Vitamin E (VitE) is essential for vertebrate embryogenesis, but the mechanisms involved remain unknown. To study embryonic development, we fed zebrafish adults (>55 days) either VitE sufficient (E+) or deficient (E–) diets for >80 days, then the fish were spawned to generate E+ and E– embryos. To evaluate the transcriptional basis of the metabolic and phenotypic outcomes, E+ and E– embryos at 12, 18 and 24 h post-fertilization (hpf) were subjected to gene expression profiling by RNASeq. Hierarchical clustering, over-representation analyses and gene set enrichment analyses were performed with differentially expressed genes. E– embryos experienced overall disruption to gene expression associated with gene transcription, carbohydrate and energy metabolism, intracellular signaling and the formation of embryonic structures. mTOR was apparently a major controller of these changes. Thus, embryonic VitE deficiency results in genetic and transcriptional dysregulation as early as 12 hpf, leading to metabolic dysfunction and ultimately lethal outcomes. Vitamin E (VitE) is a potent lipophilic antioxidant and is localized in membranes to protect against lipid peroxidative damage . VitE co-/-Ttpa mice, which show degeneration of cerebellar Purkinje [The vertebrate embryonic VitE requirement is time-dependent and is needed between embryonic days (E) 9.5 and 11.5 in rats ,7. DurinPurkinje  and spinPurkinje .In addition to morphologic derangements, E– zebrafish embryos also experience lethal dysregulation of glycolytic metabolism in the first 120 hpf , which cZebrafish development is coordinated by temporal and spatial gene expression networks. Transcriptional regulation is a key component of embryonic fate . The mecAll experimental protocols and methods were carried out in accordance with the animal use and care protocol # 5068) approved by the Institutional Animal Care and Use Committee at Oregon State University. Tropical 5D strain zebrafish were reared in the Sinnhuber Aquatic Research Laboratory at Oregon State University under standard laboratory conditions of 28 °C on a 14 h light/10 h dark photoperiod according to standard zebrafish breeding protocols [ approvedn = 3 measurements), respectively. In the E– diet, α- and γ-tocopherols were 1.1 ± 0.0 and 0.5 ± 0.0 mg/kg (n = 3 measurements), respectively. E– and E+ embryos were obtained by group spawning of adult fish fed either E– or E+ diets for a minimum of 80 days up to 9 months. Embryos were collected, staged and incubated until use in standard embryo media ). Embryos at each time point  were staged according to the appropriate developmental landmarks [VitE was extracted from the diets prior to feeding and assayed by HPLC-UV prior to feeding, as described . In the n = 10 embryos/sample) and homogenized, with 4 sample pools per VitE status. RNA integrity  was assessed using the 2100 BioAnalyzer Instrument (Agilent) at the Center for Genome Research and Biocomputing at Oregon State University  with current annotations (Ensembl 100) using STAR aligner, v2.7.3a. Raw gene counts were summarized with edgeR, Bioconductor v3.24.3 in R, v4.0.2. Data were normalized by the TMMwsp  method.Data processing was performed using the default parameters provided by the program manuals unless stated otherwise. Sequence read quality was first assessed using FastQC v0.11.5. Adapters and poly(A) tails were trimmed using bbduk v35.92. Trimmed reads were then aligned to the current reference genome . Heatmaps were generated using z-score transformed counts per million (CPM) of differentially expressed (DE) genes identified by adjusted p-value, or Benjamini–Hochberg False Discovery Rate (FDR) < 0.1.Multidimensional scaling plots (MDS) were computed to assess the similarity of group effects, identified as the interaction of diet and time, and the sequencing lane effects between samples. edgeR generalized linear model (glm) was used to fit the data and estimate the common, trended and tagwise dispersions given the experimental design. A quasi-likelihood F-test (QLF) was used to compute differential gene expression between E+ and E– embryo gene expression at each time point measured  were identified and described by predicted anatomical regions using ZFIN [FDR < 0.25. Metabolomic data from 24 hpf embryos previously acquired [DE genes were subject to Gene Ontology (GO) enrichment analysis using the Bioconductor R package GOseq, v.1.40.0. Over representation analysis (ORA) was performed with DE genes and DE genes with absolute value logbGestalt . Gene seing ZFIN . All netacquired  were intacquired .n = 30) E+ and E– embryos at 24 hpf in RIPA buffer supplemented with protease and protein phosphatase inhibitors . Rat liver extract was used as a positive control for the assay. Extracted protein (25 μg) was subjected to SDS-Page and processed by immunoblotting. All gels and blots were run simultaneously. The proteins were visualized by SuperSignalTM West Pico Chemiluminescent Substrate  and quantified using the Bio-Rad Image System . The information for antibodies is listed in the Protein was extracted from pooled  at the three time points, 12, 18 and 24 hpf. Multidimensional scaling analysis indicated strong separation between the gene expression profiles by time point and further by embryo VitE status (p < 0.05) across all time points and by VitE status. From this selected group of genes, 12% (n = 285) were considered differentially expressed (DE) under a false discovery rate threshold (FDR < 0.1). A heatmap of DE genes was clustered with z-score averages according to VitE status, age and gene (FDR < 0.25) associated with organic acid binding (GO:0043177), iron ion binding (GO:0005506), and carbohydrate binding (GO:0030246). Vitamin binding  refers to genes associated with L-ascorbic acid binding capacity, including egln2, p4ha1b, and plod1a. Cluster 2 was significantly associated with peptidase activity (GO:0008233) and unfolded protein binding (GO:0051082). Cluster 3, identifiable by increased gene expression in E+ embryos at 12 and 18 hpf, was significantly associated with protein-containing complex binding (GO:0044877), cell adhesion molecule binding (GO:0050839) and cytoskeletal protein binding (GO:0008092). Cluster 4 genes were defined by GO terms vacuole (GO:0005773) and extracellular region part (GO:0044421). The term “vacuole” was associated with endolysosomal trafficking-associated genes abcc6a, galca, and zgc:110239.To determine the VitE-dependent effects on global transcriptomic changes during embryo development, RNA was isolated from E+ and E– whole embryo lysates , which is predicted to be involved in G protein-coupled receptor signaling, and cngb3.2 , which is predicted to have cGMP binding activity and is involved in cation transmembrane transport. DE genes that decreased at all three time points in E– embryos include serpina7 , a serine-type endopeptidase inhibitor implicated in mTOR-related neuropathology in zebrafish [si:dkey-73p2.2, si:dkey-15j16.6, and si:dkey-23k10.3. nitr3r.1l  was found initially (12 and 18 hpf) to be increased highly in E– embryos, but was decreased highly at 24 hpf.The top 10 DE genes  with increased or decreased expression levels in E– embryos relative to E+ embryos were identified at each of the three time points  are shown in ascending p-value order  were ranked by their fold-change in expression level between E– and E+ sample groups. Using the ranked genes, Gene Set Enrichment Analysis (GSEA) was carried out using WebGestalt (FDR < 0.25). At 12 hpf, E– vs E+ embryos have increased expression of genes associated with vitamin C binding, demethylation and demethylase activity, metabolic processes and decreased expression of genes associated with collagen synthesis and protein import  altered the mTOR signaling pathway . Top DE TFs were identified from the dataset  enrichment to determine the extent to which gene expression trends are associated with similar TF regulatory units or promoter regions. DE genes shared the consensus site for TF binding YGTCCTTGR motif . Glycolytic genes are necessary for anabolic growth and key morphogenesis pathways in embryonic development, which we previously reported are significantly altered in E– embryos [E– zebrafish embryos at 24 hpf experience metabolic dysfunction with reduced glycolytic intermediates and increased basal oxygen consumption rates . These m embryos ,19. E– r embryos . We repo embryos . Vitamin embryos . Thus, VMetabolic pathways are also critical for cellular compensation in response to lipid peroxidation (LPO). We reported previously the depletion of long chain polyunsaturated fatty acids, specifically docosahexaenoic acid (22:6n-3), in the E– embryos  and E– aZgc:101562 and mafaa, two differentially expressed transcription factors in E– embryos, are associated with gene expression related to glucose regulation and are expressed in mouse neural tissue at E12.5 [at E12.5 ,39,40 anat E12.5 ,38,39, rat E12.5 , providiat E12.5 ,42 and mat E12.5 .Overall, genes associated with metabolism and mTOR signal integration, used to regenerate the metabolic intermediates described, appear to be the mechanism for the compensatory responses to VitE deficiency. mTOR senses energy and amino acid status signals to stimulate cell survival, growth and proliferation . mTOR ismTOR contributes to ensheathment in the zebrafish embryo  and requegln3, expressed in the neural plate in 12 hpf zebrafish embryos [p4ha1b, plod1a and col1a1b [p4ha family of genes, including p4ha1b found in this study [Many other pathways were impacted by VitE deficiency, of note is collagen remodeling.  embryos , is a hy col1a1b ,50. VitE col1a1b . Zebrafi col1a1b ,53. VitC col1a1b . We obse col1a1b ,55,56. Vis study . Togetheis study . Negativis study .nr2f1b, a transcription factor decreased in E– embryos at 12 hpf  that regulates vascularization and hindbrain regionalization in zebrafish [nitr3r.1l, an immune receptor significantly increased in E– embryos at 12 and 18 hpf  but decreased at 24 hpf (log2FC = −2.31) [cngb3.2, a cation transmembrane transporter increased in E– embryos at 18 and 24 hpf  implicated in the pathology of retinitis pigmentosa [In addition, there were numerous genes differentially expressed at 12, 18 and 24 hpf in E– embryos that might suggest multiple developmental pathways disturbed, including—ebrafish ; nitr3r.= −2.31) ; and cnggmentosa , a symptgmentosa . We alsogmentosa ,65,66,67In summary, we show that VitE deficiency as early as 12 hpf disrupts pathways underlying growth and development. This study also points to major metabolic dysfunctions occurring as early as 12 hpf. Simultaneously, E– embryos experience LPO disruption to membrane structure and signaling pathways, both of which are known secondary effects due to VitE deficiency. Errors occurring as early as 12 hpf signal catastrophic decline and impairment to neurodevelopment that relies on early signaling capacity for cellular migration, proliferation and overall tissue and organ development. There remains a need to further investigate the origins of VitE deficiency-induced embryonic death as a means of preventing developmental defects and improving prenatal health span.


