Immunoglobulin E (IgE)-associated allergy affects more than 25% of the population. Can f 1 is the major dog allergen associated with respiratory symptoms but the epitopes recognized by allergic patients IgE on Can f 1 are unknown. To characterize IgE epitopes of Can f 1 recognized by dog allergic patients, six overlapping peptides spanning the Can f 1 sequence were synthesized. In direct IgE epitope mapping experiments peptides were analyzed for IgE reactivity by dot blot and Enzyme-linked immunosorbent assay (ELISA) with sera from dog allergic patients. For indirect epitope-mapping, rabbits were immunized with the peptides to generate specific IgG antibodies which were used to inhibit allergic patients’ IgE binding to Can f 1. IgE binding sites were visualized on a model of the Can f 1 three-dimensional structure. We found that Can f 1 does not contain any relevant sequential IgE epitopes. However, IgE inhibition experiments with anti-peptide specific IgGs showed that Can f 1 N- and C-terminal portion assembled a major conformational binding site. In conclusion, our study is the first to identify the major IgE epitope-containing area of the dog allergen Can f 1. This finding is important for the development of allergen-specific treatment strategies. Dogs are kept as household pets worldwide but dog allergens were also found in homes without dogs, in schools and other public places and it is therefore almost impossible to avoid dog contact4. Dog-induced allergic symptoms range from rhinoconjuctivitis to severe asthma attacks2. In a large study conducted in a European population of patients with suspected allergic disease 27% showed positive skin prick test reactions with dog extract5. In a cross-sectional population based study in Germany up to 9% of children and adolescents showed sensitization to dog dander which is in good agreement with data obtained for the Swedish birth cohort BAMSE7. Most of the mammalian-derived allergens, including several dog allergens, belong to the lipocalin protein family2. Can f 1 is the major dog allergen with an IgE recognition prevalence of 49–76% in dog allergic populations10. Can f 1 shows partial IgE cross-reactivity with its human homologue tear lipocalin (hTL)11 and with the cat allergen Fel d 712. A cross-sectional and longitudinal study involving 779 randomly collected serum samples from children of the BAMSE birth cohort showed that sensitization to Can f 1 at the age of 4 years is a strong risk factor for the development of respiratory allergy at the age of 16 years13. In that study rCan f 1 showed a higher positive predictive value for the development of dog allergy than dog allergen extract.IgE-associated allergy is the most common hypersensitivity disease affecting more than 25% of the population worldwide. The symptoms of allergy include hayfever, asthma, skin inflammation, food allergy and life-threatening systemic anaphylactic reactions. Allergy to furry animals, in particular to cat and dog, is a major risk factor for the development of asthma and rhinitis15 almost no information is available regarding the binding of allergic patients’ IgE to Can f 1. However, the detailed knowledge of IgE- and T cell epitopes is important for the development of new forms of allergen-specific immunotherapy (AIT)16. AIT is the only allergen-specific form of treatment with disease-modifying and long-lasting effects18. Furthermore there is evidence that it is clinically more effective than pharmacotherapy and also cost effective19. However, AIT for dog allergy is less efficient than AIT for cat allergy20 possibly due to the poor quality of the dog allergen extracts21. Recently a new strategy for AIT based on carrier-bound B cell epitope containing allergen peptides has been described22. As exemplified for a recombinant grass pollen allergy vaccine, non-allergenic peptides derived from the IgE binding sites of the major four grass pollen allergens have been grafted onto the PreS protein from hepatitis B as carrier23. This vaccine, BM32, has been shown to have no allergenic activity in vitro and in vivo by skin testing of allergic patients24. Recently the clinical efficacy of BM32 has been shown in an AIT study performed in a grass pollen exposure chamber26. The engineering of new B cell epitope- as well as of T cell-epitope based allergy vaccines requires the knowledge of IgE and T cell epitopes of major allergens16. We therefore investigated the IgE binding sites of the major dog allergen Can f 1. Using six overlapping peptides of 30 to 36 amino acids that cover the Can f 1 sequence peptide-specific antibodies were raised in rabbits. Each peptide was synthesized in two forms with cysteine residues for coupling to Keyhole Limpet Hemocyanin (KLH) either at their N- or C-terminus to analyze if the coupling orientation may influence the immunogenicity of the peptides. With the synthetic peptides and dog allergic patients’ sera the presence of sequential/linear IgE epitopes in Can f 1 was investigated. Furthermore, peptide-specific antibodies were used for competing with the allergic patients’ IgE binding to Can f 1 to search for conformational/discontinuous IgE epitopes. These two approaches combined with a molecular modelling approach enabled us for the first time to identify the major IgE binding site as a conformational epitope on the surface of a 3-dimensional model of Can f 1.While the T cell response of allergic patients to Can f 1 has been studied in great detail using overlapping peptidesE.coli and purified by affinity chromatography to more than 90% purity  except for one single amino acid difference (W instead of R at position 130 in the Can f 1 sequence)  Fig.  were synIn a first set of experiments, the IgE binding capacities of the 10 Can f 1-derived peptides were compared with that of rCan f 1 in a non-denaturing, dot blot assay. Figure In order to test whether the residual IgE reactivity of the KLH-coupled peptides may lead to allergenic activity of the conjugates we performed basophil activation studies. We found that Can f 1 induced release of β-hexosaminidase from basophils loaded with serum IgE from Can f 1-allergic patients whereas the equimolar mix of P6 and P1A and an equimolar mix of the KLH-coupled peptides did not activate the basophils (data not shown).In a first set of experiments we studied if directional coupling of Can f 1-derived peptides via cysteine residues placed either at the N- or C-terminal end of the peptides to KLH may affect the binding of antibodies induced by immunization with the peptide-KLH conjugates to Can f 1. For this purpose we synthesized each of the Can f 1-derived peptides with cysteine residue on the N-terminus and C-terminus Fig. , coupledILELAQ…GQC in peptide 6A and ILKAQK…GQC in peptide 2A) in these peptides  as that achieved with an antiserum raised against the complete folded Can f 1 protein indicating that the area defined by peptides 1A and 6 contains the majority of Can f 1 IgE epitopes. We then located the region defined by peptides 1A and 6 on a three-dimensional model of the Can f 1 structure built on the basis of the homologous human tear lipocalin. Surprisingly, we found that peptides 1 A and 6 which in the primary sequence of Can f 1 comprise the N- and C-terminus of the molecule appear in close proximity on the surface of Can f 1 suggesting that during the folding of the protein in solution the peptides fold together and form a conformational IgE epitope consisting of the two ends of the protein. A similar finding has been made earlier for birch pollen profilin, a highly cross-reactive allergen from birch in which also the N- and C-terminus of the protein form a conformational IgE epitope33. Also many other important respiratory allergens contain mainly such conformational IgE epitopes35. It is known that mast cell and basophil activation by allergens requires engagement of at least two IgE antibodies. We have shown that patients IgE binding to the major IgE epitope of Can f 1 can be partially inhibited by IgG antibodies directed either against peptide 6 (54% inhibition) or against peptide 1A (40% inhibition). When IgG antibodies against these two peptides are used in combination the inhibition of patients’ IgE binding increased to 73%. This additive effect indicates that IgE antibodies with at least two different specificities can bind to the region defined by peptides 6 and 1A in close vicinity. We have previously shown for the birch allergen Bet v 1 that indeed several different Abs can bind simultaneously to a relatively small patch of the Bet v 1 structure32. Likewise, we have visualized simultaneous binding of four IgE antibodies to four small IgE-reactive peptides inserted into the N-terminal part of horse myoglobulin36. Therefore we think that the area identified on Can f 1 by peptides 6 and 1A is large enough to accommodate binding of at least two different IgE antibodies.Can f 1 is not only a major dog allergen but was also found to be the key allergen associated with the risk of developing of allergic respiratory symptoms in adolescence in a large birth cohort study12. There is no relevant sequence homology of peptides 1A and 6 with the corresponding regions in the other lipocalin allergens  which actually show no relevant IgE cross-reactivity with Can f 1, except some cross-reactivity between Can f 1 and Can f 2. However, these cross-reactivity data are based on IgE inhibition experiments performed with few sera37 or only on correlations of IgE reactivities to both allergens38. The second major clinical implication of our work is that the non-allergenic Can f 1 peptides 1A and 6 can induce blocking IgG antibodies upon immunization and thus should be useful for the construction of a hypoallergenic B cell epitope-based vaccine for therapeutic and eventually prophylactic vaccination against dog allergy22.Our finding is clinically important for at least two reasons: First, peptides 1A and 6 show close sequence similarity with the cat allergen, Fel d 7 which shows partial cross-reactivity to Can f 1 and thus we can assume that similar epitopes are involved in IgE recognition of the two allergensA/L) to dog dander extract (e5) as measured by ImmunoCAP  and IgE antibodies to Can f 1 as determined by ELISA and ImmunoCAP ISAC 10. Table IgE reactivity testing and allergenic activity testing by basophil activation assay were conducted with blood samples of adult dog allergic patients from Austria (n = 19) after having obtained informed consent of the patients with the approval of the local ethics committee . All experiments were performed in accordance with relevant guidelines and regulations. Dog allergic patients reported exacerbation of at least one of the following clinical symptoms upon exposure to dogs: conjunctivitis, rhinitis, asthma and allergic reactions of the skin  amplification (primer Can f 1_fwd: 5′-gcgcatatgcaggataccccagccttgggaaagg-3′ and primer