Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon  and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection. Piscine orthoreovirus (PRV) belongs to the genus Orthoreovirus in the family Reoviridae [Salmo salar), detected both in apparently healthy and diseased fish [oviridae ,2. The ooviridae ,4,5,6,7.sed fish ,9,10,11.sed fish . HSMI issed fish , after wsed fish . In expesed fish .Onchorhynchus kisutchi). The level of anemia in EIBS affected fish corresponded with the level of viral replication in blood [The study of molecular mechanisms linked to PRV infection has been limited by the lack of susceptible cell lines. Studies of the viral infection have therefore been performed in vivo or by infecting erythrocytes ex vivo . Piscinein blood . In addiin blood .Orthoreovirus genome consists of ten double-stranded RNA (dsRNA) segments enclosed in a double protein capsid. The genomic segments are classified according to size with three large (L), three medium (M) and four small (S) segments encoding the λ, µ and σ class proteins, respectively [Orthoreovirus, the viral transcription machinery is located in the inner core and consists of λ1, λ2, λ3, µ2 and σ2 [The ectively ,21. In m2 and σ2 . The out2 and σ2 ,24,25. T2 and σ2 ,27. Alth2 and σ2 . The N-t2 and σ2 ,30. Furt41) while in ARV, post-translational cleavage in the N-terminal region releases µNSN [Structures resembling viral factories have also been observed in PRV-infected erythrocytes, and recombinant expression of the protein in fish cell lines indicate that PRV µNS has an analogous role in factory formation ,19,31. Tses µNSN ,35,36. Ises µNSN .Considering the emerging occurrence of HSMI, PRV exhibits a considerable risk for the aquaculture industry and proper disease control is highly desired. To understand the association between PRV infection and disease outcome, and also to limit further disease outbreaks, more information regarding PRV protein kinetics is essential. In the present study, the kinetics of viral RNA, viral protein and antiviral immune response in blood cells from experimentally PRV-infected Atlantic salmon were investigated. We hypothesized that PRV causes an acute infection in blood cells correlating with innate antiviral gene expression, before the infection subsides to a low persistent level.® vector (Invitrogen). The pENTR µNS construct was used in a recombination reaction to generate the recombinant baculovirus DNA. Sanger sequencing  confirmed the sequence of the construct. Spodoptera frugiperda (Sf9) insect cells  cultured in Grace Insect Medium (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum , 100 U/mL Penicillin, 100 µg/mL Streptomycin and 0.25 µg/mL Fungizone (Life Technologies), were transfected with recombinant baculovirus DNA. Passage 1 (P1) viral stock was harvested 11 days post transfection and used to produce high titer viral stocks according to the supplier’s protocol. The BacPAK quantitative PCR (qPCR) Titration kit  was used to determine the viral titer. Finally, Sf9 insect cells were infected with Passage 2 (P2) or higher passage of recombinant baculovirus stock (>1 × 108 copies/mL) and incubated at 27 °C for 96 h for expression of the recombinant µNS protein containing a C-terminal 6xHis-tag.Following the supplier’s protocol, the BaculoDirect™ Baculovirus Expression System  was used to generate recombinant µNS. The μNS open reading frame (ORF) (acc. no. KR337478) was obtained by polymerase chain reaction  and expressed with a N-terminal 6xHis-tag, following the manufacturer’s instructions. Protein expression was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).The ORF of PRV structural protein λ1 (acc. no. KR337475) encoded by gene segment L3 was amplified . Purification of recombinant proteins was carried out using ProBond Purification System (Life Technologies) following the manufacturer’s instructions. The recombinant µNS protein was eluted with an elution buffer containing 8 M Urea, 20 mM Na2H2PO4 (pH 4.0), and 500 mM NaCl. The purity of the recombinant protein was monitored by SDS-PAGE using a 4%–12% Bis–Tris Criterion XT gel . To purify λ1, the Ni-NTA agarose was run on a SDS-PAGE where a band matching the size of λ1 was excised. The gel sample containing λ1 protein was solubilized in 250 mM Tris-HCl with 0.1% SDS, pH 6.8, sonicated 3 × 5 s and incubated at 4 °C with shaking overnight. The sample was centrifuged at 10,000× g for 10 min and the supernatant was dialyzed using the Slide-A-Lyser® Dialysis cassette with 20,000 molecular weight cut-off (MWCO) and 0.5–3.0 mL capacity  following the manufacturer’s protocol. SDS-PAGE confirmed the purity of the recombinant λ1 protein. Protein concentrations for both μNS and λ1 were determined using the DC Protein Assay Reagent Package (Bio-Rad), with bovine serum albumin  as protein standard.The Sf9 insect cells and the The purified recombinant proteins were used for immunization of rabbits and generation of antisera named anti-µNS #R320684 and anti-λ1 #K273. In the first injection, Freund’s complete adjuvant was added, thereafter the rabbits were boosted three times with Freund’s incomplete adjuvant weekly. The amount of µNS and λ1 antigen used per immunization was in the range of 45–500 μg. The rabbit sera produced were tested by Western blotting (WB) and fluorescent microscopy after transfection of epithelioma papulosum cyprini  cells with pcDNA3.1 µNS N-FLAG  or pcDNAl-glutamine, 0.04 mM mercaptoethanol and 0.05 mg/mL gentamycin-sulphate (Life Technologies), and seeded at a density of 1.5 × 104 cells/well in a 24-well plate 24 h prior to transfection. Plasmids pcDNA3.1-µNS N-FLAG and pcDNA3.1-λ1 N-HA were transfected using Lipofectamine LTX reagent (Life Technologies) according to the manufacturer’s instructions. The cells were fixed and stained 48 h post-transfection with an Intracellular Fixation and Permeabilization Buffer Set  following the manufacturer’s protocol. Antisera against µNS (1:1000) and λ1 (1:500); secondary antibody against rabbit IgG conjugated with Alexa Fluor 488 (Life Technologies) and Hoechst trihydrochloride trihydrate (Life Technologies) were used for staining. Images were captured on an inverted fluorescence microscope (Olympus IX81). Transfected EPC cells were also used to further verify anti-µNS and anti-λ1 in WB. A total of 3 × 5 million EPC cells were pelleted by centrifugation, resuspended in 100 μL Ingenio Electroporation Solution  and transfected with 4 μg pcDNA3.1 µNS N-FLAG or pcDNA3.1 λ1 N-HA. The transfected cells were transferred to 75 cm2 culture flasks containing 20 mL pre-equilibrated L-15 growth medium (described above) and collected 72 h post-transfection. The cell pellets were lysed in Nonidet-P40 lysis buffer  containing Complete ultra mini protease inhibitor cocktail . The mix was incubated on ice for 30 min, and then centrifuged at 5000× g for 5 min at 4 °C. The supernatant was mixed with Sample Buffer (Bio-Rad) and Reducing Agent (Bio-Rad), denatured for 5 min at 95 °C and run in SDS-PAGE, using 4%–12% Bis–Tris Criterion XT gel (Bio-Rad). Magic MarkTM XP Standard (Invitrogen) was used as a molecular size marker. Following SDS-PAGE, the proteins were blotted onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad) and anti-µNS and anti-λ1 were used as primary antibodies and anti-Rabbit IgG-HRP  as secondary antibody. Protein bands were detected by chemiluminescence .EPC cells were cultivated in Leibovitz-15 medium  supplemented with 10% heat inactivated FBS, 2 mM 3, and were kept in 0.4 m3 tanks supplied with filtered and UV-radiated fresh water, 12 °C ± 1 °C with a 12 h light/12 h dark regime. Water discharge of the tanks was provided by a tube overflow system with 7.2 L/min flow rate. The fish were acclimatized for two weeks prior to challenge, fed according to standard procedures and anesthetized by bath immersion (2–5 min) in benzocaine chloride  before handling. Briefly, the experimental study included one group of shedder fish (50%) marked at the time of PRV-injection by cutting off the adipose fin and one naïve cohabitant group (50%). The PRV inoculum was prepared from a batch of pooled heparinized blood samples from a previous PRV challenge experiment [A cohabitation challenge experiment was performed at VESO Vikan aquatic research facility, . The fish had an average weight of 30 grams at the onset of the experiment with a maximum stocking density of 80 kg/mperiment . On day 0 of the challenge, the heparinized blood was diluted 1:2 in PBS and 0.1 mL of the inoculum was intraperitoneal (i.p.) injected into the shedders. The inoculum was confirmed negative for salmon viruses such as infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmonid alphavirus (SAV) and piscine myocarditis virus (PMCV) by reverse transcription quantitative PCR (RT-qPCR). Samples from six fish were collected before initiation of the experiment to provide time-0 uninfected control material for protein assays. Heparinized blood was collected from six cohabitant fish at each sampling point; 3, 4, 5, 6, 7 and 8 weeks post challenge (wpc). In addition, a second cohabitation challenge experiment lasting 10 weeks was performed at the same facility following a similar experimental design. In this study, six fish sampled prior to PRV challenge were used to provide uninfected control material for protein and RT-qPCR assays, and heparinized blood was collected from six cohabitant fish at 4, 6, 8 and 10 wpc. The second challenge experiment was otherwise performed under the same conditions as the first experiment. Both experiments were approved by the Norwegian Animal Research Authority and followed the European Union Directive 2010/63/EU for animal experiments.2, 1:100 RNase Out (Invitrogen) and 1 × ROX reference dye with the following cycle parameters: 30 min at 50 °C, 15 min at 94 °C, 40 cycles of 94 °C/15 s, 54 °C/30 s and 72 °C/15 s in an AriaMx . Similar conditions and cycle parameters were also used targeting M2 and M3, although primer concentration was adjusted to 600 nM and annealing temperature to 58 °C. All samples were run in duplicates, and a sample was defined as positive if both parallel samples had a Ct <35. The fluorescence threshold for S1, M2 and M3 was set at ΔRn 0.261, 0.028 and 0.021, respectively. The primers and probes are listed in ® Green Supermix, and 500 nM forward and reverse primers  using 5 mm steel beads, TissueLyser II (Qiagen) and RNeasy Mini spin column (Qiagen) as recommended by the manufacturer. RNA was quantified using a NanoDrop, ND-1000 spectrophotometer . The Qiagen OneStep kit (Qiagen) was used for RT-qPCR with a standard input of 100 ng (5 μL of 20 ng/μL) of the isolated total RNA per reaction in a total reaction volume of 12.5 µL. The template RNA was denaturated at 95 °C for 5 min prior to RT-qPCR targeting PRV gene segments S1, M2 and M3. The following conditions were used for S1: 400 nM primer, 300 nM probe, 400 nM dNTPs, 1.26 mM MgCl primers . The qPCSamples consisting of 1.25 µL heparinized blood (diluted 1:20 in PBS) from each of the cohabitant fish in the first challenge experiment were plated into 96-well plates for intracellular staining as previously described  using anFollowing flow cytometry analysis, the cells were prepared for immunofluorescence microscopy. The nuclei were stained with Hoechst trihydrochloride trihydrate (Life Technologies) and the cells were mounted to glass slides using Fluoroshield  and cover slips. Images were captured on an inverted fluorescence microscope (Olympus IX81).g for 5 min at 4 °C, washed twice in PBS and fixed in 3% glutaraldehyde overnight at 4 °C. All samples were further washed twice in PBS and prepared for transmission electron microscopy (TEM) as described earlier [Samples consisting of 20 µL heparinized blood from each cohabitant fish in the first experimental challenge were diluted in 1 mL PBS, centrifuged at 1000×  earlier . The secg and the blood pellets was lysed in Nonidet-P40 lysis buffer containing Complete ultra mini protease inhibitor cocktail and prepared for WB as described above. Anti-µNS (1:1000), anti-µ1C (1:500) [Heparinized blood from each cohabitant fish in the first challenge experiment was analyzed separately and as pooled samples from the different time-points. The samples were centrifuged at 5000×  (1:500)  and antiBlood from six cohabitants in the first challenge experiment sampled at 4, 5 and 8 wpc were pooled and lysed in Nonidet-P40 lysis buffer containing Complete ultra mini protease inhibitor cocktail as described above. The supernatants were transferred to new tubes and added anti-µNS or anti-µ1C (1:50) and incubated at 4 °C overnight with rotation. The Immunoprecipitation Kit Dynabeads Protein G  was used for protein extraction and the beads were prepared according to the manufacturer’s protocol. The cell–lysate–antibody mixtures were mixed with the protein G-coated beads and incubated 2 h at 4 °C. The beads–antibody–protein complexes were washed according to the manufacturer’s protocol and run in SDS-PAGE. The SDS-gel was blotted onto PVDF membranes (Bio-Rad) and the proteins were detected using anti-µNS, anti-µ1C [Five and three fragments immunoprecipitated with anti-µNS (4 and 5 wpc) and anti-µ1C (5 wpc), respectively, that were not observed at 0 wpc, were excised and in-gel digested with 0.1 µg of trypsin in 20 µL of 50 mM ammonium bicarbonate, pH 7.8 for 16 h at 37 °C . The peptides were purified with µ-C18 ZipTips , and analyzed using an Ultimate 3000 nano-UHPLC system  connected to a Q Exactive mass spectrometer . Liquid chromatography and mass spectrometry was performed as previously described [Theoretical molecular weights for proteins were calculated using the Compute pI/Mw tool . PSI-blaDifferences in gene expression levels of innate antiviral genes was analyzed using one-way Anova with Tukey’s multiple