Previous work has shown that the conspicuous glucose-induced hyperaccumulation of the glycolytic metabolite fructose-1,6-bisphosphate  in tps1Δ cells triggers apoptosis through activation of the Ras-cAMP-protein kinase A (PKA) signaling pathway. However, the molecular cause of this Fru1,6bisP hyperaccumulation has remained unclear. We now provide evidence that the persistent drop in intracellular pH upon glucose addition to tps1Δ cells likely compromises the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a major glycolytic enzyme downstream of Fru1,6bisP, due to its unusually high pH optimum. Our work highlights the potential importance of intracellular pH fluctuations for control of major metabolic pathways.Glucose catabolism is the backbone of metabolism in most organisms. In spite of numerous studies and extensive knowledge, major controls on glycolysis and its connections to the other metabolic pathways remain to be discovered. A striking example is provided by the extreme glucose sensitivity of the yeast  Saccharomyces cerevisiae shows great preference for glucose as a carbon source, a deletion mutant in trehalose-6-phosphate synthase, tps1Δ, is highly sensitive to even a few millimolar glucose, which triggers apoptosis and cell death. Glucose addition to tps1Δ cells causes deregulation of glycolysis with hyperaccumulation of metabolites upstream and depletion downstream of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The apparent metabolic barrier at the level of GAPDH has been difficult to explain. We show that GAPDH isozyme deletion, especially Tdh3, further aggravates glucose sensitivity and metabolic deregulation of tps1Δ cells, but overexpression does not rescue glucose sensitivity. GAPDH has an unusually high pH optimum of 8.0 to 8.5, which is not altered by tps1Δ. Whereas glucose causes short, transient intracellular acidification in wild-type cells, in tps1Δ cells, it causes permanent intracellular acidification. The hxk2Δ and snf1Δ suppressors of tps1Δ restore the transient acidification. These results suggest that GAPDH activity in the tps1Δ mutant may be compromised by the persistently low intracellular pH. Addition of NH4Cl together with glucose at high extracellular pH to tps1Δ cells abolishes the pH drop and reduces glucose-6-phosphate (Glu6P) and fructose-1,6-bisphosphate  hyperaccumulation. It also reduces the glucose uptake rate, but a similar reduction in glucose uptake rate in a tps1Δ hxt2,4,5,6,7Δ strain does not prevent glucose sensitivity and Fru1,6bisP hyperaccumulation. Hence, our results suggest that the glucose-induced intracellular acidification in tps1Δ cells may explain, at least in part, the apparent glycolytic bottleneck at GAPDH but does not appear to fully explain the extreme glucose sensitivity of the tps1Δ mutant.Whereas the yeast  Saccharomyces cerevisiae and cloning of the trehalose-6-phosphate (Tre6P) synthase gene unexpectedly converged on the same gene, TPS1  and depletion of metabolites downstream of GAPDH  restores normal growth of  glucose , and tpscolysis 16, 8. Thexokinase . On the alactose , the paring otsA , the dising otsA , and thees pombe  suggest  of Mig1 , 23, ina of Mig1 , inhibit of Mig1 , and ina of Mig1 . In genei and reduction of NAD+ (tps1 mutants. One possible explanation is that GAPDH uses free phosphate (Pi) as a substrate and that the hyperaccumulation of sugar phosphates depletes the Pi level to such an extent that it compromises in vivo GAPDH activity (tps1Δ growth defect on glucose by stimulation of glycerol biosynthesis is consistent with this explanation (tps1Δ strain in the presence of glucose through activation of the Ras-cAMP-protein kinase A (PKA) pathway therefore provides an alternative explanation for the suppression of tps1Δ by stimulation of glycerol production  to 1,3-bisphosphoglycerate  at the expense of P of NAD+ . It has activity . Suppreslanation , since ioduction . This me glucose . In addi ethanol , and exte medium . This sue medium , 30. It e medium , 32.S. cerevisiae, GAPDH is a homotetramer composed of three isoforms, encoded by TDH1, TDH2, and TDH3  having the highest, Tdh2 intermediate , and Tdh3 the lowest activity , while the affinity ranks in the opposite order  as well as different glucose concentrations  more precisely, we applied a fluorescence-based technique by expressing the cytosolic pH-sensitive green fluorescent protein (GFP) variant, pHluorin, in the cells of interest  in wild-type cells corresponded to a pHi of approximately 6.0, at which specific GAPDH activity measured in vitro is minimal. Moreover, the tps1Δ suppressor strains, tps1Δ hxk2Δ and tps1Δ snf1Δ, clearly regained the ability to rapidly recover from the initial intracellular acidification after glucose addition, although recovery was somewhat less robust  (4+) shifts toward its un-ionized form, ammonia (NH3), which can diffuse through the plasma membrane. Once in the cell, where the pHi is lower, NH3 converts to NH4+ taking up one proton and raising the pHi. By using this approach, we were able to prevent intracellular acidification in both wild-type and tps1Δ cells after glucose addition together with 200 mM NH4Cl in extracellular medium buffered with 200 mM Tris-HCl at pH 8, 8.5, or 9  , ammoniu1Δ cells . Howeverells see a and b. 10.1128/mBio.02199-20.1FIG S14Cl. Intracellular pH was monitored after addition at time point zero of 100 mM glucose with (full lines) or without (dotted lines) 200 mM NH4Cl. Wild-type (a) and tps1Δ (b) cells in 200 mM Tris-HCl medium at pH 8 (light blue), pH 8.5 (dark blue), and pH 9 (purple). Cells were grown in uracil-deficient medium with 2% galactose to retain the pHluorin plasmid. Download FIG S1, EPS file, 2.5 MB.Intracellular acidification after the addition of glucose is prevented in 200 mM Tris-HCl medium at high pH in the presence of 200 mM NHCopyright © 2020 Van Leemputte et al.2020Van Leemputte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02199-20.2FIG S2595 for wild-type (blue) and tps1Δ (red) cells in regular liquid medium at pH 5.5 (a) or 50 mM Tris-HCl medium at pH 8 (b). Cells were pregrown on galactose medium. Download FIG S2, EPS file, 2.7 MB.High extracellular pH inhibits growth. Growth in complete synthetic medium supplemented with either 100 mM glucose (full lines) or 100 mM galactose (dotted lines) was followed over 48 h by measuring ODCopyright © 2020 Van Leemputte et al.2020Van Leemputte et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4Cl buffered at pH 9 together with glucose effectively abolished intracellular acidification in both wild-type and tps1Δ cells  transporter GAL2. The tps1Δ hxt6,7,2,4,5Δ strain displayed a reduction in the glucose (100 mM) transport rate of 65% , 42tps1Δ1Δ cells .i, with a very atypical optimum at pH 8 and thus reduced activity in the physiological pH range of 6.5 to 7.5  in glucose uptake but still was not able to grow on 100 mM glucose. Consistently, glucose-induced hyperaccumulation of sugar phosphates in that strain was only delayed and not prevented  supplemented with 100 mg/liter adenine. In case of minimal medium, cells were grown in complete synthetic medium  containing 0.17% (wt/vol) yeast nitrogen base without amino acids and ammonium sulfate (Oxoid), supplemented with 0.5% (wt/vol) ammonium sulfate (Sigma-Aldrich) and 100 mg/liter adenine. The same medium with the appropriate amount of essential amino acid(s) was used for growth of auxotrophic strains. As a carbon source, either 100 mM glucose, 100 mM galactose, 220 mM ethanol, or 325 mM glycerol was added to the growth media. The pH was adjusted to 5.5 for liquid medium and 6.5 for solid medium (2% agar) with KOH. Strains were grown at 30°C under continuous shaking for liquid cultures (180 rpm). For assay purposes, cells were typically pregrown overnight and inoculated the next day in fresh medium. Cells were harvested in the exponential phase unless mentioned otherwise.(i) Spot tests. For spot tests of growth capacity, cells were pregrown overnight on 3% glycerol in rich medium until the late-exponential early stationary phase. For cells harboring the p426 overexpression plasmid, 100 mg/liter Geneticin was supplemented in both liquid and solid media to retain the plasmid. Next, cells were washed and spotted at a 5-fold serial dilution  on freshly poured solid media with the carbon source of interest. Pictures were taken after 2 to 3 days of incubation at 30°C.(ii) Growth curves. To determine growth curves, cells from an overnight preculture on galactose were harvested, washed, and resuspended to an OD600 of 0.1 in a 96-well plate. OD595 was measured every 30 min for 48 h using the Thermo Scientific Multiskan FC microplate photometer. Plates were shaken every 10 min for 1 min at medium speed to keep cells in suspension.Metabolites were extracted and measured as described previously Peeters et al. . General(i) Sample collection. Cells for metabolite measurements were cultured in complete synthetic medium with 2% galactose (unless stated otherwise) to the exponential phase, harvested by centrifugation, and washed twice with 25 mM morpholineethanesulfonic acid (MES), pH 6. Cells were resuspended at a concentration of 75 mg (wet weight)/ml in complete synthetic medium with no added sugar and incubated at 30°C for 30 min of acclimatization. For metabolite determination after glucose or galactose addition, the sugars were added to a final concentration of 100 mM. Samples were taken by quenching cell suspensions in 60% methanol at −40°C  Sample processing. Thawed cell lysates were spun down at high speed and supernatants were collected. From this point on, cell lysates needed to undergo a process of neutralization. First, 50 μl of 5 M K2CO3 was added to 250 μl cell lysate together with 10 μl thymol blue (0.025%) to monitor the pH visually. Samples were allowed to degas on ice for 15 min. Next, samples were spun down at high speed, and 200 μl supernatant was added to 100 μl 1 M HCl and 10 μl Tris-HCl (pH 7.5). Samples were stored at −20°C.