Can f 1_rev:E. coli strain BL21 (DE3)  and purified via its His tag by Nickel affinity chromatography under denaturing conditions according to the manufacturers protocol . Purified rCan f 1 was refolded by dialysis against distilled water. The purity of the recombinant protein was analysed by SDS-PAGE, and the molecular mass was determined by matrix-assisted laser desorption/ionization time of- flight mass spectrometry . To verify the proper fold of rCan f 1 a circular dichroism analysis was performed as described39.5′-cgctcgagaactcagtgatgatgatgatgatgctgtcctcctggagagcaggtttcgctc-3′) from a dog salivary gland cDNA library. The PCR product was cut with Nde I and Xho I, purified by agarose gel electrophoresis and cloned into pET 17b vector . Recombinant Can f 1 (rCan f 1) was expressed in 40    and their sizes and identities were confirmed by mass spectrometry . Can f 1-derived peptides were coupled to keyhole limpet hemocyanin (KLH)  and purified using a conjugation kit according to the manufacturer’s instructions (Pierce). The concentration of KLH-conjugated peptides was measured using the Micro BCA Assay Kit (Pierce).Six overlapping peptides spanning the Can f 1 sequence with a length between 30 and 36 amino acids were identified based on the prediction of surface exposure of amino acids as determined by ProtScale bioinformatics tool from the ExPASY servere/) Fig. . Peptide41 using the PDB model of the human tear lipocalin 1XKI42 as template (61% sequence identity). Relative surface exposures were calculated with MSMS43 in the USCF Chimera package44 using Gly-X-Gly tripeptides45 as reference states and plotted as a bar graph against the amino-acid sequence with α-helix and β-sheet forming residues marked as H or B respectively  and visualized by autoradiography (Kodak XOMAT film).IgE reactivity of uncoupled Can f 1-derived peptides was determined by dot blot. For this purpose 0.5 µg aliquots of each peptide, of rCan f 1 and as a control human serum albumin (HSA) were dotted onto Whatman Protran nitrocellulose membrane . After blocking three times for 20 min with gold buffer , membranes were incubated with dog-allergic patients’ sera (1:10 in gold buffer) or with serum from a non-allergic person (1:10 in gold buffer) overnight at 4 °C. Bound IgE antibodies were detected with 1:15 diluted IgE reactivity to Can f 1 peptides after coupling to KLH was determined by direct ELISA. Five µg/ml of KLH coupled peptides, rCan f 1 and KLH, respectively, were coated onto ELISA plates  over night at 4 °C. After washing, plates were incubated with patients’ sera diluted 1:5 in TBST  and bound human IgE was detected with HRP-coupled goat anti-human IgE antibodies diluted 1:2500 . The optical density (OD) values corresponding to bound antibodies were measured at 405 and 490 nm. All determinations were conducted as duplicates, and results were expressed as mean values. To determine the cut-off for IgE reactivity to rCan f 1, mean OD values obtained for sera from three non-allergic subjects on the same plate were multiplied by two and patients with OD values above were considered Can f 1 reactive.5/well) were loaded overnight with sera from the dog-allergic patients and, for control purposes, with the serum from one non-allergic individual at a dilution of 1: 10. Cells were washed three times with Tyrode’s buffer  and exposed to serial dilutions of allergen  for 1 h. Supernatants were analysed for β-hexosaminidase activity as described previously46. Experiments were carried out in triplicates, and results are presented as mean percentages of total β-hexosaminidase released after addition of 1%Triton X-100 +/− SE of the mean (SEM).To test the allergenic activity of the peptides, rat basophil leukemia cells (RBL) expressing human high-affinity IgE receptor FcεRI (1 × 10Peptide-specific IgG antibodies were obtained by immunizing rabbits three times (first booster injection after 4 weeks and a second booster injection after 7 weeks) with each of the KLH-conjugated peptides (200 µg/injection) and, for control purposes, with recombinant rCan f 1 (200 µg/injection) using once Freund’s complete and twice Freund’s incomplete adjuvant . Rabbit immune responses were analyzed by ELISA titrations. For the measurement of specific rabbit IgG antibodies, ELISA plates were coated overnight with 5 µg/mL rCan f 1. After blocking, the plates were incubated overnight with serial dilutions of the corresponding rabbit anti-sera, or serum from a non-immunized rabbit . Bound rabbit IgG antibodies were detected with a 1:1.000 diluted horseradish peroxidase–labelled donkey anti-rabbit IgG antiserum .IgG epitope mapping was performed by direct ELISA. Synthetic peptides , rCan f 1 or HSA as controls were coated on the ELISA plates at a concentration of 3 µg/ml and after washing and blocking, anti-Can f 1 antiserum (1:2.000 dilution) was added. Bound rabbit IgG was detected with HRP-labelled donkey anti-rabbit IgG (Amersham Biosciences). The IgG epitope mapping ELISA experiments were repeated three times giving similar results. Results of the ELISA experiments are expressed as means of duplicate determinations with a deviation of <5%.I/ODP) × 100). ODI and ODP represent optical density values after pre-incubation with the rabbit immune serum or normal rabbit serum, respectively.ELISA plates  coated overnight with 1 µg/mL of rCan f 1 were pre-incubated for 24 hours with each of the anti-peptide antisera, anti-Can f 1 antiserum, or, for control purposes, with serum from a non-immunized rabbit  and then washed. After overnight incubation with sera from dog allergic patients (diluted 1:10), bound IgE antibodies were detected with horseradish peroxidase–labelled goat anti-human IgE antibodies . The percentage reduction of IgE binding achieved by pre-incubation with rabbit antisera was calculated as follows: 100−. Sequences with homology to Can f 1 were found using BLAST.For statistical analysis, the statistical program SPSS  was used. P-values < 0.05 were considered significant. Differences between the groups were compared using Mann–Whitney U-test. For sequence alignment Clustal Omega -multiple sequence alignment program was used (The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


Here, we studied the solution structure of the parvalbumin Sco j 1, derived from the Pacific mackerel, using nuclear magnetic resonance spectroscopy. We mapped the IgE-binding epitope proposed in a recent study onto the present structure. Interestingly, three of four residues, which were elucidated as key residues of the IgE-binding epitope, were exposed to solvent, whereas one residue faced the inside of the molecule. We expect that this solution structure can be used in future studies attempting to analyze the various IgE-binding modes of these allergens.Although fish is an important part of the human diet, it is also a common source of food allergy. The major allergen in fish is parvalbumin, a well-conserved Ca The allergen sources of fish are mainly derived from meat, eggs, roe, caviar, isinglass, and gelatin2.Fish is a valuable dietary source of proteins, amino acids, fatty acids, lipids, and vitamins, and it plays an important role in not only human food and health but also food culture. However, it is also among the most common sources of food allergy. Fish allergy, caused by a pathophysiological IgE-mediated immune response to specific fish proteins, can present clinically as urticaria, asthma, diarrhea, allergic contact dermatitis, and angioedema. Fish hypersensitivity is commonly encountered in coastal areas such as many European countries and Japan3. It is well conserved in the amino acid sequence, highly water soluble, and resistant to heat, chemical denaturation, and enzymatic digestion3. The first studies to demonstrate parvalbumin identified it in Baltic cod  as the allergen Gad c 15. However, subsequent studies have identified that the major food allergen is parvalbumin derived from Atlantic cod6 , carp7 , Atlantic salmon8 , and Pacific mackerel9 . Previous studies have also established IgE cross-reactivity among different fish species10. Fish parvalbumin has several isoforms. For example, three isoforms in Atlantic herring (Clu h 1), two isoforms in carp (Cyp c 1), and four isoforms in Atlantic cod (Gad m 1) have been identified. Each parvalbumin isoform has allergenicity. Interestingly, Pacific mackerel has a single parvalbumin isoform11. The cross-reactivity of parvalbumin from cod, salmon, and mackerel has been studied in clinical trials12. According to these studies, mackerel allergy patients show allergenic reactions to not only mackerel but also other fish species. Thus, for Sco j 1 seems to have common IgE epitopes with other fish parvalbumin isoforms.The major allergen in fish is parvalbumin, a 12-kDa sarcoplasmic protein in the white muscle of many fish species containing 108–109 amino acid residues that participates in muscle relaxation2+-binding CD sites located on the central region and EF site at the C-terminus. The N-terminal AB site is a non-functional domain. Upon Ca2+ binding and release, the parvalbumin structure changes in a global-folding manner; hence, parvalbumin has a function in calcium buffering and is probably involved in muscle relaxation13. An NMR structural and dynamics study of Gad m 1 revealed that its structure and the accessibility of putative IgE epitopes are similar to those of other parvalbumin types14. In a recent study, an interaction between Gad m 1 and the scFv fragment of the antibody was established using a 1H-15N HSQC chemical shift perturbation method15.The molecular structure of parvalbumin has been well characterized by X-ray crystallography and NMR spectroscopy. Parvalbumin contains three EF-hand motifs with two Caet al. purified Sco j 1 from fish meat, tested its reactivity with IgE, using ELISA, and cloned its nucleotide sequence from the cDNA library9. Furthermore, Yoshida et al. elucidated that the region containing the 21–40 amino acid residues of Sco j 1 is a major IgE epitope using chemically synthesized oligopeptides. These studies hypothesized that IgE can recognize the linear  epitope of Sco j 116. Kubota et al. reported that heat treatment of Sco j 1 at 140 °C completely destroys its IgE reactivity17. In contrast, Kobayashi et al. showed that Ca2+ binding of Sco j 1 maintains its IgE reactivity by using assays in the presence or absence of Ca2+ chelating agents18. These studies showed that IgE recognizes the conformational  epitope of Sco j 1. Given that both linear and conformational epitopes were found, the molecular basis underlying Sco j 1 reactivity to IgE is highly complicated and difficult to interpret. Thus, to analyze the details of recognition between Sco j 1 and IgE, further investigations are needed based on the precise three-dimensional structure of Sco j 1. However, the three-dimensional structure of Sco j 1 has not yet been reported; it