comparison test. Correlation analysis between PRV S1/M3 RNA levels and antiviral and immune gene expression were performed using nonparametric Spearman correlation.RT-qPCR targeting PRV genomic segments S1, M2 and M3 revealed high viral RNA loads in blood cells from 3 to 8 wpc . RNA froThe innate antiviral immune response in blood following PRV infection was studied by RT-qPCR targeting Atlantic salmon type I interferon (IFNab), viperin, interferon-stimulated gene 15 (ISG15), dsRNA-activated protein kinase (PKR) and IFNγ. All innate antiviral genes analyzed were statistically significantly upregulated during the peak phase of PRV infection from 4 to 6 wpc, increasing 5- to 20-fold compared to the level at 3 wpc . The Ct Blood cells stained intracellularly with anti-µNS and anti-σ1 were analyzed by flow cytometry . A PRV pBoth µNS and σ1 were detected by immunofluorescence as cytoplasmic globular inclusions in erythrocytes at 4, 5 and 6 wpc b. The inTEM of PRV infected blood cells sampled at 0, 4, 5 and 6 wpc are shown in Blood cells from six fish sampled at 3, 4, 5 and 6 wpc were analyzed by WB using anti-µNS and compared to Ct-values targeting the corresponding genomic segment M3 of the same samples . No fishThe load of structural proteins λ1, µ1, σ1 and σ3, and the non-structural protein µNS, displayed a similar transient peak at 4–6 wpc in blood cells . All fivInteraction between µNS and other PRV proteins was studied by IP and WB . At 4 wp85, M94, M115 or M169 would provide proteins with predicted sizes of 74.5, 73.6, 71.1 and 65.5 kDa, respectively. M115 is the most likely candidate due to its size and presence in all PRV strains.WB of infected blood cells consistently produced two µNS bands of approximately 83.5 and 70 kDa . Due to 42P43 (MW 69.8 kDa). Of the fourteen peptide spectrum matches identified from the 70 kDa band, two were found to overlap N42P43  was detected at 4 wpc . In contp N42P43 . This isDa bands . Among tF387S388 . The resScreening of farmed Atlantic salmon has indicated that PRV is ubiquitous in seawater and causes a persistent infection ,11,41,42Erythrocytes are major target cells for PRV . PiscineThe salmon does not appear to be able to eliminate PRV. Challenge experiments have shown that PRV RNA can be detected at a steady level in heart and liver until 36 wpc (end of experiment) , and in PRV infection in erythrocytes has previously been shown to induce expression of type I interferon and interferon-regulated genes ,18. In t115 residue was determined to be the best candidate as M94 is not conserved among all PRV isolates, and M85 and M169 are unlikely due to the sizes of the proteins generated. Post-translational cleavage to generate µNSC as shown for ARV µNS cannot be excluded, although the specific proteolytic cleavage site in the ARV protein is not conserved in PRV [115, may differ in their interactions with other PRV proteins. At 4 wpc, when only the 70 kDa variant of µNS was detected following IP, all targeted structural proteins co-precipitated. However, at 5 wpc, when full-length µNS was dominant, only the σ3 protein and the assumed µ1 fragment δ co-precipitated. Studies previously performed on aquareoviruses and ARV indicate that recruitment of viral proteins into viral factories occurs in a predefined order through direct or indirect association with µNS [Orthoreoviruses generate viral factories in the cytoplasm of infected cells ,50,51,52d in PRV ,53. The with µNS .42P43 cleavage site, both regarded as crucial for reovirus µ1-mediated membrane penetration [Four different molecular sizes of the µ1 protein were observed in the infected blood cells. Previous multiple sequence alignments of the µ1 amino acid sequence showed absolute conservation of the G2-myristoylation site and the autolytic N42P43 site were observed from the 70 kDa band following LC–MS, peptides ending in P43 were present in equal amount. We conclude that the presence of the N42P43 overlapping peptides originate from carryover of the slightly larger full-length µ1 following gel excision. In addition, proteins can exhibit different abilities to separate in SDS-PAGE. This explains the presence of a minor fraction of peptides from the δ fragment  in the φ fragment  and vice versa. No peptide sequences overlapping N42P43 were identified from the 37 and 32 kDa bands. Rather, a higher number of peptides with an N-terminal P43 generated by non-tryptic cleavage were identified, providing additional support for cleavage at N42P43.Although peptides containing amino acid sequences overlapping the N43 and F387. From 7 wpc and onwards, the only PRV protein detected was a ~35 kDa protein which could represent the δ proteolytic fragment.MRV µ1 contains a second cleavage site in its C-terminal region which, upon cleavage by exogenous proteases, generates the additional fragments δ and φ . In the Production-related diseases are often multifactorial and the outcome of a PRV infection is influenced by viral strain, age of the fish, production and environmental factors. Recently, PRV was demonstrated to be the etiologic agent of EIBS, causing anemia and mass mortality in juvenile Coho salmon . The levIn this study, we show that PRV infection has an acute phase in blood cells with high virus production before the infection subsides to a low persistent level. The continued transcription of viral RNA in the persistent phase suggests that the innate antiviral immune response may act to inhibit the virus infection post transcriptionally.


Heat shock protein 90 is a chaperone that plays an essential role in the stabilization of a large number of signal transduction molecules, many of which are associated with oncogenesis. An Hsp90 isoform (Hsp90α) has been shown to be selectively phosphorylated on two N-terminal threonine residues (threonine 5 and 7) and is involved in the DNA damage response and apoptosis. However, the kinase that phosphorylates Hsp90α after ionizing radiation (IR) and its role in post-radiation DNA repair remains unclear. Inasmuch as several proteins of the DNA damage response machinery are Hsp90 clients, the functional consequences of Hsp90α phosphorylation following IR have implications for the design of novel radiosensitizing agents that specifically target the Hsp90α isoform. Here we show that ATM phosphorylates Hsp90α at the T5/7 residues immediately after IR. The kinetics of Hsp90α T5/7 phosphorylation correlate with the kinetics of H2AX S139 phosphorylation (γH2AX). Although Hsp90α is located in both the cytoplasm and nucleus, only nuclear Hsp90α is phosphorylated by ATM after IR. The siRNA mediated knockdown of Hsp90α sensitizes head and neck squamous cell carcinoma cells, lung cancer cells and lung fibroblasts to IR. Furthermore, MEF cells that are Hsp90α null have reduced levels of γH2AX indicating that Hsp90α is important for the formation of γH2AX. Thus, this study provides evidence that Hsp90α is a