(iii) Metabolite measurements. Metabolite concentrations were determined by endpoint measurement of the absorption of NADH or NADPH at 340 nm through coupled enzymatic reactions. The increase or decrease of absorption correlates linearly with the metabolite’s concentration and was calculated by applying Lambert’s law. The general reaction buffer was composed of 50 μl sample incubated with 150 μl 100 mM Tris-HCl, pH 7.5. Other components were added to the reaction buffer depending on the metabolite to be measured. For measurement of Glu6P, basal absorption was measured at 340 nm from samples added to reaction buffer supplemented with 0.8 mg/ml NADP+ and 10 mM MgCl2. After addition of Glu6P dehydrogenase at a final concentration of 50 μg/ml, the difference in OD340 values was used to calculate the absolute concentration of Glu6P. For measurement of ATP, the stabilized absorption spectrum of the Glu6P samples was taken as the new basal level. Next, hexokinase was added to a final concentration of 100 μg/ml together with 0.5 mM glucose. Stabilized spectra were used for calculating the corresponding ATP concentration.340 values was used to calculate Fru1,6bisP levels. To express metabolite levels in terms of cytosolic concentration, an intracellular volume of 12 μl/mg protein was assumed.For measurement of Fru1,6bisP, the general reaction buffer was supplemented with 0.8 μg/ml NADH, 25 μg/ml triosephosphate isomerase, and 25 μg/ml glycerol-3-phosphate dehydrogenase. After measuring basal absorption, 200 μg/ml aldolase was added, and the difference in OD(i) Preparation of permeabilized spheroplasts. The protocol for preparation of spheroplasts was adapted from previous reports (2HPO4 plus KH2PO4), 1 mM EDTA (pH 8), 10 μl/ml β-mercaptoethanol, and 100 U/ml lyticase. Cell wall digestion at 30°C was followed by sampling the cell suspension for decrease in OD600 when treated with 5% SDS. When the OD600 dropped to around 80%, spheroplasts were considered appropriate for use, and lyticase digestion was stopped by addition of four volumes ice-cold 1.2 M sorbitol buffer. Spheroplasts were washed twice with ice-cold 1.2 M sorbitol and resuspended in spheroplast buffer . Protein concentration was measured in order to dilute the spheroplast suspension to a final protein concentration of 2 mg/ml. To permeabilize the spheroplasts, they were incubated for 10 min at 30°C with 20 μl/ml nystatin prior to measuring enzyme activity. reports . Strains(ii) Determination of specific GAPDH activity. To measure GAPDH activity using permeabilized spheroplasts, the reaction buffer was composed of spheroplast buffer, 2 mM GA3P, 4 mM NAD+, and 20 mM Tris-HCl. The pH of the reaction buffer was adjusted with HCl/KOH to obtain the desired pH values. The influence of Tre6P and Fru1,6bisP on GAPDH activity was examined at pH 6.8. The reaction was initiated by adding 5 μl spheroplast suspension (2 mg/ml) to 145 μl reaction buffer. The initial reaction velocity was calculated from the measured linear increase of absorbance at 340 nm using the Synergy H1 Hybrid reader. Specific GAPDH activity was expressed as nanomoles per minute per milligram protein.Ex: 485 nm) and 390 nm (510Ex: 390 nm). The ratio (510Ex: 485 nm/510Ex: 390 nm) was used to extrapolate the corresponding pH value using a calibration curve obtained by incubation of cell suspensions in calibration buffer at various pHs . For experiments involving addition of NH4Cl, LoFlo medium was buffered with 200 mM Tris-HCl at the desired pH. Prior to treatment, baseline fluorescence was measured for 5 min. Using an integrated dispensing system that allows for quasisimultaneous pipetting and measurement (Thermo Scientific Fluoroskan Ascent FL with 390/510 and 485/510 filter sets), glucose and NH4Cl at final concentrations of 100 mM and 200 mM, respectively, were added at time point zero, after which, fluorescence was measured for the desired time periods. Per strain, emission spectra were measured for at least 9 technical repeats to consider the variability between experiments.For determination of intracellular pH, the pHluorin plasmid was transformed into strains of interest. The cells were grown in uracil dropout medium to retain the plasmid but with LoFlo formulation (ForMedium) to minimize background fluorescence. After reaching the exponential phase on 2% galactose, cells were harvested, washed, and resuspended in LoFlo medium at pH 5.5. Emission spectra were monitored at 510 nm after excitation at the wavelengths of 485 nm . Cells were additionally washed on the filter two more times with water, after which, the loaded filter was transferred to a scintillation vial containing 3 ml liquid scintillation cocktail . To account for background signal originating from filter-attached radiolabeled glucose, three blank measurements per strain were included, for which cells where quenched prior to adding the labeled glucose. In experiments in which the effect of NH4Cl was assessed (4Cl addition. The final concentration of Tris-HCl and NH4Cl was 200 mM. Scintillation counting was performed using the Hidex 300 SL.Transport of [U-r et al.  and Özcar et al. . In shorassessed , Tris-HC


To investigate the protective effects and possible mechanisms of action of resina draconis (RD) on acute liver injury and liver regeneration after 2/3 partial hepatectomy (PH) in mice.  2/3 PH was used to induce acute liver injury. Mice were divided into three groups: sham, vehicle + 2/3 PH, and RD + 2/3 PH. Resina draconis was administered intragastrically after 2/3 PH into the RD + 2/3 PH group, and the same volume of vehicle (1% sodium carboxymethyl cellulose) was injected into the vehicle + 2/3 PH group and sham group mice. The index of liver to body weight (ILBW) and proliferating cell nuclear antigen (PCNA) were assayed to evaluate liver regeneration. Blood and liver tissues were collected for serological and western blotting analysis. α, and EGFR levels were increased in the RD group at postoperative days 2 and 4 compared with the those in the 2/3 PH group.  Resina draconis protected against 2/3 PH-induced acute severe liver injury and promoted liver regeneration as shown by significantly increased ILBW compared with that of controls. 2/3 PH increased serum AST and ALT levels, which were significantly decreased by RD treatment, while 2/3 PH decreased serum TP and ALB, which were increased by RD treatment. In the RD + 2/3 PH group, PCNA expression was significantly increased compared with the 2/3 PH group. Further, hepatocyte growth factor (HGF), TNFα, and EGFR signaling. Our results suggest that RD ameliorates acute hepatic injury and promotes liver cell proliferation, liver weight restoration, and liver function after 2/3 PH, probably via HGF, TNF The liver plays a central role in detoxification and protein synthesis and produces digestive biochemicals. It also possesses the unique ability to regenerate after surgical resection or injury to maintain these essential functions –4. The cα, EGF, and HGF are highly expressed after PH and are thought to be important in regulating hepatocyte activation, proliferation, migration, and survival during liver regeneration [Growth factors such as TGFneration –15. Hepaneration , and HGFneration , 17. Oveneration . Mice laneration .α is an important regulator of the “priming phase” of liver regeneration, and blocking TNFα signaling in rats prior to PH significantly reduces the proliferation of hepatocytes and nonparenchymal liver cells [α, together with its downstream effector IL-6, initiates hepatocyte transition from G0 to G1 phase of the cell cycle [α also activates transcription factors like STAT3 and NF-κB, which in turn promote the expression of cell cycle regulator genes such as cyclin D [TNFer cells , 21. TNFll cycle . TNFα alcyclin D , 25.α because other EGFR ligands compensate for the absence of TGFα [The EGF receptor (EGFR) also plays a vital role in liver regeneration, since liver regeneration is not impaired in mice lacking TGF of TGFα , 26. EGF of TGFα . Transge of TGFα , 28.Dracaena cochinchinensis (Lour.) S. C. Chen, is a Chinese Herbal medicine with a well-established safety and bioactivity record [α, and EGFR signaling in RD-stimulated liver regeneration was also investigated.Resina draconis (RD), a bright resin extracted from y record , 30. RD y record , and proy record . It can y record  and has y record , 35. Fury record , 37. RD y record  and antiy record , 40. Of Resina draconis powder  was dispersed in 1% sodium carboxymethyl cellulose (Na-CMC) at 10 mg/mL, 20 mg/mL, and 40 mg/mL before intragastric injection into mice.n = 8 in each group): sham, vehicle + 2/3 PH, and RD + 2/3 PH. The RD + 2/3 PH group received intragastric injections of 0.2 mL RD solution at doses of 0.1 g/kg, 0.2 g/kg, or 0.4 g/kg body weight once daily for 10 days after 2/3 PH surgery. All experiments were conducted under the institutional guidelines of the Animal Ethics Committee, Kunming Medical University.One-hundred forty-four 6-8 week-old male C57BL/6 mice weighing between 20 and 25 g were used. Animals were divided into three groups  recorded.Peripheral blood was collected and centrifuged. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), albumin (ALB), and total protein (TP) were measured in U/L with a serum multiple biochemical analyzer .β-actin , HGF , EGFR (Abcam), and TNF-α (Abcam). Super Signal West Dura Extended Duration Substrate  was used to visualize antibody-antigen complexes.Liver tissue proteins were prepared as previously described . Westernt test was used to analyze data between two groups. All values were expressed as mean ± SEM, and a p value <0.05 was considered statistically significant.Three-way ANOVA was applied to analyze BW, ILBW, TP, ALB, ALT, ALP, and AST using the factors 2/3 PH operation, RD treatment, and time after injury. Tukey's post hoc tests were applied as appropriate. Student's To address the effect of RD on general mouse health after PH, mouse body weights were monitored after 2/3 PH. The body weights of mice reduced on days 2 and 4 after 2/3 PH followed by weight gain on day 6. RD treatment at different doses induced faster and longer-lasting weight gain than vehicle group over 10 days .The ILBW from days 2 to 10 was measured after 2/3 PH to address the effect of RD on liver recovery. The ILBW was significantly higher on days 2, 4, 6, 8, and 10 in RD-treated mice versus vehicle controls . Of the Resina draconis treatment prevents decreases in serum total protein (TP) and albumin (ALB) after 2/3 PH.p < 0.01)  and albumin (ALB). TP and ALB levels were attenuated in both the 2/3 PH group and the RD-treated 2/3 PH group on days 2, 4, 6, 8, and 10 after PH. However, TP and ALB levels were significantly higher in the RD-treated 2/3 PH group compared with the vehicle + 2/3 PH group    . InteresLiver regeneration is closely related to hepatocyte proliferation. As RD promoted liver recovery, we hypothesized that RD enhanced hepatocyte proliferation. To address this hypothesis, we compared proliferating cell nuclear antigen (PCNA) levels in liver tissues between the different groups. PCNA expression was significantly higher on days 2, 4, and 6 in the RD-treated group compared with the 2/3 PH group Figures , with noα are important growth factors that stimulate hepatocyte mitogenesis in liver regeneration [α expressions were measured in liver tissue by western blotting. In the vehicle + 2/3 PH group and RD + 2/3 vehicle group, HGF, EGFR, and TNFα increased significantly on day 2 after 2/3 PH and decreased progressively to preoperative levels by the end of the observation period. However, the levels of HGF  in the RD + 2/3 PH group than in vehicle + 2/3 PH group and sham group. Further, trends in these increases continued to day 6 and day 8.HGF, EGFR, and TNFneration , 45. To  Figures , EGFR  in RD-treated mice.The exact mechanisms of liver regeneration after 2/3 PH are complicated and have not been fully defined, although a number of growth factors are implicated in the process. HGF, EGFR, and TNFSerum AST and ALT liver function biomarkers are inversely related to the severity of liver damage –68. We fα are involved in this process. These results indicate that RD may be useful in the treatment of acute liver injury.Taken together, our results confirm that intragastric injection of RD after 2/3 PH prevents acute liver failure and promotes liver regeneration. HGF, EGFR, and TNF