has only been modeled using a computational method. This study aimed to determine the three-dimensional structure of Sco j 1 at atomic-scale resolution using NMR spectroscopy.Hamada All of the backbone amide resonances of Sco j 1 were assigned except for Asp80  and carp19  were inputted into the DALI server . The identity of amino acid sequences among fish parvalbumin is relatively high at 67–85%, suggesting that their three-dimensional structures are also similar. Sco j 1 showed high structural similarity with both Gad m 1 and Cyp c 1  also reduces IgE reactivity18. These studies indicate that depletion of the Ca2+ binding ability results in a conformational change of parvalbumin; thus, IgE must recognize Sco j 1 by some type of conformational epitope. Second, it was previously reported that the IgE reactivity of muscle extracts from Pacific mackerel gradually decreased as the temperature was increased to 120 °C and was completely lost at 140 °C17. This also indicates that IgE recognizes the three-dimensional structure of parvalbumin.As mentioned above, IgE recognizes Sco j 1 by its specific peptide region that acts as the sequential epitope. However, some evidence suggests that the IgE reactivity of Sco j 1 is maintained by its three-dimensional structure. First, substitutions of Asp51 and/or Asp90, i.e., residues consisting of CaIn conclusion, this study investigated the solution structure of Sco j 1, a Pacific mackerel parvalbumin allergen, which was found to be similar to that of Gad m1 and Cyp c 1 derived from other fish species. The IgE-binding epitopes of Sco j 1 are located on one side of the molecular surface. Further investigation into the interaction mechanism between IgE and IgE-binding epitopes for both sequential and conformational recognition modes will be addressed using purified monoclonal antibodies for Sco j 1.Escherichia coli, was cloned into a pGEX-6P-1 expression vector. The actual synthesis and purification of the vector was outsourced . The expression vector was transformed into BL21(DE3) competent cells. The cells were grown in 1 L MP media containing 2 g of 15NH4Cl  and 4 g of 13C-labeled glucose  at 37 °C. Protein expression was induced at OD600 = 0.7 by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM. The induced cells were cultured at 37 °C for 3 h. After harvest, disruption, and centrifugation, the expressed protein was purified using a glutathione Sepharose 4B column  in a bed volume of 5 mL and eluted with 10 mM reduced glutathione-containing buffer. The GST tag was excised from Sco j 1 by incubation with HRV3C protease for 12 h at 4 °C. The 10 mL of isolated Sco j 1 was purified using an ÄKTA Purifier and a HiLoad 26/600 Superdex 75 gel-filtration column . The protein was concentrated to 0.5 mM using Vivaspin 15 R  in NMR buffer containing 7% D2O, 20 mM MES, 150 mM NaCl, and 10 mM CaCl2 at pH 6.8.The amino acid sequence of Sco j 1 was retrieved from the UniProt database (accession code P59747). The DNA encoding full-length Sco j 1 (1–109), optimized in the codon usage for 22, and the data analysis was performed using the Sparky program. 1H-, 13C-, and 15N-resonances were assigned using the following set of spectra: [1H-15N] HSQC, [1H-13C] HSQC, HNCO, HN(CO)CA, HNCA, CBCA(CO)NH, HNCACB, HN(CA)HA, C(CO)NH, HCCH-TOCSY, HbCbCgCdHd, and HbCbCgCdCeHe. All chemical shift values were referenced to DSS and the following frequency ratios: (15N/1H) = 0.101329118, (13C/1H) = 0.25144951923. Inter-proton distance restraints for structural calculation were obtained from 13C-edited NOESY-HSQC and 15N-edited NOESY-HSQC spectra, using 75 msec of mixing time. The restraints for backbone phi and psi torsion angles were predicted from chemical shifts of backbone atoms, using the TALOS + program24. The structure was calculated using the CYANA 2.1 software package25. For calculation with calcium ions we used the canonical EF-hand Ca2+ -binding topology. Sco j 1 has two typical EF-hands regions: D52QDKSGFIEEEE63 and D91SDGDGKIGIDE102. Two calcium ions were coordinated one by one at each binding site. The upper limit distance from the atomic group involved in coordination was 2.8 Å.NMR spectra were obtained at 25 °C using the Avance III HD 800 MHz  and Unity INOVA 600 and 500 MHz  spectrometers. Two- and three-dimensional NMR spectra were processed using NMRPipe26 from a carp parvalbumin structure (PDB ID: 1CDP), all NOE signals were manually assigned. Among these assigned signals, we extracted the restraints between the medium- and long-range residues, and used a pseudo-atom for all optical isomeric protons. Consequently, we used 889 distance restraints with a fixed upper limit of 6 Å. At each “noeassign” cycle, 100 structures were calculated using 30,000 steps of simulated annealing, and a final ensemble of 20 structures was selected based on the CYANA target function values.NOE distance constraints were automatically assigned and calculated using seven cycles under the “noeassign” macro of CYANA. To correct for the automatic NOE signal assignment, we used additional distance restraints derived from manual NOE assignments. Based on a model structure of parvalbumin, constructed using MODELLARThe resonance assignments have been deposited to BMRB (code: 36086).The atomic coordinates have been deposited to PDB (code: 5XND).


Streptococcus suis is a previously neglected, newly emerging multidrug-resistant zoonotic pathogen. Mobile genetic elements (MGEs) play a key role in intra- and interspecies horizontal transfer of antimicrobial resistance (AMR) determinants. Although, previous studies showed the presence of several MGEs, a comprehensive analysis of AMR-associated mobilome as well as their interaction and evolution has not been performed. In this study, we presented the AMR-associated mobilome and their insertion hotspots in S. suis. Integrative conjugative elements (ICEs), prophages and tandem MGEs were located at different insertion sites, while 86% of the AMR-associated MGEs were inserted at rplL and rum loci. Comprehensive analysis of insertions at rplL and rum loci among four pathogenic Streptococcus species  revealed the existence of different groups of MGEs, including Tn5252, ICESp1108, and TnGBS2 groups ICEs, Φm46.1 group prophage, ICE_ICE and ICE_prophage tandem MGEs. Comparative ICE genomics of ICESa2603 family revealed that module exchange and acquisition/deletion were the main mechanisms in MGEs' expansion and evolution. Furthermore, the observation of tandem MGEs reflected a novel mechanism for MGE diversity. Moreover, an in vitro competition assay showed no visible fitness cost was observed between different MGE-carrying isolates and a conjugation assay revealed the transferability of ICESa2603 family of ICEs. Our statistics further indicated that the prevalence and diversity of MGEs in S. suis is much greater than in other three species which prompted our hypothesis that S. suis is probably a MGEs reservoir for other streptococci. In conclusion, our results showed that acquisition of MGEs confers S. suis not only its capability as a multidrug resistance pathogen, but also represents a paradigm to study the modular evolution and matryoshkas of MGEs. Streptococcus pneumoniae, and the “Concerning” level erythromycin-resistant Streptococcus pyogenes and clindamycin-resistant Streptococcus agalactiae  reservoir that can contribute to the spread of resistance genes to the above-mentioned streptococci  declared the top 18 drug-resistant threats in the United States, including the “Serious” level drug-resistant http://faculty.washington.edu/marilynr/)  and macrolides (up to >70%) have been reported worldwide since the 1980s - and aadE-carrying 89-kb (89 K) pathogenicity island (PAI), which was found to be unusual in streptococcal toxic shock syndrome (STSS)-causing S. suis strains, was responsible for full bacterial virulence in two major outbreaks in China -carrying prophage ΦSsuD.1 was found in a S. suis strain isolated from a patient with meningitis and is highly similar with the main S. pyogenes Φm46.1 and S. agalactiae λSa04  to explore the diversity and evolution of MGEs. This is the first study that provides the landscape of the prevalence and diversity of MGEs at rplL and rum loci in S. suis, S. pneumoniae, S. pyogenes, and S. agalactiae to comprehend the underlying evolutionary mechanism of MGEs. Our work further confirms the hypothesis that S. suis is an important AMR reservoir and suggest that S. suis might be a MGEs reservoir for other streptococci which promoted the worldwide emergence of antibiotic-resistant streptococcal infection.Although, some MGEs have been identified in S. suis isolates from our routine surveillance on antimicrobial resistance were chosen and sequenced in this study as they displayed different resistance genotype  were determined by a standard broth micro dilution method.A total of seven pe Table . The compe Table . IsolateS. suis strain P1/7 (AM946016), using the Mauve v2.4.0 software . These scaffolds of each genome were ordered according to the reference genome of the http://www.mlst.net/). Acquired antimicrobial resistance genes were identified using ResFinder 2.1 . For rplL and rum loci, sequences between rplL and hdy genes as well as rum and glf genes were analyzed, respectively. Three conditions will be considered: (1) no insertion, sequence length will be ~128 or ~561 bp; (2) with insertion and gap, no sequence will be acquired; (3) with full length insertion, sequence length will be insertion length plus ~128 or ~561 bp. The obtained sequences with insertion were further annotated by an online PATRIC server  and length . If all three proteins were present, the elements were considered ICEs, elements that only harbored integrase and partially or no relaxase and VirB4 were considered defective ICEs (dICEs). Similar analysis of insertions was also done in 637 S. agalactiae, 5063 S. pneumoniae, and 268 S. pyogenes.For ICE identification, signature proteins of integrase, relaxase and VirB4 were searched by BLASTp comparison. Filters were defined by the coverage (>25% and cover function domain), Phylogenetic tree of integrases, relaxases and VirB4 proteins were further analyzed. Protein identity of less than 60% was considered as different clade. ICEs were classified by the presence of signature integrases, relaxase, VirB4, and AMR profiles.Sa2603 families of ICEs were retrieved from insertions at rplL and rum sites. BLAST atlas maps were built by comparing ICEs with reference sequences of 89 K (CP000407) using BLASTn with a >80% identity threshold within the CGView Comparison Tool  and SS-1RF (tetracycline- and erythromycin-susceptible but rifampin- and fusidic acid-resistant) were used as recipient and S. suis strains (tetracycline- and erythromycin-resistant but rifampin- and fusidic acid- susceptible) which harbored different MGEs were utilized as donors. Filter mating assays were