component of the signal transduction events mediated by ATM following IR, and that Hsp90α loss decreases γH2AX levels. This work supports additional investigation into Hsp90α T5/7 phosphorylation with the goal of developing targeted radiosensitizing therapies. Hsp90AA1) and Hsp90β (Hsp90AB1), are 86 per cent homologous in amino acid sequence, are expressed from separate genes and are together referred to as Hsp90 [in vivo function [The 90 kDa heat shock protein (Hsp90) is an abundant and essential stress protein expressed in all eukaryotic cells that aids in the post-translational folding and stabilization of a wide variety of proteins called Hsp90 “clients”, and it is an important regulator of protein homeostasis , 2. The as Hsp90 . The funas Hsp90 . In contas Hsp90 . These dHsp90 is primarily located in the cytoplasm with only about 3% being in the nucleus; nevertheless, the chaperone regulates a number of nuclear events . MultiplAll three of the PIKKs can phosphorylate Hsp90α, but DNA-PK is the sole PIKK responsible for Hsp90α phosphorylation during TRAIL-induced apoptosis , and it Hsp90 inhibitors compromise DNA repair after radiation , 15, sugFigure DNA-PK has been shown to phosphorylate Hsp90α at the threonine 5 and 7 positions following TRAIL-induced apoptosis  and treaTo determine the cellular location of phospho-T5/7 Hsp90α we performed immunostaining in MRC5 cells. Specifically, cells were fixed at the 30 minute, 6 hour and 24 hour time points. As expected, phosphorylation was detected at 30 minutes and decreased to normal levels within 24 hours. Antibodies against phospho-T5/7 Hsp90α (red) and total Hsp90α (green) were utilized in this experiment. As shown in Figure To determine the effects of Hsp90α deficiency on cell survival and radiosensitivity, we knocked down Hsp90α using siRNA in UMSCC1, NCI-H1975, and MRC5 cells. Figure Given that γH2AX is important for the formation of repair foci following DNA damage, we sought to determine if γH2AX levels are affected following IR in Hsp90α deficient cells. To accomplish this, we obtained wild-type MEF cells and MEF cells that are Hsp90α null. In Figure In the present study, we show that IR induces Hsp90α threonine 5 and 7 phosphorylation by ATM and that Hsp90α deficiency reduces γH2AX levels and sensitizes cells to IR. This work provides evidence that Hsp90α is a component of the complex signaling cascade mediated by ATM and is directly involved in the DNA damage response following IR.We demonstrate that ATM plays a major role in the threonine 5 and 7 phosphorylation of Hsp90α following IR of AT5BIVA cells. In a recent study, Solier et al.  reportedThe phosphorylation of Hsp90α on threonine 5 and 7 was observed in all the cell lines used here as well as in studies previously published , 13. TheOur study supports further research into the implications of Hsp90α threonine 5 and 7 phosphorylation upon IR and other DNA damaging agents. Investigation into selective inhibitors of Hsp90α activity may lead to promising radiosensitizing agents because of its direct role in the DNA damage response.AA1 Smartpool) was purchased from Dharmacon .Antibodies to detect phospho-T5/7 Hsp90α, heat shock protein 70 (Hsp70), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technologies . Antibodies to detect Hsp90α and Hsp90β were purchased from Enzo Life Sciences . The antibody to detect γH2AX was acquired from Millipore  and the antibody to detect phospho-S254 Hsp90β was acquired from Abcam . Protease inhibitor cocktail was purchased from Sigma . Small interfering RNA (siRNA) used in this study against Hsp90α  provided the human head and neck squamous cell carcinoma cell line, UMSCC1. Dr. J.A. Engelman  provided the lung cancer cell line, NCI-H1975. Hsp90α null MEF cells and wild-type MEF cells were provided by Dr. Heiichiro Udono . AT5BIVA, AT5BIVA kinase dead, and AT5BIVA-ATM cells were acquired from Dr. Mira Jung, . The lung fibroblast cell line, MRC5, was acquired from the American Type Culture Collection. UMSCC1 and NCI-H1975 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. MRC5 and MEF cells were grown in DMEM medium supplemented with 10% fetal bovine serum. All AT5BIVA cells were grown in RPMI 1640 medium supplemented with 20% fetal bovine serum. In all experiments performed in this study, cells were released from flasks with 0.25% trypsin and 0.2 mM EDTA and plated onto cell culture dishes the day prior to any treatment.Cells were scraped into a lysis buffer containing 50 mM HEPES.KOH, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM NEM, 1 mM NaF, 100 μM sodium orthovanadate, 10% glycerol, 10 mM β-Glycerophosphate, 0.1% NP40, and protease inhibitor cocktail. All samples were briefly sonicated and protein lysates were centrifuged at 13,200 RPM for 5 minutes at 4°C. The supernatant was subject to protein estimation using the Bradford method at 595 nanometers. Loading buffer containing 250 mM Tris (pH 6.8), 40% Glycerol, 4% SDS, 12.5 mM EDTA, 10% β-Mercaptoethanol, and 0.08% bromophenol blue was added to each sample before it was loaded onto a 4–12% pre-cast Bis-Tris gel (Invitrogen). Protein was transferred to PVDF membranes and was blocked with 5% BSA with 1% normal goat serum in 1X tris-buffered saline and 0.1% tween 20 at room temperature for 45 minutes. Membranes were incubated with 1 μg/ml primary antibody overnight at 4°C. Following overnight incubation, membranes were washed in 1× tris-buffered saline and 0.1% tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody  for 60 minutes at room temperature. Membranes were washed 3 additional times and bound antibody was detected using an enhanced chemiluminescence agent .Immunofluorescence staining was performed using standard protocols. Briefly, MRC5 fibroblasts grown on coverslips were washed in PBS, fixed in methanol at −20°C for 15 minutes, permeabilized with 0.2% Triton-x-100 on ice for 5 minutes, blocked for 60 minutes, and incubated in primary antibody at 4°C overnight. The primary antibody dilution for phospho- Hsp90α and Hsp90α was 1:100 in 5% BSA. The following morning the coverslips were washed using PBS, incubated with the fluorescence-conjugated secondary antibody for 60 minutes, washed again using PBS, and prepared with a coverslip using a drop of ProLong Gold anti-fade reagent with 4, 6-diamidino-2-phenylindole (Molecular Probes). Fluorescence images were obtained using a DS-Fi1  camera fitted on an Olympus 1×-71 microscope.Hsp90α knockdown was accomplished in UMSCC1, NCI-H1975, and MRC5 cells by using the lipofectamine-based protocol (Invitrogen) to transfect 50 nM of Hsp90α siRNA or non-specific siRNA in Opti-mem (Gibco). The Opti-mem was replaced with cell culture media 24 hours following transfection, and the cells were irradiated and processed for further analysis 48 hours after transfection.Clonogenic survival assays were performed by plating 75,000 cells in 60 mm cell culture dishes. Following the transfection (48 hours for Hsp90a knockdown) cells were radiated, trypsinized, and plated in 6-well cell culture dishes in triplicate. The UMSCC1, NCI-H1975, and MRC5 cells were plated based on a predetermined plating efficiency in 6-well cell culture dishes. Six to nine days later, cell colonies were fixed with acetic acid/methanol (1:7 ratio) and stained with a crystal violet  solution. A stereomicroscope was used to count cell colonies. The fraction of cell colonies surviving each treatment was normalized to the survival of the control cells in each experiment. The cell survival enhancement ratio was calculated as the ratio of the mean inactivation dose in control cells divided by the mean inactivation dose in treated cells . Experim


