis virulent strains and can be used as a predictor of virulence, other factors may also be necessary for disease development [Nevertheless, some discordant results were also found within the LS-PCR data. We found that a small number of isolates from typically non-disease-associated serovars , and classified as non-virulent by LS-PCR, were recovered from systemic sites. In this case, other environmental factors or coinfections could be responsible for creating the necessary conditions for less virulent strains to invade systemically , 31, 40.elopment , 32, 40.G. parasuis serovars remains predominantly constant over the past 20 years, with few serovars representing most of the strains isolated from Glasser’s disease-affected pigs and noting the confirmation of serovar 7 as a globally relevant serovar. Even though the serovar and virulence relationship is not definitive, serotyping is still suitable for the epidemiological characterization of G. parasuis in pig farms, helping implement more adequate control measures, such as selecting isolates to be included in vaccines. When serotyping is not available, the site of isolation can become crucial for screening disease-associated isolates since systemic strains are likely the ones that possess the necessary virulent mechanisms for disease development. To date, vtaA genes are the most well-characterized G. parasuis virulence factors. This survey of global G. parasuis isolates confirms that the LS-PCR is useful for G. parasuis virulence prediction.In conclusion, the overall distribution of Additional file 1. Metadata of Glaesserella parasuis isolates including isolation site, year of isolation, and country of origin.Additional file 2. Distribution of Glaesserella parasuis serovars in North America.Additional file 3. Distribution of Glaesserella parasuis serovars in Europe.Additional file 4. Distribution of Glaesserella parasuis serovars in Asia.