performed as previously described  was determined in competition experiments in triplicate and repeated at least twice, as previously described  in fitness assay were analyzed using student t-test. A P < 0.05 was considered statistically significant.Statistical analysis was performed and illustrated with GraphPad Prism 5 . Insertions analysis of the prevalence of each MGEs groups in these four species and statistics of growth rate and relative fitness (AYSB00000000. Sequences of 17 MGEs identified have been deposited at GenBank (Accessions: KX077882-KX077898).The Whole Genome Shotgun (WGS) sequence of YY060816 has been deposited at GenBank under the accession number erm(B), mef (A), and msr(D)], tetracyclines , and aminoglycosides  were carried by corresponding MGEs , and one ICE_ICE type tandem ICEs Figure . MLST tyEs Table .rplL gene with the exception of ICESsuJH1308-1 (in SSU0468) and ICESsuZJ20091101-1 (in SSU1262)  Figure . Conserv1 Figure , which c3 Figure .mef (A)-carrying prophages ΦJH1301-2 and two CMGEs (CMGEYY060816 and CMGETZ080501) were inserted in the 3′ terminal of rum gene  of the strains contained insertions. Among them, 194 (59.51%) were fully sequenced with no gap between rplL and hdy genes  and 73 (36.32%), respectively  consisted of the largest number of ICEs in both sites. Three subgroups were divided by the chimeric of Tn916 or Tn5397. The ICESa2603 subgroup contained six ICEs at rplL site and one ICE in rum site. Five ICEs at rplL site encode a Clade Vb Int and a Clade IIIb VirB4, and one ICE encoded a Clade Va Int, a Clade IVa relaxase, and a Clade IIIa VirB4. However, the ICESa2603_rum contained a Clade III Int, a Clade II relaxase, and a Clade IIIb VirB4. The ICESa2603-Tn5397 subgroup consisted of 12 ICEs at rplL site and 45 ICEs in rum site. This group ICEs resulted from the integration of Tn5397 in I-2 site of ICESa2603  included six ICEs at rplL site and one ICE in rum site. ICEs of this group encoded a Clade I relaxase and a Clade II VirB4 associated with a Clade IV (rplL site) or Clade I (rum site) Int (Table SsuTYPE3 family (TnGBS2 group) was only identified at rplL site. ICEs of this group encoded a Clade Vb Int which was identical to IntICESa2603, but the conjugation module, a Clade IV relaxase and a Clade I VirB4, was distinct from those of ICESa2603 family (Table Sp1108 group encoded multiple AMR genes including tet(W), tet(L), erm(B), lnu(B), aadE, and cat. The schematic of the genetic organization of the three groups of ICEs are shown in Figure The ICEily ICESp108 groupSa2603 family ICEs were compared to 89 K reference ICE using BLAST to detect genetic variability and rearrangements within core and variable regions. Figure The 210 identified ICEabiGI/abiGII genes presented in VR4 of some ICEs but absent in other ICEs; Figure Streptococcus suis serotype 2 . Phylogenetic analysis of relaxases and VirB4 proteins showed different combination in subgroups, which suggested that exchange of DNA processing modules may occur although the evolution of relaxases into different Clade cannot be ruled out (Table rplL site) or serine recombinase (at rum site) families but harbor a common conjugation module  and CMGEYY060816/CMGETZ080501 (ICE_Prophage). Further insertion analysis at rplL and rum sites identified 17 tandem MGEs displaying various tandem combinations , S. agalactiae (6.78%) and S. pneumoniae (2.34%), while at rum site the percentage was 65.75% (S. suis), 2.06% , and 0.28% (S. pneumoniae), respectively. The ICESp1108 group presented at rum site of S. pyogenes (68.18%), S. agalactiae (18.56%), S. suis (1.37%), and S. pneumoniae (0.62%), but at rplL site only presented in S. suis (3.09%)  and S. agalactiae (0.85%). Collectively, the prevalence and diversity of MGEs  is much greater than other three species  Figure . The TnGs Figure .S. suis, in which exchange of integration/excision module and tandem accretion of MGEs play important roles in ICE diversity and evolution, deletion of modules was predominant in ICEs of S. agalactiae, S. pneumoniae, and S. pyogenes at rplL and rum sites, which, as a result, generated defective ICEs (dICEs) probably with no function (Table S. pyogenes at the rum site which only had 8.33% (22/268) of the isolates harbor insertions  into at least three groups on the basis of their conjugation modules: Tn5252, ICESp1108, and TnGBS2  and 67.12% (at rum site) of the strains with insertions harbored ICEs, while dICEs were more prevalent at both sites in S. agalactiae, S. pneumoniae, and S. pyogenes  was shown in S. agalactiae, S. pneumoniae, and S. pyogenes. The ICESp1108 group, first reported at rum site of S. pyogenes  was shown to have the transferability to S. agalactiae, S. pneumoniae, and S. pyogenes  and SSU0468 (lysS) in this study. Although, Tn5252 group of ICEs encodes a DDE transposase was not found in S. suis, this type of ICEs may occur during interaction of the ICEs. Further, exchange of integration module also occurred in ICESp1108 group at least in S. suis if not other streptococci  in S. suis  in depth to speculate their evolution mechanism. The original host might be S. suis since the most majority of ICEs have been identified in this species, including those within other insertion sites -carrying Tn916, erm(B)- and/or tet(O)-carrying Tn5397, and aadE-carrying ISSsu5 composite transposon generating the “chimeric” or “composite” MGEs: ICESa2603-Tn5397 and ICESa2603-Tn916. ICESa2603, ICESa2603-Tn5397, and ICESa2603-Tn916 can undertake exchanging of modules between with MGEs or conjugating transfer to other streptococci. ICEs in their host continue to acquire AMR and virulence genes in order to adapt to the host or degrade into dICEs by deletion of core modules of ICEs. Figure Sa2603 core structure. Notably, by acquiring of SalK-SalR constituent, the tet(M)- and aadE-carrying 89 K PAI caused two major human outbreaks in China  and S. agalactiae (~16%). The prevalence of tandem MGEs in S. suis as well as other streptococci indicating that tandem accretion plays a role in evolution of MGEs  was shown to be transferable, which was consistent with other studies (Li et al., St1 was observed (Bellanger et al., Sa2603-Tn916, ICESa2603-Tn5397, and tandem MGEs are widely distributed in S. suis.Acquisition of MGEs was thought to impose an immediate biological cost, the initial costs of MGE carriage may be mitigated during growth (Starikova et al., S. suis, and illustrates the role of module exchange, acquisition/deletion, and tandem accretion in diversity and evolution of MGEs among four pathogenic Streptococcus species which suggests that S. suis is probably a MGEs reservoir for other streptococci.In summary, the study provides an overview of AMR-associated mobilome and their insertion hotspots in JH, LW developed the concept and designed experiments. JH, JM, KS, YL, and LC performed the experiments and collected the data. JH, XH, and ZW conducted all bioinformatics analyses. JH, DL, LD, and LW prepared the manuscript. All authors have contributed to, seen and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ICD signal was used for evaluation of the association constant, demonstration of the reversibility of the interaction and determination of the binding site, revealing that divanillin has preference for Sudlow’s site I in BSA. This property was confirmed by displacement of the fluorescent markers warfarin (site I) and dansyl-L-proline (site II). Molecular docking simulation confirmed the higher affinity of divanillin to site I. The highest scored conformation obtained by docking  was used for calculation of the circular dichroism spectrum of divanillin using Time-Dependent Density Functional Theory (TDDFT). The theoretical spectrum showed good similarity with the experimental ICD. In summary, we have demonstrated that by interacting with the chiral cavities in BSA, divanillin became a atropos biphenyl, i.e., the free rotation around the single bound that links the aromatic rings was impeded. This phenomenon can be explained considering the interactions of divanillin with amino acid residues in the binding site of the protein. This chiroptical property can be very useful for studying the effects of divanillin in biological systems. Considering the potential pharmacological application of divanillin, these findings will be helpful for researchers interested in the pharmacological properties of this compound.Vanillin is a plant secondary metabolite and has numerous beneficial health applications. Divanillin is the homodimer of vanillin and used as a taste enhancer compound and also a promissory anticancer drug. Here, divanillin was synthesized and studied in the context of its interaction with bovine serum albumin (BSA). We found that divanillin acquires axial chirality when complexed with BSA. This chiroptical property was demonstrated by a strong induced circular dichroism (ICD) signal. In agreement with this finding, the association constant between BSA and divanillin (3.3 x 10 Vanillin, a component of vanilla, is one of the most widely used flavors in the world and has numerous applications in food, beverage and pharmaceutical industries . VanilliDifferently of vanillin, its homodimer, divanillin was much less investigated. However, the pharmacological potential of this compound is quite promisor, as can be verified in a very recent publication that showed its capacity to decrease the metastatic potential of human cancer cells by inhibiting the FAK/PI3K/Akt signaling pathway . Other aFrom the point of view of its molecular structure, vanillin has great similarity with apocynin, and its dimer, diapocynin , is simiDivanillin is an ortho-substituted biphenyl system, and, as such, it is reasonable to suspect the existence of axial chirality for this compound, i.e., the existence of energetic barrier that could impede the rotation around the central single bond that connect the aromatic rings. The consequence would be the existence of stable axial stereoisomers. This phenomenon is known as atropisomerism or axial chirality and divides the biaryl system in two categories: atropos systems, from a (not) tropos (to turn), when the rotation is restricted due to steric hindrance; and tropos system, when racemization is observed due to the low energy barrier. In other words, only atropos biphenyls may exist as a pair of stable enantiomers.Human serum albumin (HSA) is the most abundant protein in the human bloodstream and has many physiological functions, including a significant contribution for the colloidal osmotic pressure and as a carrier and depot protein for metabolites and xenobiotics . It is a-1) were prepared in dimethyl sulfoxide. Working solutions were prepared by dilution the stock solution in 50 mmol L-1 phosphate buffer at pH 7.0. BSA was dissolved in 50 mmol L-1 phosphate buffer at pH 7.0 to give a 1 mmol L-1 stock solution and its concentration measured by its absorbance   is the