In this paper, comparisons of damage evolution between 2D C/SiC and SiC/SiC ceramic-matrix composites (CMCs) under tension–tension cyclic fatigue loading at room and elevated temperatures have been investigated. Fatigue hysteresis loops models considering multiple matrix cracking modes in 2D CMCs have been developed based on the damage mechanism of fiber sliding relative to the matrix in the interface debonded region. The relationships between the fatigue hysteresis loops, fatigue hysteresis dissipated energy, fatigue peak stress, matrix multiple cracking modes, and interface shear stress have been established. The effects of fiber volume fraction, fatigue peak stress and matrix cracking mode proportion on fatigue hysteresis dissipated energy and interface debonding and sliding have been analyzed. The experimental fatigue hysteresis dissipated energy of 2D C/SiC and SiC/SiC composites at room temperature, 550 °C, 800 °C, and 1100 °C in air, and 1200 °C in vacuum corresponding to different fatigue peak stresses and cycle numbers have been analyzed. The interface shear stress degradation rate has been obtained through comparing the experimental fatigue hysteresis dissipated energy with theoretical values. Fatigue damage evolution in C/SiC and SiC/SiC composites has been compared using damage parameters of fatigue hysteresis dissipated energy and interface shear stress degradation rate. It was found that the interface shear stress degradation rate increases at elevated temperature in air compared with that at room temperature, decreases with increasing loading frequency at room temperature, and increases with increasing fatigue peak stress at room and elevated temperatures. Ceramic materials possess high strength and high modulus at elevated temperatures. However, their use as structural components is severely limited due to the brittleness. Continuous fiber-reinforced ceramic-matrix composites (CMCs), are fabricated by incorporating fibers into ceramic matrices, and provide an alternative to conventional ceramics in high temperature applications. CMCs retain the strength, low weight, and high temperature capability while exhibiting less brittleness than ceramics. The CMCs exceed the capability of current nickel super alloys used in high-pressure turbines and provide increased efficiency .The CMCs are subject to fatigue upon cyclic mechanical loads at fixed temperature with both room temperature and elevated temperature examined . BesidesThe objective of this paper is to compare the damage evolution in 2D C/SiC and SiC/SiC composites under tension–tension cyclic fatigue loading at room and elevated temperatures. The fatigue hysteresis loops models considering multiple matrix cracking modes in 2D CMCs have been developed based on the damage mechanism of fiber sliding relative to matrix in the interface debonded region. The relationships between fatigue hysteresis loops, fatigue hysteresis dissipated energy, fatigue peak stress, matrix multiple cracking modes, and interface shear stress have been established. The effects of fiber volume fraction, fatigue peak stress and matrix cracking mode proportion on fatigue hysteresis dissipated energy and interface debonding and slipping have been analyzed. The experimental fatigue hysteresis dissipated energy of 2D C/SiC and SiC/SiC composites at room temperature, 550 °C, 800 °C, and 1100 °C in air, and 1200 °C in vacuum corresponding to different fatigue peak stresses and cycle numbers have been analyzed. The interface shear stress degradation rate has been obtained through comparing the experimental fatigue hysteresis dissipated energy with theoretical computational values. The damage evolution in 2D C/SiC and SiC/SiC composites have been compared using damage parameters of fatigue hysteresis dissipated energy and interface shear stress degradation rate.σmax, which is greater than the initial cracking stress of transverse and longitudinal ply or yarn, it is assumed that transverse cracks and matrix cracks would extend throughout the entire laminate cross-section. The multicracking modes in the cross-ply or 2D woven CMCs can be classified into five different modes, as shown in Cracking mode 1: Transverse cracking in the transverse tow, with debonding at tow boundary.Cracking mode 2: Transverse cracking and matrix cracking with perfect fiber/matrix bonding, and fracture of fibers occurs in the longitudinal tow.Cracking mode 3: Transverse cracking and matrix cracking with fiber/matrix debonding and sliding in the longitudinal tow.Cracking mode 4: Matrix cracking with perfect fiber/matrix bonding, and fracture of fibers occurs in the longitudinal tow.Cracking mode 5: Matrix cracking and fiber/matrix interface debonding and sliding in the longitudinal tow.Upon first loading to fatigue peak stress rf, and the matrix radius is R (R = rf/Vf1/2). The length of the unit cell is L/2, which is half matrix crack spacing, and the interface debonded length is Ld. Upon unloading, counter slip occurs in the interface debonded region. The interface debonded region can be divided into two regions, i.e., interface counter-slip region and interface slip region, as shown in y. Upon reloading, new slip occurs in the interface debonded region. The interface debonded region can be divided into three regions, i.e., interface new-slip region, interface counter-slip region, and interface slip region, as shown in z. Based on the damage mechanism of fiber sliding relative to matrix upon unloading and subsequent reloading, the hysteresis loops can be divided into four different cases, including:Case 1: Interface partially debonds, and fiber completely slides relative to matrix.Case 2: Interface partially debonds, and fiber partially slides relative to matrix.Case 3: Interface completely debonds, and fiber partially slides relative to matrix.Case 4: Interface completely debonds, and fiber completely slides relative to matrix.The hysteresis loops develop as a result of energy dissipation through frictional sliding between fibers and the matrix upon unloading and subsequent reloading. In the matrix cracking modes mentioned above, the interface debonding and sliding occur in the matrix cracking mode 3 and mode 5. The shape, location and area of hysteresis loops of 2D woven CMCs depend on the interface debonding and sliding in cracking in cracking mode 3 and mode 5. The schematic figure for fiber sliding relative to matrix upon unloading and reloading is illustrated in ɛcu and reloading strain ɛcr corresponding to the interface slip Case 1 and Case 2 are determined by Equations (1) and (2), respectively [Vf_axial denotes the fiber volume fraction along the loading direction; Ef denotes the fiber elastic modulus; τi denotes the interface shear stress; L denotes the matrix cracking space; Ld denotes the interface debonded length; y and z denotes the interface counter-slip and new-slip length, respectively; rf denotes the fiber radius; αf, and αc denote the fiber and composite thermal expansion coefficient, respectively; and ΔT denotes the temperature difference between the fabricated temperature T0 and testing temperature T1 (ΔT = T1 − T0).The unloading strain ectively .