The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson’s disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies. Parkinson’s disease (PD) belongs to the family of synucleinopathies, whose hallmark is the accumulation of misfolded aggregates of the protein alpha-synuclein (αSyn) in neuronal and non-neuronal brain cells . The hetRecent studies have further investigated the structure–toxicity relationship in vitro of short human αSynOs (H-αSynOs) ,16. ThesHere, we assessed for the first time the neurotoxic action in vivo in rats of such purified toxic H-αsynOs, by investigating whether unilateral inoculation into the rat substantia nigra pars compacta (SNpc) would recapitulate, in a single model, the crucial hallmarks of PD, including cell loss, pathological deposits of p129-αSyn, and neuroinflammation, as well as motor impairment or non-motor symptoms.Previous studies have investigated PD pathology after inoculation of preformed fibrils of αSyn (PFFs) into dopaminergic areas ,29,30,31Since the progressive neurodegeneration in PD may be caused by pathological events that evolve crosswise disease stages, we here assessed, at progressive time-points, changes in H-αSynO-induced neuropathological hallmarks and associated symptoms. Specifically, we studied the formation of neuronal and microglial p129-αSyn in the injection area as well as in the striatum, as the main projection field of nigrostriatal dopamine neurons; we measured the nigral neuroinflammatory response as Iba-1, MHC-II, TNF-α, and IL-10 immunoreactivity (IR), in order to evaluate microglial activation and changes in cytokine profile induced by the infusion of H-αsynOs; in addition, to better characterize the microglial behavior in response to H-αsynOs, we performed a phagocytosis assay in vitro after pre-exposure of microglia cells to H-αsynOs. We investigated the toxic effect of H-αsynOs on dopaminergic neurons by measuring tyrosine hydroxylase (TH) IR and mitochondrial abnormalities and numbers in TH-positive cells in the SNpc, as well as by assessing striatal dopamine levels. Finally, we investigated whether the H-αsynOs infusion induced any motor or cognitive deficits.In this study we infused oligomers purified in vitro using H-αsynOs that induce significant toxicity when incubated with neuronal cultures . These oWe evaluated whether the infusion of H-αsynOs into the SNpc induced the formation of pathological p129-αSyn within microglia and neurons in the infused SNpc, and whether the p129-αSyn pathology spread via dopaminergic fibers to the striatum; as this is the main SNpc projection area. p129-αSyn was analyzed as a frequently used marker for the pathological form of αSyn highly represented in protein aggregates.p < 0.01 and p < 0.001, respectively by Tukey post hoc)  cells and with TH positive (TH+) cells in the SNpc one, three, and five months after the H-αsynOs infusion. Qualitative and quantitative evaluation revealed the presence of few and small deposits of p129-αSyn inside Iba-1+ cells one month post infusion in the H-αsynOs-infused SNpc, and that significantly increased in number and became larger three and five months post infusion, as compared to all control groups  . p129-αSost hoc) A,D. Thioost hoc) .p < 0.01 vs. vehicle-injected animals (Veh), * p < 0.001 vs. the contralateral SNpc, by Tukey post hoc), suggesting that this was an earlier event preceding microglial uptake of p129-αSyn   C,F, and infusion . We did infusion B,C,E,F, infusion E,F.We investigated neuroinflammatory responses in the SNpc as a neuropathological hallmark of PD. Since a persistent and dysregulated microgliosis has been reported in the brain of PD patients, we measured Iba-1 and major histocompatibility complex (MHC)-II IR as a marker of reactive microglia in the SNpc. Moreover, we measured the levels of cytokines TNF-α and IL-10 within Iba-1 positive cells, in order to investigate whether the microglia acquired a pro-inflammatory, or anti-inflammatory phenotype, respectively.The H-αsynOs infusion induced an early and persistent microglial activation in the SNpc. Iba-1 IR was significantly increased compared to the contralateral side one month post infusion, and further increased three and five months post infusion A see Fi. SpecifiThe infusion of H-αsynOs into the SNpc induced a substantial increase of TNF-α levels within Iba-1-positive cells, compared to all control groups  see B. In conp < 0.001 and p < 0.0001, respectively, by Tukey post hoc)  to H-αsynOs for 24 and 48 h, but not for 6 h, reduced their phagocytic activity against fluorescent beads (ost hoc) A,C. Thisost hoc) B and corost hoc) D, suggesp < 0.0001 by Tukey post-hoc) (p < 0.0001 by Tukey post-hoc).Stereological analysis showed a gradual and persistent loss of dopamine neurons in the SNpc. One month post infusion of H-αsynOs the density of TH+ neurons in the infused SNpc was similar in all experimental groups, confirming that the infusion procedure did not cause any acute cell damage, as also shown by H&E staining performed three days after surgery A,B. Neurost-hoc) A,B. As s3,20 = 0.26, p = 0.85), and a significant reduction after three months  and five months  . The Tukey post-hoc analysis showed that dopamine tissue levels were significantly lower in the ipsilateral striatum of H-αsynOs-injected rats compared to the contralateral side .Since one month post infusion the presence of inflammatory microglia was not associated with any dopamine loss in the H-αsynOs-infused SNpc, we performed mitochondrial-mass measurement, specifically within TH-positive cells, and TEM morphometric analysis to investigate the potential loss or damage of mitochondria at this early time-point. COX IV-stained mitochondria were numerically reduced within TH-positive cells in the H-αsynOs-injected SNpc compared to the contralateral SNpc A–C; more1,126=13.0 p < 0.0005), width , and time  factors. Considering two-way ANOVA among each different time point, while at one month post-infusion of H-αsynOs the rats did not show any motor impairment in any tested beam   and five  months post-infusion  . In addition, an increased number of errors was made by five month infused rats as compared to one and three month infused rats in the beam of 15 mm, suggesting a progression in the development of motor deficits  A, they dinfusion A. Intere < 0.05) B. Specif1,28 = 11.50, p < 0.01). Multiple comparisons did not reveal significance between subjects. However, the subsequent Student’s t-test within each time point, showed significance at three and five months, when comparing vehicle vs. H-αSynOs-infused rats .As shown in 2,28 = 22.88, p < 0.0001). Bonferroni’s multiple comparisons test showed that in both vehicle and H-αSynO-infused rats the distance travelled decrease at five months after infusion, suggesting an age-dependent decrease on locomotor activity.The open field was used to measure the spontaneous locomotor activity of H-αSynO-infused and vehicle-infused rats at progressive time-points following the infusion D. Two-waC. elegans models of αSyn toxicity [We exploited the availability of well-characterized human H-αSynOs, which are kinetically trapped in a toxic conformation associated with a high homogeneity in size and structural properties . We showtoxicity ,24. HereThe presence of fibrillar aggregates of αSyn within LBs is a main hallmark of PD and synucleinopathies, however several pieces of evidence have suggested that the most toxic species of αSyn are small soluble aggregates in the form of oligomers, which form as intermediates during the process of amyloid fibril formation ,10,11,33We showed here that the inoculation of purified human H-αSynOs induced the intraneuronal deposition of p129-αSyn in the infused SNpc as well as in the striatum, analyzed as the main dopaminergic projection area. The phosphorylation process accelerates the deposition of insoluble protein aggregates, and p129-αSyn is highly expressed within Lewy bodies . TherefoBesides neuronal deposits, we found aggregates of p129-αSyn within microglial cells of the H-αSynO-infused SNpc, indicating that p129-αSyn was phagocyted by microglia. Interestingly, the colocalization volume of p129-αSyn within the microglia, and the size of the aggregates, were increased from one to three months post-oligomer infusion, but did not display any further increase at the later time point, suggesting that microglia phagocytosis of p129-αSyn was limited. Since a pathological interaction of αSyn with the phagocytosis machinery has been proposed in PD ,54,55, wH-αSynOs triggered an intense neuroinflammatory response in the SNpc that preceded any frank cell loss and persisted in some aspects up to five months after the oligomer infusion, as shown by the early and increasing Iba-1 staining, early MHC-II staining, and changes in the cytokine profile. Specifically, we observed an early rise of the pro-inflammatory cytokine TNF-α and MHC-II, but not the anti-inflammatory cytokine IL-10, which instead displayed some reduction, indicating a disbalance in cytokine production, and suggesting that microglia acquired a pro-inflammatory profile after the interaction with H-αsynO, resembling PD neuropathology. Interestingly, MHC-II expression in the oligomer-infused SNpc was drastically decreased at three and five months, in line with previous reports , and desH-αSynOs induced gradual damage and degeneration of dopaminergic cells within the SNpc, and a consistent decrease of dopamine content. Hence, dopamine neurons were not numerically decreased one month after the oligomer infusion, but displayed a reduced number and morphologically defective mitochondria, while they underwent cell death at later time points. Therefore, all together, the results revealed mitochondrial damage in dopamine neurons associated with an intense neuroinflammatory response in the SNpc that preceded neurodegeneration, suggesting that H-αSynOs may follow a double toxic pathway, directly interacting with mitochondria as previously shown in vitro ,24,72, aReflecting the evolution of nigrostriatal degeneration, H-αSynOs-infused rats developed a significant motor impairment, that was unveiled by the beam challenging test. This is a highly sensitive test for the detection of sensorimotor deficits in the presence of partial nigrostriatal degeneration in αSyn-based models ,74, and PD pathology following exogenous inoculation of PFFs has been intensively investigated in recent years ,47,78,79Escherichia coli using the plasmid pT7-7 [-1 ampicillin to an OD600 of 0.6.Recombinant H-αSyn was purified following overexpression in id pT7-7 . After tg . The cell pellets were resuspended in lysis buffer  and lysed by sonication. The cell lysate was centrifuged at 22,000× g for 30 min to remove cell debris. In order to precipitate the heat-sensitive proteins, the supernatant was then heated for 20 min at 70 °C and centrifuged at 