concentration of divanillin. Before fitting the data to Stern-Volmer equation, the fluorescent intensities were corrected for the inner filter effect as presented in the material and methods section. It is worth of note that the inner filter effect is provoked by attenuation of the excitation and emission signals due to the absorption of the study compounds [ompounds .5 mol-1 L at 298 K) revealed the strong association between divanillin and BSA , the quenching mechanism can be considered a static process [The magnitude of the obtained Stern-Volmer constant  using a double logarithmic equation , which is comparable to those obtained for pharmaceutical compounds that are well-stablished as ligand of albumin, for instance: warfarin (4.2 x 105 mol-1 L), naproxen (3.8 x 105 mol-1 L), phenylbutazone (1.3 x 105 mol-1 L) and mesalazine (2.5 x 105 mol-1 L) [4 mol-1 L) [4 mol-1 L, which is in agreement with the reported value to HSA [n value for the complexation of divanillin was close to one, which is indicative of a 1:1 stoichiometry. However, this result does not discard the possibility of more than one binding site and/or sites with less affinity could be also occupied using a large excess of divanillin.mol-1 L) –46. Moremol-1 L) . It is we to HSA , and reiKa) against the absolute reciprocal temperature (1/T) using the Van`t Hoff equation (− 1 K− 1) and T is the absolute temperature.Aiming a better comprehension of the non-covalent forces involved in the complexation of divanillin, the thermodynamic parameters for binding were obtained by plotting the natural logarithm of the association constants Ka) againequation . Where R-1), which have been demonstrated to be correlated with the formation of hydrogen bonds and Van der Waals forces and by the positive ΔS° (40.8 J mol-1 K-1), an indicative of the importance of hydrophobic interactions for the formation of the complex [-1) and ΔS° (5.8 J mol-1 K-1) [ complex ,49. A col-1 K-1) . These rl-1 K-1) .Divanillin is an ortho-substituted biphenyl system; hence, it is reasonable to suspect the existence of axial chirality. To evaluate this putative chiroptical property, its CD spectrum was measured as indicated in θ mdeg) signal was taken as proportional to the concentration of bound ligand  was obtained from the non-linear fitting of K is a proportionality constant, Pt and Lt are the added concentrations of protein and ligand, respectively.Regarding the interaction between proteins and ligands, the existence of ICD signal may contribute significantly for the elucidation of the binding process. Firstly, as described previously, the ICD signal is a direct evidence of the complexation; secondly, the ICD signal can also be used to evaluate the binding affinity. The last feature has advantage upon other spectroscopic techniques. Indeed, as a chiroptical technique, measurements based on ICD is less susceptible to superposition of bands or inner filter effects as usually observed using alterations in fluorescence. For these reasons, we also used the concentration dependent alteration in the ICD signal as an analytical parameter for evaluate the association constant between divanillin and BSA. For that, an analytical rationalization and mathematical derivatization presented by Zsila and collaborates was used . In this LP, Eqs  and 7. TKa (1.3 x 104 mol -1 L) was obtained. As can be observed, this value is about one order of magnitude lower compared to the constant obtained by fluorescence quenching experiment (3.3 x 105 mol-1 L). An explanation for this lower value has its fundament in the origin of the ICD signal of divanillin. In fact, it is reasonable to consider that the observed ICD is the result of an enantiomeric excess of a specific conformation of divanillin fixed in the binding sites of BSA. In other words, it is possible that more than one conformation can bind, but one has preference. Hence, the ICD signal of a conformer could be partially concealed by its enantiomeric pair. This fact could explain the lower association constant obtained using this technique. The results depicted in Another potential application of the ICD phenomenon is in the elucidation of the binding site of the studied ligand . Thus, wConsidering that divanillin is a site I ligand. Then, the addition of warfarin provoked a partial remotion of divanillin from the protein cavity, indicating a direct competition for this site.The increased divanillin ICD signal provoked by addition of the site II ligands might be an evidence that divanillin does not occupy or occupy with less efficiency this site. In this case, the binding of ibuprofen or naproxen at site II could affect the capacity of divanillin as a ligand of site I, leading to an increased and/or altered signal. Corroborant with that, an increase in the ICD signal of bilirubin (site III) provoked by addition of ethacrynic acid (site II) was interpreted as an allosteric interaction leading increased affinity of the bilirubin at site III .The observed effects of the pharmaceutical drugs on ICD signal of divanillin suggested a tendency to a higher affinity of this compound at site I, however, it was not totally conclusive. Hence, two experiments were idealized to further confirmation of the binding site of divanillin in BSA. One of then was the use a fluorescent probe specific for site II. This is the case of dansylproline, a dansylated amino acid that binds in site II and has fluorescence quantum yield increased and shift to a lower wavelength when complexed to albumin . In thisFollowing the same approach, the alteration in the intrinsic fluorescence of warfarin due to its binding in BSA and the subsequent displacement by the addition of divanillin was also studied . The resAs well stablished, the interaction between protein and ligands is usually weakened, as the temperature is increased . Therefo-1, compared to site II, -59.7 kJ mol-1. Despite the difference in the absolute values, these score values are consistent with the experimentally determined binding energy ΔG°, -31.5 kJ mol-1) and reinforce the thermodynamic favorability of the complexation. The higher score for site I is also in agreement with our experimental results, which showed the preference of divanillin for this site. The existence of an ICD spectrum for divanillin implies that it must have a non-zero dihedral angle around the single bond that links the phenyl rings. Therefore, our next step was to perform docking studies to obtain the conformations of divanillin bound to site 1 and 2 of BSA. For these studies, the crystallographic structure used was that of BSA in complex with naproxen available in PDB (access code 4OR0). In this crystallographic structure, naproxen occupies three different sites in BSA, named naproxen site 1 (NPS1), naproxen site 2 (NPS2) and naproxen site 3 (NPS3). As stablished by the authors, NPS3 corresponds to Sudlow's site I and NPS1 to Sudlow's site II, which are the binding sites of interest here. NPS 2 is an alternative site for naproxen and was not considered in our study . From thAn ICD signal provoked by the binding of divanillin in BSA is related to the induction of axial chirality in this di-ortho substituted biphenyl. It means that divanillin, a tropos biphenyl, behaves as an atropos when attached into the site I and, more importantly, some degree of stereoselectivity must be present, otherwise no ICD signal should appear, since a conformation could be concealed by its enantiomer. Therefore, there must be a preferential stabilized conformation with a non-zero dihedral angle around the single bond that links the two phenyl rings. Aiming to elucidate this minimum energy conformation, theoretical studies were performed searching for the most stable conformations of divanillin and the results were compared with those obtained by docking simulations. Density Function Theory (DFT) using the M06-2X functional along with the 6-31G(d) basis set was initially used to search for the most stable conformations adopted by divanillin in the gas phase. The energy for each conformation of divanillin was obtained and plotted as a function of the dihedral angle . The potential energy surface (PES) which consists of single point energy evaluations over a rectangular grid involving the Cartesian coordinates of divanillin atoms was obtained. The interval and step size were 0 to 360 and 10 degrees, respectively. In this methodology of calculation, the solvent, water, was included through the Polarised Continuum Medium (PCM) model, and the harmonic vibrational frequency for characterisation of the stationary points was obtained . Fig 11 The four minima energy conformations for divanillin were verified at dihedral angles of about 60°, 120°, 230° and 300° . InteresOnce found the most stable conformation of divanillin in site I of BSA, the next step was the simulation its ICD spectra. The computer simulation was performed at the TDDFT level of theory using CAM-B3LYP, the hybrid exchange-correlation functional Coulomb-attenuating method, and the 6–311++G basis set . The solIn the By interacting with the chiral cavities of the BSA, divanillin became a atropos biphenyl, i.e., the free rotation around the single bound that links the aromatic rings was impeded. This phenomenon can be explained considering the interactions with amino acid residues in the binding site of the protein. The chirality of divanillin complexed to BSA was evidenced by the generation of an ICD spectrum. The efficiency of complexation was proven by the high association constant between the divanillin and BSA. The ICD signal in divanillin was useful for determination of site I as the main binding site of this ligand in BSA. The ICD signal was also useful for confirmation of the high association constant and to demonstrate the reversibility of the binding of divanillin with BSA. The conformation of divanillin obtained for docking simulation  was used to calculate the ECD spectrum using TDDFT. The theoretical spectrum showed good similarity with the experimental. Considering the potential pharmacological application of divanillin, these findings will be helpful for researchers interested in the pharmacological properties of this compound.S1 FigVanillin and divanillin 100 μM in 0.05 M phosphate buffer 0.05 M pH 7.0.(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig-1 BSA in the absence or presence of vanillin (0–30 μmol L-1) in 0.05 mol L-1 phosphate buffer pH 7.0 at 298 K .Double logarithmic fitting for determination of the association constant. The results are the average and SD of experiments performed in triplicate. Experimental condition: 5 μmol L(DOCX)Click here for additional data file.S4 FigVan`t Hoff plot.(DOCX)Click here for additional data file.S5 Fig-1 BSA in the absence or presence of pharmaceutical drug (30 μmol l L-1) in 0.05 mol L-1 phosphate buffer pH 7.0 at 298 K.Experimental condition: 30 μmol L(DOCX)Click here for additional data file.