(1)εcu=ɛcu and reloading strain ɛcr corresponding to interface slip Case 3 and Case 4 are determined by Equations (3) and (4), respectively [The unloading strain ectively .(3)εcu=εcu and reloading strain εcr corresponding to interface slip Case 1 and Case 2 are determined by Equations (5) and (6), respectively [k denotes the proportion of transverse yarns in the entire composite; and σto denotes the axial stress in the transverse yarns.The unloading strain ectively .(5)εcu=εcu and reloading strain εcr corresponding to interface slip Case 3 and Case 4 are determined by Equations (7) and (8), respectively [The unloading strain ectively .(7)εcu=εcu and εcr denote the unloading and reloading strain, respectively. Substituting the unloading and reloading strains of Equations (1)–(4) into (9), the fatigue hysteresis loss energy U3 of matrix cracking mode 3 can be obtained for different interface slips cases; substituting the unloading and reloading strains of Equations (5)–(8) into (9), the fatigue hysteresis loss energy U5 of matrix cracking mode 5 can also be obtained for different interface slip cases. The composite fatigue hysteresis loss energy Uc is determined by Equation (10) [η denotes the composite damage parameter, i.e., the proportion of matrix cracking mode 3 in the entire matrix cracking modes.The fatigue hysteresis dissipated energy corresponding to different cycle number is determined by Equation (9) .(9)U=∫σion (10) .(10)Uc=ψ of interface shear stress can be determined by the Equation (11).Ninitial and Nfinal denote the initial and final cycle number for estimating interface shear stress, respectively; and τi and τi denote the estimated interface shear stress at the initial and final cycle number, respectively.By comparing experimental fatigue hysteresis dissipated energy with theoretical computational values, the interface shear stress of CMCs can be obtained . The degUnder cyclic fatigue loading, the material properties, i.e., fiber volume content, peak stress, and damage state, i.e., matrix cracking mode proportion, affect the shape, location and area of hysteresis loops. The effect of these factors on interface slip and fatigue hysteresis dissipated energy evolution of matrix cracking mode 3 and mode 5, and composite would be analyzed.The effect of fiber volume content on fatigue hysteresis dissipated energy evolution and interface slip of matrix cracking mode 3, mode 5 and the composite is illustrated in Ld/L versus interface shear stress curves of matrix cracking mode 3 and mode 5 are illustrated in 3 at the interface shear stress of 50 MPa to the peak value of 63.3 kJ/m3 at the interface shear stress of 7.6 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.32 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 16.4 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in 3 at the interface shear stress of 50 MPa to the peak value of 31.9 kJ/m3 at the interface shear stress of 3.8 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.10 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 6.5 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in When the fiber volume fraction is 40%, the fatigue hysteresis dissipated energy and interface debonded length 2Ld/L versus interface shear stress curves of matrix cracking mode 3 and mode 5 are illustrated in 3 at the interface shear stress of 50 MPa to the peak value of 51.4 kJ/m3 at the interface shear stress of 6.1 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.25 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 12.8 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in 3 at the interface shear stress of 50 MPa to the peak value of 26 kJ/m3 at the interface shear stress of 3.1 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.07 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 4.8 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in When the fiber volume fraction is 45%, the fatigue hysteresis dissipated energy and interface debonded length 2Ld/L versus interface shear stress curves of matrix cracking mode 3 and mode 5 are illustrated in 3 at the interface shear stress of 50 MPa to the peak value of 41.8 kJ/m3 at the interface shear stress of 5 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.19 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 9.9 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in 3 at the interface shear stress of 50 MPa to the peak value of 21.2 kJ/m3 at the interface shear stress of 2.5 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.05 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 3.4 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in When the fiber volume fraction is 50%, the fatigue hysteresis dissipated energy and interface debonded length 2η is 0.2, the composite fatigue hysteresis dissipated energy versus interface shear stress curves when the fiber volume fraction is 40%, 45% and 50% are illustrated in 3 at the interface shear stress of 50 MPa to the peak value of 35.4 kJ/m3 at the interface shear stress of 4.4 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa; when the fiber volume fraction is 45%, the composite hysteresis dissipated energy increases with decreasing interface shear stress from 3.6 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 28.8 kJ/m3 at the interface shear stress of 3.6 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa; and when the fiber volume fraction is 50%, the composite hysteresis dissipated energy increases with decreasing interface shear stress from 2.5 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 23.6 kJ/m3 at the interface shear stress of 2.9 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa.When matrix cracking mode 3 proportion When fiber volume content increases, the hysteresis dissipated energy of matrix cracking mode 3, mode 5 and the composite at the same interface shear stress decrease, due