22,000× g. Subsequently, streptomycin sulphate was added to the supernatant to a final concentration of 10 mg·mL−1, to stimulate DNA precipitation. The mixture was stirred for 15 min at 4 °C followed by centrifugation at 22,000× g. Then, ammonium sulphate was added to the supernatant to a concentration of 360 mg·mL−1, in order to precipitate the protein. The solution was stirred for 30 min at 4 °C and centrifuged again at 22,000× g. The resulting pellet was resuspended in 25 mM Tris-HCl, pH 7.7 and dialyzed against the same buffer in order to remove salts. The dialyzed solutions were then loaded onto an anion exchange column  and eluted with a 0–1 M NaCl step gradient, and then further purified by loading onto a size exclusion column . All the fractions containing the monomeric protein were pooled together and concentrated by using Vivaspin filter devices . The purity of the aliquots after each step was analyzed by SDS-PAGE, and the protein concentration was determined from the absorbance at 275 nm, using an extinction coefficient of 5600 M−1 cm−1.The expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h, and the cells were harvested by centrifugation at 6200× −1. The solution was passed through a 0.22 μm cut off filter and subsequently incubated at 37 °C for 24 h in stationary mode, and without agitation in order to avoid acceleration of fibril formation. Residual fibrillar species were removed by ultracentrifugation for 1 h at 288,000× g using a TLA-120.2 Beckman rotor . The excess of αS monomers in the sample was then removed by means of several filtration steps using 100 kDa cutoff membranes, which resulted in the enrichment of the oligomeric αS species. Samples of the toxic αSyn oligomers prepared in this manner have been found to be stable for many days, but in this study were used within two days of their production. In the case of the fluorescently labelled αSyn oligomers, labelled monomers carrying the AF488 dye  were obtained by using an N122C mutational variant, allowing the dye molecules to react with the thiol moiety of Cys122. The labeled protein was then purified from the excess of free dye by a P10 desalting column with a Sephadex G25 matrix  and concentrated using Amicon Ultra Centricons . Fluorescent oligomers were generated by mixing 90% and 10% of unlabeled and labeled α-syn, respectively. The low ratio of labeled to unlabeled monomers, and the C-terminal position of Cys122 ensuring that both the core of the oligomers and the membrane interaction elements were unperturbed, guaranteed the absence of significant modifications to the properties of the oligomers.Toxic oligomeric samples were prepared from purified recombinant H-αSyn as previously described . These oMale Sprague Dawley rats  were housed in groups of six in a climate-controlled animal room  on a 12 h light/dark cycle (lights on at 7:00 a.m.) with standard chow and water ad libitum. All efforts were made to minimize animal discomfort and to reduce the number of animals used. All experimental procedures complied with the ARRIVE guidelines and were in accordance with the guidelines and protocols approved by the European Community (2010/63UE L 276 20/10/2010). Experimental protocols have been approved by the Italian Ministry of Health (Aut. 766-2020/PR).In order to find the optimal volume and concentration of H-αsynOs, male Sprague Dawley rats weighing 275–300 g were deeply anesthetized with Fentanyl (3 mg/kg), and infused with 5, 10, and 20 μL of fluorescent oligomers at two different concentrations (1 mg/mL and 0.50 mg/mL) in the SNpc  via a steel injector. Three days after surgery, the animals were transcardially perfused with PFA 4%, and the brains were collected and vibratome cut in 40 μm slices containing the SN. A set of slices across the SN were observed under a fluorescence microscope  to verify the infusion site, and to detect the extent of H-αsynOs diffusion within the SN or along the injector trace, and the deposition of large fluorescent aggregates that would prevent diffusion A. Moreovn = 45) or vehicle  at the infusion rate of 1 μL/min  were transferred to the test room 30 min before testing for acclimation, to avoid any alteration in behavioral parameters induced by the novel environment. Tests were carried out during the light time (9:00–14:00 h).One, three, and five months post-surgery, behavioral tests were performed in order to detect motor deficits and cognitive impairment. Rats , and spontaneous motor activity was monitored for 10 min (test period).The total distance travelled was analyzed and expressed in cm, which represented the horizontal distance travelled by an animal.A NOR test was carried out according to a previously published protocol . BrieflyAfter the behavioral tests, the rats were anesthetized and transcardially perfused with 4% paraformaldehyde. The brains were post-fixed and 40 µm thick coronal midbrain and striatum sections were vibratome-cut.n = 8 per group), midbrain sections were pre-incubated with a blocking solution with normal serum, and then immunoreacted with polyclonal rabbit anti-TH  primary antibody. TH-positive cells were visualized using the classic avidin-peroxidase complex  protocol, using 3,30-diaminobenzidine  as a chromogen.For stereological counting (n = 4–5 per group), 3 sections per each animal, from the midbrain or the striatum, were pre-incubated with a blocking solution with normal serum/BSA, and then immunoreacted with primary antibodies for single or double immunolabelling: goat polyclonal anti Iba-1 ; rabbit polyclonal anti-TNF-α ; rabbit polyclonal anti IL-10 ; rabbit monoclonal anti p129-αSyn ; mouse monoclonal anti-TH ; rabbit polyclonal anti COX IV ; mouse monoclonal anti MHC-II . Control slices were incubated without primary antibodies, and all slices were thereafter incubated with the appropriate fluorochrome-conjugated secondary antibodies. For fluorescence visualization of Iba-1 a two-step indirect labelling protocol was used, while a three-step detection was performed to increase the signal of TNF-α, IL-10, and p129-αSyn, as previously described [For immunofluorescence  dissolved in 50% EtOH, and then washed three times in EtOH 80% in order to eliminate unspecific dye residues, as described . Section2) were obtained. Sampling fraction was delimited at low power and cells were sampled with a ×40 oil immersion objective through a defined depth with a 2 μm guard zone. The coefficient of error (CE) for each estimation and animal ranged from 0.05 to 0.1.All immunohistochemical reactions were analyzed by an operator blind to the experimental groups, and different from the experimenter that performed the behavioral tests and histology. TH-immunoreactive neurons were counted bilaterally in the SNc, as previously described . A dedicQualitative and quantitative analysis for markers of neuroinflammation and p129-αSyn was performed on immunofluorescence-stained sections by an operator blind to the experimental groups, using a spinning disk confocal microscope  with a 63X oil objective. The analysis for p129-αSyn colocalized with Iba-1 and TH was performed using a laser scanning confocal microscope Zeiss with a 63X oil objective. Each frame was acquired eight times and then averaged to obtain noise-free images. Surface rendering, maximum intensity, colocalization, and simulated fluorescence process algorithms were used (ImageJ and Imaris 7.3). 3D reconstruction of neuron body and fibers, as well as microglia, containing p129-αSyn is shown in supp. The volumes occupied by Iba-1, MHC-II, TNF-α/Iba-1, IL-10/Iba-1, p129-αSyn/Iba-1, p129-αSyn/TH, COX IV/TH colocalization in SNpc, and p129-αSyn/TH in the dorsal striatum, were determined. For colocalization analysis, a colocalization channel was automatically generated by Imaris 7.3. A stack was obtained from each dataset (20–40 images). In the resulting stacks, ten regions of interest for the SNpc  and five regions of interest for the dorsal striatum  in each acquired section and for each animal were randomly chosen, and volume of the elements calculated. Values were expressed as a volume. The sides contralateral to the stereotaxic infusion were analyzed as inner controls across studies.n = 6 per group) was sonicated in 0.25 mL of 0.2 M perchloric acid and then centrifuged at 9391× g for 15 min at 4 °C. The supernatant was transferred and filtered in a Spin-X centrifuge tube filters (0.45 mm). The filtrate was diluted 1:10. Twenty microliters were injected into an HPLC apparatus, equipped with a rever se-phase column  and a coulometric detector  to quantitate DA. Electrodes were set at +150 mV (oxidation) and 250 mV (reduction). The mobile phase  was pumped  at 1 mL/min flow rate. The assay sensitivity for DA and DOPAC was 10 fmol/sample.Striatal tissue (n = 4) were transcardially perfused with a mix of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.15 M cacodylate buffer. After fixation and rinsing in the same buffer, brains were post-fixed with 1% osmium tetroxide in distilled water for 2 h, and then stained overnight with 0.5% uranyl acetate at 4 °C. The brains were then dehydrated in a graded acetone series and embedded in EPON resin. To identify the SNpc, semi-thin coronal sections of the SN were cut with a Reichert Supernova ultramicrotome and stained with toluidine blue. Ultrathin sections  were observed under a JEOL JEM 1400 Plus electron microscope, equipped with a CCD camera, at an acceleration voltage of 80 kV.One month post infusion, H-αsynOs-infused rats (2) within the SNpc infused with H-αsynOs and the contralateral SNpc as a control. A total of 40-50 unitary area fields were considered in each hemisphere. More than 6700 mitochondria were randomly sampled on 505 non-overlapping micrographs at a final magnification of 10,000×. Swollen cristae were recognized when the individual cristae size  doubled the average cristae size.The total number of mitochondria, the percentage of swollen cristae mitochondria  was evaluated in the unitary area  were added 2, 26, and 44 h after seeding. Control cells were supplemented with equivalent volume of vehicle at the same times. Then, 48 h after the first treatment all the cells were tested for phagocytotic capacity as indicated below. Alternatively, the cells were incubated with different concentrations of α-synOs the day after seeding for 24 h as previously described [2, and 1 mM CaCl2, and pre-opsonized by addition of 50% FBS and incubation for 30 min in 5% CO2 at 37 °C. 12 × 106 pre-opsonized microspheres were resuspended in 12 mL of DF culture medium without FBS, distributed on cells (around 5 beads/cell), and incubated for 2.5-3 h. Cells were washed with PBS, trypsinized for 4 min and collected with DF medium in conical tubes, washed with PBS, and suspended in 0.3 mL PBS containing 1% FBS and 0.05% EDTA. Samples were acquired in the green channel (502 nm) by FACS Aria  within 20 min. The whole experiment was repeated three times.The murine microglial cell line MMGT12 was used. Cells were cultured in DF culture medium comprising DMEM/F12 , supplemented with 10% fetal bovine serum (FBS) without antibiotics, grown in humidified atmosphere of 5% COescribed . Briefly® 6 for Windows (GraphPad software). Histological, HPLC, and in vitro data were statistically compared by one-way ANOVA followed by Tukey post-hoc test, or by Student’s t-test for electron microscopy, as data did not follow a normal distribution. The level of significance was set at p < 0.05.Statistical analysis was carried out by Statistica 8 . Behavioral data were presented as mean ± SEM and were analyzed by three-, two- or one-way analysis of variance (ANOVA), with groups, months, and width as factors. Post-hoc comparisons were made using Tukey, Bonferroni multiple comparison test, or t test, where appropriate. Results were analyzed using GraphPad Prism