Here we sought to apply such “clean” sampling techniques with in silico decontamination approaches used elsewhere to investigate microorganisms archived in ice at ∼41  and ∼49 m  depth in an ice core (GS3) from the summit of the Guliya ice cap in the northwestern Tibetan Plateau. Four “background” controls were established – a co-processed sterile water artificial ice core, two air samples collected from the ice processing laboratories, and a blank, sterile water sample – and used to assess contaminant microbial diversity and abundances. Amplicon sequencing revealed 29 microbial genera in these controls, but quantitative PCR showed that the controls contained about 50–100-times less 16S DNA than the glacial ice samples. As in prior work, we interpreted these low-abundance taxa in controls as “contaminants” and proportionally removed them in silico from the GS3 ice amplicon data. Because of the low biomass in the controls, we also compared prokaryotic 16S DNA amplicons from pre-amplified (by re-conditioning PCR) and standard amplicon sequencing, and found the resulting microbial profiles to be repeatable and nearly identical. Ecologically, the contaminant-controlled ice microbial profiles revealed significantly different microorganisms across the two depths in the GS3 ice core, which is consistent with changing climate, as reported for other glacier ice samples. Many GS3 ice core genera, including Methylobacterium, Sphingomonas, Flavobacterium, Janthinobacterium, Polaromonas, and Rhodobacter, were also abundant in previously studied ice cores, which suggests wide distribution across glacier environments. Together these findings help further establish “clean” procedures for studying low-biomass ice microbial communities and contribute to a baseline understanding of microorganisms archived in glacier ice.Microorganisms in glacier ice provide tens to hundreds of thousands of years archive for a changing climate and microbial responses to it. Analyzing ancient ice is impeded by technical issues, including limited ice, low biomass, and contamination. While many approaches have been evaluated and advanced to remove contaminants on ice core surfaces, few studies leverage modern sequencing to establish  The cryosphere covers approximately 20% of the Earth’s surface, and includes glaciers, snow, ice sheets, permafrost, lake ice, river ice, and sea ice . Althoug2 to 104 cells ml-1  within Proteobacteria and Firmicutes might be capable of post-depositional biological production of N2O in situ at some depths of the North Greenland Eemian Ice Drilling ice core  at some depths in the ice cores are due to ongoing in situ production by microorganisms. However, microbial activity is presumed to be very low in the deep glacial ice  emission from various sources  and transportation in the air over the ice sheet by atmosphere currents; (ii) deposition onto the glacier ice surface; and (iii) gradual incorporation into the deeper ice layers as snow accumulates continuously  during tlls ml-1 . Bacterills ml-1 . Most oflls ml-1 , which hlls ml-1 . A growiice core . The excr growth . Iron-reice core . Some doice core . These rcial ice . Further2 to 104 cells ml-1) and often only small volumes of ice are available  for collecting low-contaminant ice samples. Considerable effort has been put forth to develop clean sampling technology and a number of surface decontamination strategies have been proposed and summarized in detail . Brieflyocedures . Open aiocedures . Two “baocedures . A totalocedures . In anotocedures .All of these studies removed suspected “contaminants” in ice samples by conducting “background” controls and obtained decontaminated data for further microbial analysis. It is a challenge to determine if the removed microorganisms were from ice or contaminants, and it has been suggested to not remove OTUs identified in “background” controls due to cross-contamination if they are biologically expected in the given sample type .in silico decontamination methods use them to identify and quantify microbial diversity at two depths in an ice core from the Guliya ice cap in northwestern Tibet. The Tibetan Plateau is a mountainous area  that covers about 2.5 million km2 of the Eurasian continent (2), thickest (308.6 m), and coldest (-18.6°C) ice cap among all the ice caps in middle-low latitude regions . This ice core was 10 cm in diameter, 50.80 m in length , and the bedrock temperature was about -15°C. Ice core sections were sealed in plastic tubes, put into cardboard tubes covered with aluminum, and transferred at -20°C by truck from the drill site to freezers in Lhasa, by airplane to freezers in Beijing, by airplane to Chicago, and then by freezer truck to the Byrd Polar and Climate Research Center at Ohio State University where it is stored at -34°C.The Guliya summit 3 (GS3) ice core was drilled in October 2015 from the summit of the Guliya ice cap , for microbial analysis. Dust, chemical ions, and oxygen isotopes were analyzed as described previously  and the clean room (Air_CleanRoom), respectively. Cell collection in the air started at the same time as the processing of the GS3 ice core sections, and continued after ice core processing for a total of 4 days of sampling. The air samples were passed through sterilized polycarbonate 0.8-μm-pore-sized filters , as well as a Button Aerosol Sampler (SKC Inc.), which is reported to have higher recovery efficiency of bacteria (specific recovery efficiency not provided) in indoor and outdoor air compared to three other samplers including the IOM Inhalable Dust Sampler, the NIOSH Personal Bioaerosol Cyclone Sampler, and the 37-mm Filter Cassette sampler  and autoclaved (121°C for 30 min) water was frozen (-34°C for 12–24 h) and then processed in parallel with the GS3 ice core samples through the entire analysis. This control facilitated evaluation of contamination from the instruments used to process the ice. Finally, a blank control was established by extracting DNA directly from 400 ml of 0.22-μm filtered and autoclaved water (as above). This control allowed evaluation of contamination downstream of the ice processing, including the molecular procedures .Four “background” control samples were used to investigate possible sources of background contamination during processing. First, we assessed what microorganisms were in the air from the cold and clean rooms used for ice core processing. Specifically, cells from 28.3 and 28.8 m sampler . These tA total of 400 ml of artificial ice , 400 ml of the blank control (Blank), and 50 ml each of the two ice samples (D41_50_F and D49_50_F) were filtered through sterilized polycarbonate 0.22-μm-pore-sized filters  to collect microorganisms including all bacterial/archaeal cells, with cell sizes exceeding 0.22 μm. The filters were used to isolate DNA. DNA was also isolated from cells concentrated from 100 (D41_100_A) and 20 ml (D41_20_A) of Sample D41 to 0.6 ml by 100 kDa Amicon Ultra Concentrators , with a pre-filtration by 3.0-μm-pore-size filters to remove big dust particles to avoid clogging the concentrators. Community DNA was isolated from these four ice samples  and the four “background” controls  with a DNeasy Blood & Tissue Kit  according to the manufacturer’s instructions, with an additional step of beating with beads to disrupt bacterial spores and Gram-positive cells before cell lysis by homogenizing at 3,400 rpm for 1 min with 100 mg of autoclaved (121°C for 30 min) 0.1-mm-diameter glass beads  in a MiniBeadBeater-16 . DNA was stored at -80°C. DNA denaturants  and 70% ethanol were used to eliminate potential naked DNA and cell contaminants on the surface of gloves, lab benches, and some tools used in this study.Cellulophaga baltica strain 18 . All reactions were performed in triplicate, using an Illumina Eco cycler (Cat No. 1010180).Total bacterial and archaeal biomass was estimated using real-time qPCR for the four ice samples and the four “background” controls after isolating DNA. Primer sets 1406f (5′-GYACWCACCGCCCGT-3′) and 1525r (5′-AAGGAGGTGWTCCARCC-3′) were used to amplify bacterial and archaeal 16S rRNA genes . Each 202), 0.4 μl 10 mM dNTP, 1 μl of each primer , 0.2 μl high-fidelity DNA polymerase, 2 μl template DNA, and 11.4 μl of water. For all eight samples, the first round amplification consisted of a 40-s denaturing step at 98°C, followed by 28 cycles of 8 s at 98°C, 20 s at 48°C, and 15 s at 72°C, with a final extension of 8 min at 72°C. To recondition the PCR products, the amplified reactions were diluted fivefold into a fresh reaction mixture of the same composition and cycled eight times using the same conditions as the first round PCR. All reactions were conducted in triplicate, which were combined as one sample after each PCR. The combined reaction mixtures after reconditioning PCR were purified by Agencourt AMPure XP Beads  and collected in 50 μl of buffer, according to the manufacturer’s instructions.Reconditioning PCR, reported to reduce PCR artificial bias , was conBar-coded primers 515f/806r  were selSequences with an expected error >1.0 or length <245 nt were excluded from the analyses . The remt-test. A heatmap was generated based on the number of sequences per OTU per 30,000 sequences using functions in the Pheatmap package version 1.0.8  based on weighted UniFrac distances, which accounts for changes in relative taxon abundance  of the 16S rRNA gene. The selected samples included two to four samples from each of three Tibetan Plateau ice caps   and the bundance . PrincipSRP114723.The nucleotide sequences discovered during this study have been deposited in the NCBI Sequence Read Archive under accession number Figures 1A,B) to monitor past climate change and archived microbial profiles. In this study, four “background” controls including a sterile water artificial ice core , two air samples collected from the ice processing laboratories (Air_ColdRoom and Air_CleanRoom), and a blank sterile water sample (Blank) were co-processed with four real ice samples to check “background” microbial profiles and their abundances, and establish “clean” sampling and amplicon sequencing protocols. Subsequently the procedures, together with published in silico decontamination methods, were used to investigate the microbial profiles archived in ice at two depths in the GS3 ice core .A GS3 ice core, which was 50.80 m in length and contained ice up to ∼30,000 years old, was retrieved from the Guliya ice cap, China in 2015 . The DNA extraction process could also introduce contaminations, such as those from investigators , tools, and reagents  were in the four “background” controls. Total microbial cell abundance was first measured by epifluorescence microscopy after the cells were concentrated on a 0.22-μm-pore-sized filter  and stained by SYBR Gold as described previously . A totalreagents . Thereforeagents .Figure 2). These genera belonged to five phyla, Proteobacteria, Firmicutes, Cyanobacteria, Bacteroidetes, and Actinobacteria, which contained 16, 4, 2, 3, and 4 genera, respectively . Many exogenous sequences assigned as unexpected taxa from contamination have been detected during the analysis of low-biomass environmental microbiota . These findings indicate that many microbial taxa are common contaminants in microbial community studies. Two additional genera, Cellulophaga and Synechococcus, were detected and interpreted as contaminants in this study . Many isolates belonging to these two genera have been used previously as type strains to investigate virus–host interactions in our laboratory  for further analysis. These sequences were affiliated with 169 bacterial genera, 94 of which had recognized names , we pre-amplified the targeted region (V4) of bacterial and archaeal 16S rRNA genes by reconditioning PCR  . The similar community composition in each pair of original and pre-amplified samples indicates that the reliable microbial profile values were obtained for both original and pre-amplified “background” controls, and that reconditioning PCR captures a microbial community that is almost identical to the original samples with low biomass. Gasterosteus aculeatus). They reported that 24% of the clones were artificial allele chimeras generated by the hybrids of two or three different alleles that occurred in the same individual, using standard PCR, while the number of artificial chimeras was reduced 10-fold by reconditioning PCR , effectively removes the contaminant sequences derived from the “background” controls in the samples of interest  as described in the section “Materials and Methods” . Total sequences belonging to eight OTUs, including OTU_1, OTU_4, OTU_5, OTU_9, OTU_3, OTU_953, OTU_188, and OTU_12, accounted for 93.4–98.9% of all sequences in the 32 OTUs for each ice sample, but only made up 0.3–2.9% of the “background” controls . In contrast, the other 24 OTUs contributed 1.1–6.4 and 97.1–99.7% of the sequences in the 32 OTUs from the ice samples and “background” controls, respectively . These results indicate that the most abundant OTUs in the ice samples were notably different from those in the “background” controls, and that the latter 24 OTUs are probably contaminants and should be in silico removed from the ice samples before taxonomic analysis. Sequences belonging to the most abundant OTUs in the ice samples  were also detected in small amounts in the “background” controls . Similar results were also observed in a study that investigated the bacterial community in mock and control samples . We also pre-amplified the targeted region (V4) of prokaryotic 16S rRNA genes by reconditioning PCR . In addition, the results from ANOSIM analysis . These results indicate that similar microbial profiles were captured from the ice samples regardless of the differences in sample volume and concentrating methods used for collecting cells and DNA extraction.With the “clean” reads after ning PCR  in all fMethylobacterium within the family Methylobacteriaceae was the most abundant taxon in the three D41 samples and had a relative abundance of 67.3–76.6%. An unclassified genus belonging to the same family, Methylobacteriaceae, was also abundant (relative abundance 11.7–14.3%) in these three samples . Members belonging to the genus Methylobacterium were also reported to dominate the microbial community in ancient ice cores from many previous studies  within the family Methylobacteriaceae were also abundant making up 18.3 and 5.2%, respectively, of the total microbial population . The most abundant genus in this sample, however, was Sphingomonas with a relative abundance of 75.2%. Three other genera, including Lactobacillus and two unclassified genera in the phyla Bacteroidetes and Actinobacteria, accounted for 0.1–0.7% of the sequences . Thus there is a notable difference in the microbial profiles between samples D41 and D49. The results from ANOSIM analysis confirmed that the microbial communities were significantly different between samples from D41 and D49 .For sample D49_50_F, the genus Figure 1B and Supplementary Table 3- and NH4+ in D49 were lower than those in D41 while higher concentrations of dust and all other tested ions including Cl-, SO42-, Na+, K+, Mg2+, and Ca2+ occurred in D49 . The first and second dimensions of PCoA showed that the distribution of all samples accounted for 51.5 and 18.9% of community variability, respectively. The weighted UniFrac tree (UPGMA) also showed that most of the samples from a given ice cap formed a lineage . Samples from GLDD and NJKS glaciers clustered together, indicating a closer relationship of their microbial communities. This finding agrees with the original report and might be attributed to the fact that both NJKS and GLDD glaciers are strongly influenced by the same westerly jet stream . This result can be attributed to the fact that both samples were dominated by the genus Sphingomonas with a relative abundance of 94.4 and 75.5% for NEEM-1858 and D49, respectively (data not shown).As noted above the Guliya ice cap is the highest, largest, thickest, and coldest ice cap among all the ice caps in middle–low latitude regions . Considet stream . Samplesp = 0.04) with the age of ice samples, suggesting that the variation in microbial community composition among these ice samples probably reflects unique climate conditions at the time they were deposited. Unfortunately, the relationships between microbial community composition and environmental parameters were not investigated in this study, because of the absence of relevant data in the other studies. In addition, the other ice samples were collected and analyzed in three different projects and laboratories. Although the microbial communities of all samples were analyzed with the overlapped gene region using a next-generation sequencing strategy, the difference in other experimental steps and/or methods  likely also influences the microbial communities reported. Conflicting results of microbial content were reported in several papers investigating microorganisms in glacier ice (Aquabacterium was detected using 16S rRNA gene amplification and sequencing in the Lake Vostok ice samples (Aquabacterium was considered to be a contaminant because it was present in both the Lake Vostok ice sample and its negative control from another study (The Guliya ice cap also shared some bacterial groups with other more distant glaciers, which supports the perspective that microorganisms are distributed everywhere in the world . The twocier ice . For exa samples , Howeverer study . As methin silico “contaminant” removal protocols for our work with low-biomass ice samples. Our effort expands prior work that established in silico contaminant removal procedures (Microbial communities in glacier ice with low biomass have been studied previously. However, as a laboratory new to this science, we sought to establish robust “background” controls and ocedures ; howeverocedures . This wiocedures  and singocedures , we willZ-PZ, NS, MG, DK, EM-T, VR, JVE, LT, and MS conceived and designed the research, analyzed the data, and critically reviewed the manuscript. Z-PZ, NS, MG, and DK performed the laboratory experiments. Z-PZ, EM-T, VR, JVE, LT, and MS wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ticagrelor (30 mg/kg) improved neurological deficits, reduced the infarct volume, and improved CBF. It promoted the phosphorylation of endothelial nitric oxide synthase (eNOS) and extracellular signal-regulated kinase 1/2 (ERK1/2) during the early phase after reperfusion. Increased phosphorylation of eNOS and ERK1/2 were also observed in HBMVECs after OGD stress. Ticagrelor attenuate ischemia reperfusion injury possibly via phosphorylation of eNOS and ERK1/2 in endothelial cells. This suggests that ticagrelor has neuroprotective effects via mechanisms other than its antiplatelet action.Ticagrelor is a direct acting and reversibly binding P2Y12 antagonist approved for the prevention of thromboembolic events. Its potential benefits in ischemic stroke have not been investigated sufficiently. Mice were subjected to 2 hours of transient middle cerebral artery occlusion (MCAO). Mice were orally treated with ticagrelor (10 or 30 mg/kg), aspirin (60 mg/kg), or vehicle at 3 and 24 hours before MCAO and 0 and 6 hours after reperfusion. The infarct volume and neurological deficits 22 hours after reperfusion were evaluated. Cerebral blood flow (CBF) within 24 hours after MCAO was monitored. We performed western blotting and  In patients at high risk for occlusive vascular events, including myocardial infarction, ischemic stroke, angina and peripheral arterial disease, antiplatelet therapy is a key treatment to prevent the including myocardial infarction, ischemic stroke, angina and peripheral arterial disease including myocardial infarction, ischemic stroke, angina and peripheral arterial disease. Ticagrelor is an oral P2Y12 receptor antagonist with a distinct mechanism of action compared with the thienopyridines . Unlike the thienopyridines which are all prodrug with active metabolites binding irreversibly ticagrelor is direct acting and reversibly binding. In the Platelet Inhibition and Patient Outcomes trial (PLATO), which is a phase III trial in patients with acute coronary syndrome (ACS), ticagrelor reduced the composite endpoint of cardiovascular death, myocardial infarction, and stroke when compared to clopidogrel without increasing overall major bleeding2. Based on the clinical evidence, ticagrelor has been approved for the prevention of thromboembolic events in ACS patients. Although the benefit of ticagrelor is likely primarily related to the antiplatelet effect, additional mechanisms have been proposed3. Several authors have reported that ticagrelor inhibits the cellular uptake of adenosine. Armstrong et al. reported that ticagrelor inhibits the cellular uptake of adenosine by inhibition of equilibrative nucleoside transporter-I (ENT-1)4. In rat and pig ischemia reperfusion models, chronic treatment with ticagrelor reduced myocardial infarct volume via an adenosine-dependent mechanism5. These reports implicate that ticagrelor has other effects than P2Y12 inhibition.Stroke is one of the most common causes of death and disability worldwide6. In an experimental study using a permanent rat middle cerebral artery occlusion (MCAO) model, Gelosa et al. reported that the neuroprotective effects of ticagrelor occur via P2Y12-mediated inhibition of microglial activation7.Although ticagrelor is expected to provide benefit in the settings of cerebral ischemia, experimental and clinical evidence are limitedin vivo using an experimental murine transient MCAO model and in vitro using an oxygen-glucose deprivation (OGD) model of human brain microvascular endothelial cells (HBMVECs).To investigate the additional effects of ticagrelor, we investigated the effects of ticagrelor against cerebral ischemia reperfusion injury (IRI) The protocol used in this experiment is illustrated in Fig. Ticagrelor at 30 mg/kg significantly improved neurological deficits as measured using the Garcia score Fig.  and the Figure To investigate the mechanisms underlying the protective effects of ticagrelor against IRI, western blotting was conducted at 2, 6, and 22 hours after reperfusion using peri-infarct brain tissues Fig. . Thirty in vitro OGD stress model using HBMVECs. The protocol used in this study is illustrated in Fig. in vivo study, 1 μmol/L of ticagrelor promoted the phosphorylation of eNOS and ERK1/2 two hours after reoxygenation  to confirm its pharmacological activity. Ticagrelor inhibited platelet aggregation at 10 and 30 mg/kg. These data are consistent with those from previous reports indicating that ticagrelor inhibits platelet aggregation8. We also investigated the effects of aspirin as a control antiplatelet drug. The dose of aspirin was determined based on previous reports that investigated the inhibition of collagen-induced platelet aggregation by aspirin9. Ticagrelor at 3 mg/kg has been reported to have neuroprotective effects in a permanent MCAO rat model via inhibition of P2Y12 depending microglial inhibition8. Importantly, at this dose, ticagrelor did not inhibit platelet aggregation in the same rat study. Here we tested the effects of aspirin and ticagrelor in a mouse model of cerebral IRI at doses providing inhibition of platelet aggregation. Ticagrelor at 30 mg/kg, but not 10 mg/kg, nor 60 mg/kg of aspirin significantly reduced the infarct volume and improved neurological deficits 24 hours after the induction of MCAO, as shown in Fig. In this study, we evaluated the effects of ticagrelor against IRI 10. NOS has three isoforms: eNOS, inducible NOS, and neuronal NOS (nNOS). Among these isoforms, eNOS is constitutively expressed in the vascular endothelium and the choroid plexus. Therefore, we confirmed the promotion of phosphorylation of eNOS after ischemia by ticagrelor in in vitro analysis using HBMVECs under a clinical relevant condition. This result suggests that ticagrelor affects endothelial cells directly. P2Y12 receptor is expressed in platelets and microglia, therefore our results suggest that ticagrelor has P2Y12-independent neuroprotective effects.We investigated the putative mechanisms underlying the protective effects of ticagrelor other than its antiplatelet effects. We found that ticagrelor at 30 mg/kg induces the phosphorylation of eNOS 2 to 24 hours after the induction of MCAO when compared to control group. NOS catalyzes the oxidation of L-arginine and produces NO and L-citrulline23, while NO derived from nNOS leads to neurotoxicity after cerebral ischemia20. The phosphorylation of eNOS at Ser1177 is one of the key regulators of its activity25. The phosphorylation of eNOS may be induced by several factors, including mechanical shear stress13, estrogen14, and vascular endothelial growth factor15. In addition, simvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) is reported to induce the phosphorylation of eNOS pharmacologically16.NO derived from eNOS plays an important role in preserving and maintaining the