Participants underwent vastus lateralis muscle biopsies, a hyperinsulinemic–euglycemic clamp, oral glucose tolerance tests and body composition testing. Basal skeletal muscle ANGPTL4 mRNA was lower in men than women (p < 0.01). Peroxisome proliferator-activated receptor (PPAR) alpha (PPARα) mRNA expression was higher in men than women (p < 0.05). There were no significance changes in serum or skeletal muscle ANGPTL4  or muscle PPARα mRNA expression after AEX + WL. Muscle mRNA ANGPTL4 is correlated with serum ANGPTL4 , body fat , and glucose utilization . AEX + WL does not change basal or insulin-stimulated skeletal muscle ANGPTL4 mRNA expression, suggesting other factors contribute to improved insulin sensitivity after the loss of body fat and improved fitness.Angiopoietin-like protein 4 (ANGPTL4) is an adipokine that plays an important role in energy homoeostasis and lipid and lipoprotein metabolism. This study was designed to determine the effect of an exercise plus weight loss intervention on ANGPTL4 expression and its relationship with metabolic health. Thirty-five obese sedentary men ( Angiopoietin-like protein 4 (ANGPTL4), a member of the angiogenin-like protein family, is expressed in a wide variety of tissues, such as liver, adipose tissue, skeletal muscle, placenta, small intestine, brain, thyroid, kidney, spleen, pituitary gland, hypothalamus and heart ,6,7,8, wIt is possible that ANGPTL4 could be modified by exercise or diet as regular exercise training is an effective stimulant of muscle metabolism remodeling. Immediately after an acute bout of endurance and strength exercise, both serum and skeletal muscle ANGPTL4 mRNA increase in healthy, sedentary middle-aged men . SimilarANGPTL4 is the target of peroxisome proliferator-activated receptors (PPAR) and induced by PPARα in mice and rat models ,17,18. Mn = 35) before and after the intervention are presented in p < 0.001) with a decrease in the percentage of body fat of 11% (p < 0.001), a 17% decrease in total fat mass (p < 0.001), a 20% decrease in visceral adipose tissue (VAT), a 16% decrease in subcutaneous abdominal fat and a 12% decrease in mid-thigh subcutaneous fat (p < 0.001). There was no change in fat-free mass (FFM) or mid-thigh muscle area (p < 0.05). VO2max (mL/kg//min) increased 21% after aerobic exercise + weight loss (AEX + WL) (p < 0.001).The baseline physical and metabolic characteristics and gene expressions of the subjects  and insulin AUC120 and insulin AUC180 during the OGTT both decreased 17% (p < 0.01). Glucose utilization and insulin sensitivity increased between 18 and 40% . There were no significant changes in skeletal muscle ANGPTL4 mRNA, PPARα mRNA and serum ANGPTL4 protein level after AEX + WL (± 0.17 vs. 0.724 ± 0.150 AU) or after (0.817 ± 0.139 vs. 0.858 ± 0.218 AU) AEX + WL .At baseline, 20 subjects had normal glucose tolerance, 13 had impaired glucose tolerance, and 2 had unconfirmed diabetes . FastingAEX + WL . There wAEX + WL . There wn = 18) and women (n = 17), are presented in 2max than women . The men had a significantly lower percent of body fat than women. Women had higher total cholesterol, HDL-cholesterol and LDL-cholesterol than the men (p < 0.05). Fasting glucose, insulin and serum ANGPTL4 were not significantly different between groups. Skeletal muscle ANGPTL4 mRNA was lower in men than women (p ˂ 0.01) (n = 10) vs. NGT women (n = 10): 0.14 ± 0.19 vs. 1.07 ± 0.22, p < 0.05) and (IGT men (n = 8) vs. IGT women (n = 7): 0.63 ± 0.13 vs. 1.33 ± 0.26, p < 0.05). PPARα mRNA was higher in men than in women (p ˂ 0.01) (± 0.119 vs. 0.493 ± 0.095 AU) or women (1.174 ± 0.167 vs. 1.307 ± 0.244 AU). In addition, there were no significant changes in serum ANGPTL4 in men (343.8 ± 48.0 vs. 372.3 ± 70.4 pg/mL) or women (374.3 ± 71.4 vs. 450.5 ± 94.4 pg/mL).Baseline physical and metabolic characteristics, and the gene expressions of men ( ˂ 0.01) A. There  ˂ 0.01) B. There p < 0.05, 2max . Muscle ANGPTL4 was associated with M, only when expressed per FFM  and women . Serum ANGPTL4 was associated with BMI , percent body fat  and fat mass .Skeletal muscle ANGPTL4 mRNA was associated with serum ANGPTL4 in the total group  between men and women could explain the sex differences in the regulation of muscle ANGPLT4 levels. ANGPTL4 serum levels are significantly higher in obese patients with abnormal glucose tolerance and positively correlated with BMI, waist circumference, fat mass, HbA1c, HOMA-IR, fasting triglycerides, and with inflammatory markers [We also examined the associations between muscle ANGPTL4 and obesity, aerobic capacity, glucose tolerance and insulin sensitivity. Our results are in agreement with studies that demonstrate that ANGPTL-4 levels are positively correlated to BMI, abdominal obesity (by waist circumference) and fat mass . Our obs markers . Althoug2max, in contrast to our findings. We are unaware of any studies that have examined the relationship between muscle ANGPTL4 and VO2max. Since all the participants in our study were sedentary at baseline, the variability in fitness levels was limited. It is possible that including a wider range of VO2max might result in different findings.In young and middle-aged adults, serum levels of ANGPTL4 were inversely related to fasting glucose, and HOMA-IR levels were lower in patients with type 2 diabetes than healthy adults . Using aOur data show that the baseline levels of ANGPTL4 and PPARα are positively correlated in both genders. However, the expression of ANGPTL4 in skeletal muscle is much higher in women than in men, while the expression of PPARα is the opposite, implying that PPARα is more efficient (requires a lower concentration) in regulating the expression of ANGPLT4 in women than in men. According to other studies ,25, a PPThe study limitations include the small sample size for the measurement of skeletal muscle PPARα and the absence of measurements of PPARδ and PPARγ which could be examined in future studies. Results are generalizable to generally healthy older mostly Caucasian adults and would need to be confirmed in other racial and ethnic groups. However, we conducted a rigorous exercise and weight loss program and employed the gold-standard glucose clamp and had sophisticated methods to measure fitness and body fat. Furthermore, we provided food for two days prior to the glucose clamp and the muscle biopsy; thus, controlling for any immediate effect of dietary intake. This is important given that meal fatty acid composition affects plasma ANGPTL4, as ANGPTL4 is secreted from human muscle after a high-saturated fatty acid mixed meal , but pla2; range of 25–46 kg/m2) and sedentary (<20 min of aerobic exercise 2×/week). They were between the ages of 54–77 years, of whom 27 were Caucasian and 8 were African–American. All subjects were nonsmokers, showed no evidence of cancer, liver, renal or hematological disease, or other medical disorders by medical history and physical exam. A graded exercise treadmill test excluded those with asymptomatic coronary artery disease. Individuals with untreated hypertension (blood pressure higher than 160/90 mm Hg) or hyperlipidemia  were referred to their doctor for therapy. The women in the study were post-menopausal and had not menstruated for at least 1 year. Participants (n = 35) met all the study criteria (18 men and 17 women). Samples from the men were from a study of genetics and insulin sensitivity. Samples from the women were a subset of a prior investigation in our lab [Study entry criteria were men and post-menopausal women 50–80 years of age who were nonsmokers and had no previous diagnoses of diabetes or cardiovascular disease. The participants also had to be weight stable (<2.0 kg weight change in the past year), overweight and obese . All sessions were supervised by exercise physiologists. Exercise sessions included a warm-up and a cool-down phase for each of 5–10 min. The average compliance, defined as attending each of the exercise sessions and weight loss classes was greater than 75%.The research was conducted at the University of Maryland Baltimore, and Baltimore Veteran Affairs Medical Center from 2001–2008, and lab analysis in 2018–2019. Adults were enrolled in a dietary induced weight loss plus moderate-to-high intensity aerobic exercise intervention (AEX + WL) for 6 months. Before beginning the weight-loss intervention, all subjects completed an initial 7-day food record and then received instructions for maintaining stable weight, through a Therapeutic Lifestyle Changes (TLC) diet , from a 2max was measured using a continuous treadmill test protocol, as previously described [VOescribed . Height escribed .± 2%) for at least two weeks prior to testing before and after the AEX + WL and were provided with a eucaloric diet for two days before the clamp by an RD to control nutrient intake [All participants were weight stabilized . Plasma insulin was measured by RIA . Glucose and insulin total area under the curve at 120 and 180 min  were calculated by the trapezoidal method. The lipid levels were obtained from three separate fasting visits and the results averaged as previously described .−2.min−1  [Testing was conducted after the participants underwent a 12-h fast. Peripheral tissue sensitivity to exogenous insulin (M) was measured using the hyperinsulinemic–euglycemic clamp technique  for 180 lis, IN) . Three mlis, IN) . The PriANGPTL4 serum level was measured in duplicate by enzyme-linked immunosorbent assay (ELISA) , according to the manufacturer’s instruction. There was no serum collected for five participants.p < 0.05. Data were analyzed by SPSS statistical software  and expressed as mean ± SEM.Differences between pre-intervention and post-intervention measures of variables were determined using paired t-test. Missing data are denoted in the text or by sample sizes in the tables and figure legends. Univariate regression analyses were used to determine relationships between ANGPTL4 expression and outcome variables. Statistical significance was set at Our data indicate that skeletal muscle ANGPTL4 and PPARα exhibit differences by sex. ANGPTL4 expression in skeletal muscle is associated with serum ANGPTL4, glucose metabolism and obesity. Although six months of exercise training and weight loss did not change skeletal muscle AGNPTL4 in older adults, future investigations could explore the response of ANGPTL4 expression in adipose tissue.


As an important anti-HBV drug, pegylated interferon α (PegIFNα) offers promising clinical efficacy, but biomarkers that accurately forecast treatment responses are yet to be elucidated. Here, we evaluated whether HBV RNA could act as an early monitor of pegylated interferon responses.We analyzed a phase 3, multicenter, randomized cohort of 727 HBeAg-positive non-cirrhotic patients receiving a 48-week treatment of PegIFNα-2a or PegIFNα-2b and a 24-week treatment-free follow-up. Serum levels of HBV RNA, HBV DNA, HBeAg, and HBsAg were measured at weeks 0, 12, 24, 48, and 72.p value < 0.05). HBV RNA ≤ 1000 copies/mL was an optimized cutoff at week 12 that offered better prediction than other HBV biomarkers. This optimized cutoff plus patient age, HBV genotype B, and HBeAg offered a strong estimation of HBeAg seroconversion .HBeAg seroconversion and HBsAg loss at week 72 were observed in 217 (29.8%) and 21 (2.9%) patients, respectively. During the 48-week treatment, HBV RNA decreased more rapidly than HBV DNA and HBsAg, but HBV RNA and HBeAg shared similar dynamics with positive correlations. Multivariate regression analyses consistently revealed the significance of HBV RNA at weeks 0, 12, 24, and 48 to monitor HBeAg seroconversion but not HBsAg loss. Although baseline HBV RNA only showed a modest AUC performance, HBV RNA with a significant increase of AUC at week 12 outperformed other HBV biomarkers to forecast HBeAg seroconversion (HBV RNA at week 12 is an effective monitor of HBeAg seroconversion in HBeAg-positive patients treated with pegylated interferons.The online version of this article (10.1007/s12072-020-10015-3) contains supplementary material, which is available to authorized users. To offer effective treatment responses towards hepatitis B virus (HBV) infections, eight FDA-approved drugs belonging to two classes are currently available, including two interferon alfa drugs and six nucleos(t)ide analogs . HBeAg sHBV RNA has been recently proposed as a new biomarker to assess treatment responses, because HBV RNA transcripts produced directly from HBV cccDNA may monitor the progressive clearance of HBV cccDNA reservoirs –9. As a Despite encouraging findings above, many aspects of HBV RNA remain unclear. First, dynamic changes of HBV RNA in large-scale cohorts of interferon-treated patients remain poorly understood, since previous studies focused mostly on nucleos(t)ide analogs. Second, it is ambiguous if HBV RNA acted as an effective predictor of interferon responses, particularly in a large cohort with non-responders. Third, to offer better responses, it remains unclear whether HBV RNA in combination with known HBV biomarkers could offer better predictive performance. Last, it remains unclear whether HBV RNA could predict HBsAg loss which is generally considered as a functional “cure” that signifies a favorable outcome of antiviral treatments , 15. To ®, Roche, Switzerland) or PegIFNα-2b  at the dose of 180 μg/week for 48 weeks and a treatment-free follow-up for 24 weeks  and ClinicalTrials.gov (NCT01760122). Inclusion and exclusion criteria were described in our clinical protocol (see Supplementary).Between March 2013 and July 2015, a total of 855 HBeAg-positive non-cirrhotic patients who were infected with HBV mono-infection and treatment-naïve for at least 6 months were treated with either PegIFNα-2a or PegIFNα-2b in a phase 3, multicenter, randomized, controlled clinical trial . Patient®). More details are provided in our Supplementary.Routine physical examinations with biochemical and hematologic assessments were conducted at weeks 0, 12, 24, 48, and 72. HBV RNA was extracted from patient serum samples (200 μL) using a nucleic acid extraction kit (Sansure Biotech Inc. China) which was developed based on the magnetic bead technology . Eluted ® Amplicor HBV Test, Version 2.0 . ALT assay was conducted using Architect c8000 clinical chemistry analyzer  with the IFCC standard for enzyme determination. The NCBI HBV genotyping tool [10 values of four biomarkers above were transformed prior to statistical analyses.Elecsys tests  were used to measure HBeAg, anti-HBe, HBsAg, and anti-HBs. HBV DNA was quantified by the Roche Diagnostics Cobasing tool  was applHBeAg seroconversion, also called serological response (SR), was defined as the disappearance of HBeAg accompanied by the gain of anti-HBe throughout the treatment-free follow-up . HBsAg lFour major statistical analyses were performed in our study. First, to explore statistical differences of biomarker values between patient groups, Fisher’s exact tests and Mann–Whitney tests were conducted for categorical and continuous variables, respectively. Logistic regressions were performed to estimate the odds ratio (OR) and 95% confidence intervals (95% CI). Second, we quantified correlations between continuous biomarkers using Spearman correlation coefficients. Third, logistic linear regression analyses revealed factors significantly associated with treatment responses. Predictive performance was measured by AUC analyses, while HBV biomarker values below detection limits were replaced with their detection limits. Fourth, a standard software, Cutoff Finder, was applied to optimize biomarker cutoffs . The stan = 427, 58.7%), followed by genotypes B , D , and B/C recombinant . At baseline, the mean and standard error of patient age, body weight, and ALT were 28.2 ± 0.3 years, 63.6 ± 0.4 kg, and 193.0 ± 5.5 IU/mL, respectively . Responses of HBsAg loss also showed no difference between the PegIFNα-2a and PegIFNα-2b arms . Neither host  nor viral biomarkers  showed any significant difference between the PegIFNα-2a and PegIFNα-2b arms (Table S1). For subsequent analyses, patients who achieved serological responses and HBsAg loss were sorted into the SR and HL groups, respectively.HBeAg seroconversion and HBsAg loss at week 72 were observed in 217 (29.8%) and 21 (2.9%) patients, respectively. HBeAg seroconversion was found in 26.5% (66/249) of PegIFNα-2a-treated patients, similar to 31.6% (151/478) of patients who received PegIFNα-2b , while its correlation coefficients with HBsAg were the weakest at week 0, 12, or 24 . Similar patterns were also observed in the PegIFNα-2a or PegIFNα-2b arm. Overall, strong correlations of HBV RNA with HBeAg supported that HBV RNA might be a potential predictor of HBeAg seroconversion.First, one-sample Kolmogorov–Smirnov tests showed that HBV RNA at individual sampling timepoints did not follow a normal distribution. Second, non-parametric Spearman correlation analyses revealed positive coefficients in the pairwise correlations between HBV RNA and HBeAg, HBsAg, HBV DNA. Third, HBV RNA was strongly correlated with HBeAg and HBV DNA , whereas pairwise comparisons between other factors all exhibited significant differences  or HBeAg seroconversion (n = 4). Two (9.5%) of 21 HL patients and 8 (3.7%) of 217 SR patients showed no decline of HBV RNA at week 12. Moreover, HBV RNA stabilization after treatment cessation from week 48 to 72 was more likely maintained in the SR than non-SR patients .Three independent analyses were performed to explore if HBV RNA served as a favorable monitor to foretell interferon responses. First, HBV RNA shared a decreasing pattern similar to HBeAg in the SR group which harbored 217 patients with HBeAg seroconversion , whereas gender and PegIFNα types showed no difference. Multivariate analyses further showed that HBV RNA plus HBeAg and patient age remained significant at weeks 0, 12, 24, and 48 . Moreover, HBV RNA showed an advantage over HBeAg  at week 12, and its performance was also superior to HBV DNA and HBsAg at weeks 0, 12, and 24 . However, compared with HBsAg, HBV RNA was only a modest predictor of HBsAg loss  to week 12 (AUC = 0.77 ± 0.05), week 24 (AUC = 0.78 ± 0.04), and week 48 (AUC = 0.80 ± 0.03)  comparable to HBsAg and HBV DNA. Compared with baseline results, the optimized cutoff of HBV RNA ≤ 1000 copies/mL at week 12 offered significant increases of accuracy (76.6%), negative predictive value (85.3%), and positive predictive value (56.3%). The former two measures showed the best performance compared with HBeAg, HBsAg, and HBV DNA cutoffs (Table S3).To identify simple cutoffs for clinical use, HBV biomarker cutoffs were optimized to forecast HBeAg seroconversion. The baseline cutoff of HBV RNA at 6.0 log10 copies/mL was observed in 174 (95.6%) of 182 patients who failed to achieve HBeAg seroconversion. This cutoff also showed the best performance of accuracy and positive predictive value compared to that of HBV DNA, HBsAg, and HBeAg cutoffs at week 12 (Table S3).A negative predictive value above 95% is a useful indication to discontinue interferon treatments. At week 12, HBV RNA > 5.2 logp value < 0.01) . This hypothesis was supported by dual combinations that adding HBV RNA to individual traditional HBV biomarkers significantly increased AUC performance  offered the best performance that their stepwise combinations gradually increased the accuracy (95.2%) to predict HBeAg seroconversion  to identify the best combination that achieved the highest rate of SR with the minimum variable size. A combination of four 12-week factors  screened from two different clinical trials . Despite this, our results and literature results support that low levels and fast decreases of HBV RNA are likely associated with treatment success even in the early period [Kinetics of HBV RNA may vary due to different therapies and treatment duration, but a decline of serum HBV RNA is commonly observed during the treatment of interferons and/or nucleos(t)ide analogs , 13, 23.y period .10 copies/mL at week 12, because more than 95% of these patients were unable to achieve HBeAg seroconversion. In addition to the predictive performance of individual biomarkers, our study proved that multivariate combinations of HBV RNA plus HBeAg, patient age, and genotype B offered high levels of positive predictive value (95.2%) and specificity (99.8%). This provides clear evidence that HBV RNA can be effectively integrated with traditional biomarkers to monitor treatment responses.HBV biomarker cutoffs are commonly used to monitor treatment responses in clinical practice. Based on our cohort of 727 patients, the 12-week cutoff of HBV RNA was optimized at 1000 copies/mL that effectively predicted HBeAg seroconversion. The favorable performance of HBV RNA cutoff was also highlighted by its better accuracy (76.6%) compared with HBV DNA, HBsAg, and HBeAg cutoffs (Table S3). Furthermore, early discontinuation of interferon treatments could be considered in HBeAg-positive patients who showed HBV RNA > 5.2 logThere are limitations to our study. We analyzed a phase 3 clinical trials of 727 Chinese patients mainly infected with HBV genotype B or C (98.9%), while future studies need to recruit patients infected with other HBV genotypes. Our study focused on interferon treatments in HBeAg-positive non-cirrhotic patients, while future analyses need to clarify HBV RNA in HBeAg-negative patients and associations between HBV RNA and cirrhosis or hepatocellular carcinoma. The full-length HBV RNA could be quantified using our primers taken from previous publications , 23, butOverall, our findings suggest that serum HBV RNA may serve as an early on-treatment predictor of HBV cccDNA activity to reveal the treatment success of interferon therapies. Although standard HBV RNA toolkits are yet to be developed for clinical use , the preSupplementary material 1 (PDF 1512 kb)Below is the link to the electronic supplementary material.