brain’s microcirculation by inhibiting platelet aggregation, leukocyte adhesion and migration, and reducing smooth muscle proliferationet al. have reported that transgenic mice expressing phosphomimetic forms of eNOS display greater vascular reactivity, suppression of stroke, and improvements in CBF after 1 hour of transient MCAO when compared to transgenic mice expressing unphosphorylatable forms of eNOS17. Several agents, such as Rho kinase inhibitors18 and tetramethylpyrazine analogues19 have been reported to ameliorate brain damage after MCAO via pharmacological phosphorylation of eNOS. In contrast, pharmacological or genetic inhibition of eNOS exacerbates brain damage after cerebral ischemia21. These reports support the idea that phosphorylated eNOS plays an important role in the protection against ischemic brain injury. In this study, ticagrelor induced phosphorylation of eNOS at an earlier time than endogenous phosphorylation, which occurs 6 to 24 hours after cerebral IRI22. We also measured CBF 24 hours after the induction of MCAO. Thirty mg/kg (p.o.) of ticagrelor improved CBF 24 hours after induction of MCAO.Several authors have reported protective effects of eNOS modulation against IRI. Atochin in vivo analysis. Ticagrelor upregulated the phosphorylation of ERK 1/2 in our in vitro study. ERK1/2 is a member of the mitogen-activated protein kinase family and exists mainly in the cytoplasm. Although the role of ERK1/2 in the setting of stroke is debatable, it is involved in the neuroprotective effects of growth factors, estrogen, preconditioning, and hypothermia23. There are several reports regarding the association between ERK 1/2 activation and eNOS. Ho et al. reported that adenosine induces phosphorylation of eNOS via ERK 1/2 phosphorylation in the nucleus tractus solitarii and modulates cardiovascular function in rats24. Cheng et al. reported that simvastatin induces eNOS up-regulation via the Ras-mediated phosphatidylinositol 3-kinase-AKT and extracellular signal-regulated kinase (ERK)− 40 S ribosomal protein S6 kinase signaling pathways in the nucleus tractus solitarii and induces central hypotensive effects25. In previous in vitro studies using endothelial cells, activation of ERK has been shown to be involved in the phosphorylation of eNOS and the NO-mediated production of adenosine, estrogen, and bradykinin27. A recent study indicated that ticagrelor inhibits the cellular uptake of adenosine via ENT-1 and leads to an increase in the extracellular concentration of adenosine4. It is reported that exogenous adenosine increases coronary blood flow velocity in healthy volunteers taking 180 mg of ticagrelor orally28. It is also reported that intracerebral concentrations of adenosine are elevated under ischemic conditions29 and that pharmacological inhibition of adenosine uptake has neuroprotective effects against focal cerebral ischemia31. In myocardial IRI models ticagrelor has been shown to reduce infarct size via an adenosine mediated mechanism and to increase the phosphorylation of eNOS, ERK, Akt also via an adenosine mediated mechanism32. Taken together, the above data indicate that the activation of eNOS via ERK1/2 may underlie the neuroprotective effects of ticagrelor and that the inhibition of adenosine uptake by ticagrelor might be associated with these effects.Since phosphorylation of ERK1/2 is associated with the phosphorylation of eNOS, we measured ERK1/2 phosphorylation levels 2 hours after reperfusion in the ticagrelor-treated group in our Ticagrelor is expected as a prophylactic agent for recurrence of ischemic events. Our results, ticagrelor has P2Y12-independent neuroprotective effects, suggests that ticagrelor has additional effects as a prophylactic agent for recurrence of ischemic events.In conclusion, ticagrelor significantly improved cerebral blood flow, reduced infarct volume, and improved neurological function after cerebral IRI. The phosphorylation of eNOS and ERK 1/2 in endothelial cells may underlie the neuroprotective effects of ticagrelor. These findings indicate that ticagrelor has protective effects on cerebral IRI by mechanisms other than platelet inhibition.in Vivo Experiments guidelines. Careful consideration was given to minimizing the numbers of animals used and their suffering. All in vivo experiments were conducted using male ddY mice  purchased from Japan SLC, Inc. . All animals were kept in a controlled environment  and permitted to access filtered clean water and standard laboratory food freely.The experimental design and procedures of this study were approved by the animal experiment committees of Gifu Pharmacological University and were in compliance with Animal Research: Reporting Two-hundred and eighty-six mice were used for this study. The number of mice used in each experiment is as follows: (1) platelet aggregation test, 48 mice; (2) MCAO model, 129 mice (12 mice excluded); (3) western blotting, 109 mice (17 mice excluded).Ticagrelor  and aspirin  were purchased. Mice were randomly divided into two ticagrelor treatment groups (10 mg/kg and 30 mg/kg), an aspirin treatment group (60 mg/kg), and a vehicle treatment group. Drugs were dissolved in 0.4 mL 1.0% carboxymethylcellulose and administrated by oral gavage at 3 and 24 hours before MCAO and 0 and 6 hours after reperfusion. Mice in vehicle treatment group were administered the same amount of carboxymethylcellulose at same time point.33. Mice were treated 24 hours and 3 hours before the measurement of platelet aggregation. Under deep anesthesia with pentobarbital, whole blood was obtained by cardiac puncture using a 22-gauge needle. Four milliliters of whole blood obtained from four or five mice were collected in a 5-mL tube containing 3.0 mL of 3.8% sodium citrate. Platelet-rich plasma was obtained by centrifugation at 155 g for 12 minutes at room temperature. Twenty micromoles per liter adenosine diphosphate  was added to 225 μL of platelet-rich plasma to induce platelet aggregation. Platelet aggregation was measured using an aggregometer . During the measurement of platelet aggregation, the samples were continuously stirred and kept at 37 °C.Platelet aggregation was measured using light transmission aggregometry, as previously described34. In brief, mice were anesthetized using a mixture of 70% NO2, 30% O2, and 2.0–2.5% isoflurane  using an animal general anesthesia machine . The left common carotid artery was exposed using a midline skin incision. The origin of the middle cerebral artery was occluded using an 8–0 nylon monofilament coated with Provil novo . After 2 hours of MCAO, the filament was removed to establish reperfusion under brief anesthesia. The body temperature of the mouse was kept at 37.0–37.5 °C using a heating lamp and a heating pad during the procedure. After the procedure, the mice were kept in the same conditions as the preoperative environment  until further experiments.To induce focal ischemia, a mouse MCAO model was established, as previously describedThe following exclusion criteria were set before the experiment: (1) death during surgery due to procedural or anesthetic problems; (2) death before sampling; (3) inability to achieve reperfusion; (4) absence of a detectable ischemic core; and (5) a reduction of CBF less than 30% of baseline, as measured using laser Doppler flowmetry .34. In brief, the forebrains of mice were removed under deep anesthesia with isoflurane  24 hours after the induction of MCAO. Five 2-mm thick coronal sections were stained using a 2% TTC  solution for 30 minutes. The stained sections were digitally imaged and analyzed using image-processing software .The experimenters (K.Y. and T.I.) performing the assessments were blinded to the experimental groups. Infarct volume was quantified as previously describedet al., the following six behaviors were assessed: (1) spontaneous activity for 5 minutes, (2) symmetry of the four limbs during movement, (3) symmetry of forelimb outstretching, (4) climbing ability, (5) body proprioception, and (6) response to vibrissae touch. Each behavior was scored on a scale between 0 and 3 points and a total score was calculated. Lower scores indicated more severe neurological deficits35. As previously reported by Lee et al., the grid walk test was performed to evaluate walking skill. Mice were allowed to freely walk on a grid , and the number of step errors was counted for 2 minutes36.All behavioral tests were conducted in a quiet and low-light environment by blinded scientists (K.Y. and T.I.). As previously reported by Garcia 37. Mice were placed in the prone position under brief anesthesia with 2.0–3.0% isoflurane , and the skull was exposed using a linear skin incision. The skull was exposed to a 780-nm semiconductor laser. The reflected light, which was linearly polarized, was detected using a charge-coupled device camera placed above the head through a hybrid filter. The hybrid filter was used to preclude the detection reflected light from the surface of the tissue and to enable stable and specific measurements. The raw speckle images were videotaped to evaluate the speckle contrast, which indicates the velocity and numbers of moving red blood cells. The average of 20 consecutive raw speckle images was used to obtain one blood flow image. The red color indicates the higher cerebral blood flow. The images were analyzed using the software installed in a laser speckle imaging system . Oval regions of interest (ROIs) were created at the ipsilateral and contralateral middle cerebral artery (MCA) areas. Relative blood flow in the MCA area was calculated by averaging the blood flow of the ROIs, and the ratio of ipsilateral CBF to contralateral CBF was calculated. CBF was measured just before MCAO, just after MCAO, just before reperfusion, just after reperfusion, and 4 and 22 hours after reperfusion.Periprocedural cerebral blood flow was measured using laser Doppler flowmetry , as previously reportedin vitro, we used an OGD model in HBMVECs, as previously reported38. Briefly, the cells were cultured in 96-well culture plates until they became confluent. The cells were then washed twice with glucose-free Dulbecco’s modified Eagle’s medium (DMEM) and incubated in the same glucose-free medium in an oxygen-free incubator  for 6 hours, and under normal growth conditions for an additional 2 hours (reoxygenation). Ticagrelor  was added to the medium just before OGD and just after reoxygenation. The concentration of ticagrelor was determined according to the Cmax (1.4 ± 0.7 (μmol/L ± SD)) and Cmin (0.4 ± 0.7 (μmol/L ± SD)) levels in patients taking 4 weeks of standard doses of 90 mg BID as previously reported39. Control cells were incubated in DMEM. We performed the cell viability and western blot assays 2 hours after reoxygenation.To assess the effects of ticagrelor against IRI in endothelial cells 40. Two hours after reoxygenation, the cells were incubated with 10% 2-(2-methoxy-4-nitrophenyl)−3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt for 3 hours at 37 °C, and optical density at 450 nm was measured using a microplate reader .Cell viability after OGD was measured using Cell Counting Kit 8  as previously describedg for 30 minutes at 4 °C), supernatants from the samples were collected. Ten micrograms of the protein samples were applied onto 5–20% gradient sodium dodecyl sulfate polyacrylamide gels  and separated according to molecular weight using electrophoresis. The separated proteins were transferred to polyvinylidene fluoride membranes . The following primary antibodies were used: rabbit anti-phosphorylated eNOS Ser1177 , rabbit anti-eNOS , rabbit anti-phospho-ERK1/2 Thr202/Tyr204 , rabbit anti ERK1/2 , and monoclonal anti-β-actin . The following secondary antibodies were used: anti-rabbit immunoglobulin G (IgG)  and anti-mouse IgG . Immunoreactive bands were visualized using a Lumino imaging analyzer . Multi Gauge software  was used to analyze differences in band intensity.The forebrains of mice were removed quickly under deep anesthesia with isoflurane 2 hours, 6 hours, and 22 hours after reperfusion. Tissue from the peri-infarct area was also collected. In the cell culture experiments, the cells were collected 2 hours after reoxygenation. Ten milliliters ice-cold radioimmunoprecipitation assay lysis buffer and protease/phosphatase inhibitor per gram of tissue was used for homogenization (Sigma-Aldrich Co.). After centrifugal separation .All data are shown as means ± standard errors of the mean. We used Student’s two-paired Supplementary Information