Escherichia coli (STEC) can cause a wide range of symptoms from asymptomatic carriage, mild diarrhea to bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). Intimin, encoded by the eae gene, also plays a critical role in STEC pathogenesis. Herein, we investigated the prevalence and genetic diversity of eae among clinical STEC isolates from patients with diarrhea, BD, HUS as well as from asymptomatic STEC-positive individuals in Sweden with whole-genome sequencing. We found that 173 out of 239 (72.4%) of clinical STEC strains were eae positive. Six eae subtypes  were identified eae and its subtype γ1 were significantly overrepresented in O157:H7 strains isolated from BD and HUS patients. ϵ1 was associated with O121:H19 and O103:H2 strains, and β3 to O26:H11 strains. The combination of eae subtype γ1 and stx subtype (stx2 or stx1+stx2) is more likely to cause severe disease, suggesting the possibility of using eae genotypes in risk assessment of STEC infection. In summary, this study demonstrated a high prevalence of eae in clinical STEC strains and considerable genetic diversity of eae in STEC strains in Sweden from 1994 through 2018, and revealed association between eae subtypes and disease severity.Shiga toxin (Stx)-producing  Escherichia coli (STEC), is an enteric foodborne pathogen that can be asymptomatic or cause mild diarrhea, bloody diarrhea (BD) or even hemolytic uremic syndrome (HUS) in infected humans [Shiga toxin (Stx)-producing d humans ,2. HUS id humans . Serotypd humans . Neverthd humans . Ruminand humans . Direct d humans .stx genes located on lambdoid prophages, has two types, Stx1 and Stx2, where Stx2 shows much stronger correlation with severe symptoms [stx shedding is a main cause of secondary person-to-person  transmission, the longer duration poses a high transmission risk [eae gene located in the locus of enterocyte effacement (LEE) pathogenicity island [Pathogenicity of STEC in human is largely dependent on Stx, which is considered as the most important virulence factor. Stx, which is encoded by symptoms ,11. The ion risk . Besidesion risk ,13. Intiy island , plays ay island ,16. Alsoy island . Moreovey island .eae gene is about 2,800 base pairs (bp). eae has several subtypes, owning to its heterogeneous 3’ regions, which encoded protein that has been identified to be the intimin cell-binding domain (Int280a) [eae subtypes, i.e. α, β, γ, ϵ, ξ, z, η, θ, τ, ι, κ, λ, μ, ν, υ, ο, π, ρ and σ, that have been defined so far [et al. showed that eae subtype γ1 appeared to be the most frequent among O157:H7 and O145:H28/H25/H- strains [Escherichia coli (EPEC), where intimin β1 was suggested to be the most frequent subtype among atypical EPEC strains from diarrheal patients [eae-positive STEC strains, especially clinical strains, have rarely been described. Moreover, the relationship between eae subtypes and clinical symptoms, as well as duration of stx shedding remains to be addressed.The full length of the Int280a) . There ad so far . It has d so far . Roger e strains ,23. A prpatients . Howevereae subtypes and polymorphisms among clinical STEC strains isolated from STEC-positive individuals present with varying symptoms in Sweden, and to assess the association of eae subtypes with disease severity.The aim of this study is therefore to investigate the The study was approved by the regional ethics committees in Gothenburg (2015/335-15) and Stockholm (2020-02338), Sweden, respectively.stx-PCR-positive sample to the first negative sample, and clinical symptoms were classified into HUS, bloody diarrhea (BD) and non-bloody stool (NBS) [A total of 239 STEC strains were isolated from STEC-infected individuals from 1994 through 2018 in Sweden. Clinical data of STEC patients were collected through reviewing medical records as well as routine praxis used for the STEC surveillance performed in Sweden. The duration of bacterial shedding was determined as the time period from the first ol (NBS) .de novo assembled with SKESA (version 2.3.0) [de novo assembled with SPAdes (version 3.12.0) in “careful mode” [Bacterial DNA was extracted and whole genomes were sequenced by Illumina HiSeq X platform at SciLifeLab  and Ion Torrent S5 XL platform  at The Public Health Agency of Sweden as previously described . The Illn 2.3.0) . The Ionul mode” . The genul mode” . The asshttp://www.genomicepidemiology.org/) using BLAST+ v2.2.30 [stx subtypes were determined by ABRicae version 0.8.10 (https://github.com/tseemann/abricate). An in-house stx subtyping database was created with ABRicate by including representative nucleotide sequences of all identified stx1 and stx2 subtypes. The assemblies were then used to search against the stx subtyping database. Multilocus sequence typing (MLST) was conducted in silico using the on-line tool provided by the Warwick E. coli MLST scheme website . Sequences types (STs) were determined based on the seven housekeeping allelic genes  profile.Serotype was determined by comparing assemblies to the SerotypeFinder database   based on eae sequence polymorphism was used to determine the diversity within each eae subtype.The complete sequences of the escribed , a 95% neae-positive clinical STEC strains in this study and strains from other sources, the MLST allelic profiles of eae-positive strains isolated from animals, meat and humans reported in a recent survey [To assess the relationship of t survey  were useeae subtypes and bacterial features or clinical outcomes, the statistical significance was determined by Statistica12 , p-value <0.05 was considered statistically significant.Fisher's exact test was used to analyze the association between eae was present in 173 (72.4%) strains, including 56 HUS-associated strains, and 117 non-HUS strains . All 65 O157:H7 strains and 108 (62.1%) non-O157 strains carried eae (p<0.0001). eae was overrepresented in strains from children . The presence of eae was significantly associated with BD, HUS, and O157:H7 , gamma1 (γ1), beta3 (β3), theta (θ), zeta3 (ζ3) and rho (ρ), were assigned in 173 eae-positive STEC strains. γ1 was present in 39.3% of all strains, being the most predominant subtype, followed by ϵ1 (30.6%) and β3 (24.9%). GTs were analyzed to determine the diversity within each eae subtype. Twenty-nine unique eae sequences were obtained, among which, ϵ1 subtype had ten GTs (GT1-GT10), followed by β3 (GT1-GT8), γ1 (GT1-GT5), θ (GT1-GT4), ζ3 (GT1) and ρ (GT1) .Six  ρ (GT1) . Isolateferences . Notably strains . No assoeae-positive strains were typed into seventeen O serogroups and nine H types, which were assigned into 17 serotypes, O157:H7  was the most predominant serotype. O157:H7 strains harbored eae-γ1 , ϵ1  and θ . γ1 was found to be statistically associated with O157:H7, while ϵ1 and β3 were related to non-O157 serotypes . All O121:H19 strains carried eae with one exception. ϵ1/GT7 was found to be linked to O121:H19 (p<0.0001).In total, 173 erotypes . Among nstx subtypes and combinations were identified in 173 eae-positive STEC strains, namely, stx1a, stx2a+stx2c, stx2a, stx2c, stx1a+stx2a, and stx1a+stx2c, among which 2stx (57.2%) was more prevalent than stx1 (35.3%). We found that the presence of stx1+eae was significantly more prevalent in non-HUS STEC strains compared with HUS associated STEC strains. Notably, the presence of stx2+eae was significantly linked to HUS associated STEC strains, and stx1+stx2+eae was linked to BD-related strains .eae-positive STEC isolates (Table S1). ST11 was the most common sequence type, all 61 O157:H7 strains belonged to ST11. A minimum spanning tree was generated using 22 STs from this study and 18 STs from other sources reported previously [eae subtypes, the majority strains belonging to the most predominant eae subtype γ1 were grouped closely with a few exceptions. Similarly, strains with the same other eae subtypes were more likely to cluster closely (Twenty-two sequence types (STs) were found in the 173 eviously . Interes closely .Figure eae are suggested to be more pathogenic with a higher risk of developing HUS [eae gene in STEC strains derived from patients as well as their association with disease severity. Here, we performed molecular characterization of eae-positive STEC strains from patients with a variety of symptoms as well as asymptomatic carriers. We found that 72.4% of clinical STEC strains were eae positive, out of which 37.6% were O157 strains, and 62.4% were non-O157 strains. All clinical O157 strains were eae positive, while 62.1% of non-O157 strains carried eae, which is much higher than that of reported in a recent study where only 9.5% of non-O157 strains carried eae [eae, which was significantly higher than eae prevalence in non-HUS STEC strains (65.4%). Additionally, eae positive rate in strains isolated from patients with BD (86.3%) was higher than that of individuals with NBS (57.0%). It’s well-recognized that O157 is the primary cause of HUS [eae was significantly more prevalent in O157 strains, which may partially explain severe clinical outcomes of O157 strains.STEC strains harboring ping HUS . Little ried eae . We foune of HUS . We founeae sequences in 173 STEC strains were classified into six subtypes, namely ϵ1, γ1, β3, θ, ζ3 and ρ. eae-γ1 and ϵ1 were the most common subtypes in this study. The prevalence of eae subtypes varies among studies. In a previous study, β1 and ζ3 were the most prevalent eae subtypes among STEC strains from different sources including diarrhea patients, raw beef and mutton, cattle, and yak [eae-γ1 and β1 were reported to be the most widespread subtypes in STEC strains isolated from patients in Germany [eae alleles examined in STEC strains isolated from ruminant animals also showed great genetic diversity. β and ζ were the most common eae subtypes in strains isolated from sheep, while β and θ were more prevalent in strains from cattle [ϵ1 and γ1 were the most frequent eae subtypes among STEC strains isolated from healthy cattle [eae subtype β, ϵ, γ1, and θ are linked to more virulent strains [γ1 was associated with severe clinical symptoms such as BD and HUS, highlighting the clinical significance of eae subtype γ1. However, the underlying mechanisms how different eae subtypes modulate the pathogenicity remains to be elucidated.The  and yak . eae-γ1  Germany . eae allm cattle . ϵ1 and y cattle . The rea strains . Here, weae-positive STEC isolates. An earlier study showed association between serotypes and eae subtypes: O157 and O145 strains tended to harbor γ1, O103 and O121 harbored ϵ, O26 carried β, while O111 possessed θ and β [γ1 was significantly overrepresented in O157:H7 strains, also in line with a previous report of STEC strains derived from humans in Switzerland [ϵ1 was found to be prevalent in O121:H19 and O103:H2 strains. β3 was predominant in O26:H11 strains.A diverse range of serotypes were observed among  θ and β . In the tzerland . Similarϵ1 was found in four O157:H7 strains, which has been rarely reported before. In Germany, two O157:H16 strains isolated from diarrheal children carried eae-ϵ [eae-ϵ [eae-ϵ subtype.Notably, ed eae-ϵ . Anotherϵ [eae-ϵ . More dastx and eae, especially stx2, are more likely to enhance virulence and increase the severity of clinical outcomes in humans than those carrying stx1 alone [stx2+eae in STEC strains is strongly associated with HUS. Interestingly, the presence of stx1+stx2+eae was linked to BD, while stx1+eae was associated with NBS, supporting the evidence that the presence of stx2, rather than stx1, together with eae was associated with severe disease. Besides the finding that eae subtype γ1 was associated with HUS and O157:H7, γ1 was also found to be associated with stx2a+stx2c and stx1a+stx2c, these are high virulent stx subtypes, which could also contribute to the severity of clinical symptoms.The coexistence of x1 alone . Consiststx shedding poses higher risk for the transmission of STEC strains from person to person, STEC-infected patients below 15 years old are usually associated with longer shedding duration [eae was associated with children. However, the presence of eae and subtypes has no association with the duration of stx shedding. As the information of age and the duration of shedding for some individuals is missing, further research is needed to understand the role of eae in children with longer duration of shedding.Longer duration of duration ,43. Consduration . In thiseae genes in clinical STEC isolates from Sweden from 1994 through 2018. Our results show that the majority of the clinical STEC isolates carry eae genes, which demonstrate highly genetic diversity. We found associations between eae subtypes and certain serotypes. Furthermore, eae subtype γ1 is associated with strains causing severe symptoms. However, no correlation was observed between the presence of eae gene/subtypes and duration of bacterial shedding. Our study proposes that the coexistence of eae subtype γ1 and stx2 or stx1+stx2, could be used as a risk predictors for severe symptoms of STEC infections.In conclusion, here we describe the prevalence and genetic diversity of Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.