to less interface debonding and frictional slip between fibers and the matrix.The effect of peak stress on fatigue hysteresis dissipated energy evolution and interface slip of matrix cracking mode 3, mode 5 and composite is illustrated in Ld/L versus interface shear stress curves of matrix cracking mode 3 and mode 5 are illustrated in 3 at the interface shear stress of 50 MPa to the peak value of 44 kJ/m3 at the interface shear stress of 7 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.34 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 17.2 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in 3 at the interface shear stress of 50 MPa to the peak value of 22.2 kJ/m3 at the interface shear stress of 3.5 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.1 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 6.6 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in When the fatigue peak stress is 150 MPa, the fatigue hysteresis dissipated energy and interface debonded length 2σmax = 200 MPa, the fatigue hysteresis dissipated energy and interface debonded length 2Ld/L versus interface shear stress curves of matrix cracking mode 3 and mode 5 are illustrated in 3 at the interface shear stress of 50 MPa to the peak value of 76.7 kJ/m3 at the interface shear stress of 9.2 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.47 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 23.7 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in 3 at the interface shear stress of 50 MPa to the peak value of 39.5 kJ/m3 at the interface shear stress of 4.7 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.17 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 10 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in When σmax = 250 MPa, the fatigue hysteresis dissipated energy and interface debonded length 2Ld/L versus interface shear stress curves of matrix cracking mode 3 and mode 5 are illustrated in 3 at the interface shear stress of 50 MPa to the peak value of 118.1 kJ/m3 at the interface shear stress of 11.3 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.6 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 30.1 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in 3 at the interface shear stress of 50 MPa to the peak value of 61.7 kJ/m3 at the interface shear stress of 5.9 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa, as shown in Ld/L increases with decreasing interface shear stress from 0.24 at the interface shear stress of 50 MPa to the peak value of 1.0 at the interface shear stress of 13.5 MPa, and remains to be constant of 1.0 until the interface shear stress of 0 MPa, as shown in When η = 0.2, the composite hysteresis dissipated energy versus interface shear stress curves when the fatigue peak stress is 150 and 200 MPa are illustrated in 3 at the interface shear stress of 50 MPa to the peak value of 24.6 kJ/m3 at the interface shear stress of 4.1 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa; when the fatigue peak stress is 200 MPa, the composite hysteresis dissipated energy increases with decreasing interface shear stress from 7.2 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 43.7 kJ/m3 at the interface shear stress of 5.4 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa; and when the fatigue peak stress is 250 MPa, the composite hysteresis dissipated energy increases with decreasing interface shear stress from 13.8 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 68.3 kJ/m3 at the interface shear stress of 6.8 MPa, and decreases to 0 kJ/m3 at the interface shear stress of 0 MPa.When the proportion of matrix cracking mode 3 is When the fatigue peak stress increases, the hysteresis dissipated energy of matrix cracking mode 3, mode 5 and composite at the same interface shear stress increase, due to more interface debonding and frictional slip between fibers and the matrix.η on fatigue hysteresis dissipated energy versus interface shear stress curves is illustrated in η is 0, there is only matrix cracking mode 5 in the composite, the composite hysteresis dissipated energy increases from 2.3 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 32 kJ/m3 at the interface shear stress of 3.1 MPa, as shown in η is 1, there is only matrix cracking mode 3 in the composite, the composite hysteresis dissipated energy increases from 9.8 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 62.5 kJ/m3 at the interface shear stress of 6 MPa, as shown in η < 1, there are both matrix cracking mode 3 and mode 5 in the composite, when η is 0.2, the composite hysteresis dissipated energy increases from 3.8 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 35.4 kJ/m3 at the interface shear stress of 3.5 MPa; when η is 0.5, the composite hysteresis dissipated energy increases from 6.1 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 42.5 kJ/m3 at the interface shear stress of 4.4 MPa; and when η is 0.8, the composite hysteresis dissipated energy increases from 8.3 kJ/m3 at the interface shear stress of 50 MPa to the peak value of 54 kJ/m3 at the interface shear stress of 5.7 MPa, as shown in η, the peak value of hysteresis dissipated energy increases, as shown in The effect of matrix cracking proportion η increases, the composite hysteresis dissipated energy and the corresponding interface shear stress increase, which indicates that the hysteresis dissipated energy can approach to the peak value at high interface shear stress.When matrix cracking mode 3 proportion 3, and an open porosity less than 5%. The fatigue tests were conducted in force control with a stress ratio  of 0.1, and a sinusoidal waveform loading frequency of 10 Hz. The material properties are given by: Vf = 40%, Ef = 230 GPa, Em = 350 GPa, rf = 3.5 μm, αf = −0.38 × 10−6/°C, αm = 2.8 × 10−6/°C, and ΔT = −1000 °C.Li  investigσmax = 105 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.8. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 1st cycle lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loop of the 1st cycle corresponds to interface slip Case 2, i.e., interface partially debonds and fiber slides partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear. Staehler et al. [Under fatigue peak stress of r et al.  observedr et al. . The wea3. The fatigue tests were performed under force control at a sinusoidal loading frequency of 10 Hz, with a stress ratio  of 0.1. The material properties are given by: Vf = 45%, Ef = 230 GPa, Em = 350 GPa, rf = 3.5 μm, αf = 0, αm = 4.6 × 10−6/K, and ΔT = −1000 K.Shuler et al.  investigσmax = 335 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.2. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 1st cycle lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loop of the 1st cycle corresponds to interface slip Case 2, i.e., interface partially debonds, and fiber slides partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear. By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in Under fatigue peak stress of 3. After machining, each test specimen received a SiC seal coat via CVD. The fatigue loading was in a sinusoidal waveform with a loading frequency of 40 Hz. The fatigue stress ratio was 0.05. The material properties of 2D woven C/SiC composite are given by: Vf = 45%, Ef = 225 GPa, Em = 430 GPa, rf = 3.5 μm, αf = 1.0 × 10−6/°C, αm = 4.8 × 10−6/°C, and ΔT = −1000 °C.Staehler et al.  investigσmax = 375 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.2. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 2nd cycle lies in the left part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loop of the 2nd cycle corresponds to interface slip Case 4, i.e., the interface completely debonds, and fiber slips completely relative to matrix in the interface debonded region. By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in Under fatigue peak stress of Vf = 50%, Ef = 230 GPa, Em = 350 GPa, rf = 3.5 μm, αf = −0.38 × 10−6/°C, αm = 2.8 × 10−6/°C, and ΔT = −1000 °C.Li  investigσmax = 57 MPa, the fatigue hysteresis loops corresponding different applied cycles are illustrated in η = 0.2. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 11,104th and 33,262nd applied cycles lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loop of the 1st cycle corresponds to interface slip Case 2, i.e., the interface partially debonds, and fiber slips partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear. By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in Under fatigue peak stress of Vf = 40%, Ef = 230 GPa, Em = 350 GPa, rf = 3.5 μm, αf = −0.38 × 10−6/°C, αm = 2.8 × 10−6/°C, and ΔT = −200 °C.Li  investigσmax = 105 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.8. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 1st cycle lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loop of the 1st cycle corresponds to interface slip Case 2, i.e., the interface partially debonds, and fiber slips partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear and interface oxidation. For interface oxidation, the oxidation of fiber coating is a process such as,Under fatigue peak stress of By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in 3. The test specimens were cut from 216 mm × 216 mm composite panels using diamond grinding into a dog-bone configuration. After the machining and cutting, the test specimens were then seal coated with SiC via CVD process. The fatigue tests were conducted in the force control with a stress ratio  of 0.05, and a sinusoidal waveform loading frequency of 0.1 Hz. The fatigue stress ratio was 0.1. The material properties of 2D woven C/SiC composite are given by: Vf = 45%, Ef = 225 GPa, Em = 430 GPa, rf = 3.5 μm, αf = 1.0 × 10−6/°C, αm = 4.8 × 10−6/°C, and ΔT = −500 °C.Mall and Engesser  investigσmax = 350 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.2. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 100th cycle lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loops of the 100th, 200th, and 212th applied cycles correspond to interface slip Case 2, i.e., the interface partially debonds, and fiber slips partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear and interface oxidation. By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in Under fatigue peak stress of 3. The fatigue tests were performed under force control with a triangular loading cycle with a loading frequency of 10 Hz, and a stress ratio  of 0.1. The material properties of 2D woven C/SiC composite are given by: Vf = 40%, Ef = 225 GPa, Em = 350 GPa, rf = 3.5 μm, αf = 1.0 × 10−6/°C, αm = 4.8 × 10−6/°C, and ΔT = −100 °C.Rodrigues et al.  investigσmax = 300 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.2. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 1000th, 100,000th, and 1,000,000th applied cycle lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loop of the 1st cycle corresponds to interface slip Case 2, i.e., the interface partially debonds, and fiber slips partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear and interface oxidation. By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in Under fatigue peak stress of Vf = 40%, Ef = 270 GPa, Em = 300 GPa, rf = 7.0 μm, αf = 5.1 × 10−6/°C, αm = 4.7 × 10−6/°C, and ΔT = −1000 °C.Shi  investigσmax = 150 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.2. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 121st and 1,200,331st applied cycles lie in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loops correspond to interface slip Case 2, i.e., the interface partially debonds, and fiber slips partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear and interface oxidation. By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles applied cycles can be estimated, as shown in Under fatigue peak stress of σmax = 250 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.8. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 81st, 401st, and 641st applied cycles lie in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loops correspond to interface slip Case 2, i.e., the interface partially debonds, and fiber slips partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear and interface oxidation. By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in Under fatigue peak stress of Vf = 40%, Ef = 270 GPa, Em = 300 GPa, rf = 7.0 μm, αf = 5.1 × 10−6/°C, αm = 4.7 × 10−6/°C, and ΔT = −200 °C.Shi  investigσmax = 150 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.2. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 5th and 36,500th applied cycles lie in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loops correspond to interface slip Case 2, i.e., the interface partially debonds, and fiber slips partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear and interface oxidation. By comparing experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in Under fatigue peak stress of Vf = 40%, Ef = 230 GPa, Em = 350 GPa, rf = 7.0 μm, αf = 3.9 × 10−6/K, αm = 2 × 10−6/K, and ΔT = −100 K.Groner  investigσmax = 140 MPa, the fatigue hysteresis loops corresponding to different applied cycles are illustrated in η = 0.2. The experimental and theoretical fatigue hysteresis dissipated energy as a function of interface shear stress is shown in 3, and then decreases with decreasing interface shear stress to 0 kJ/m3. The experimental fatigue hysteresis dissipated energy of the 7249th, 15,381st, and 23,391st applied cycles all lie in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve. The fatigue hysteresis loops correspond to interface slip Case 2, i.e., the interface partially debonds, and fiber slips partially relative to matrix in the interface debonded region. With the number of applied cycles increasing, interface shear stress decreases with increasing cycle number due to interface wear and interface oxidation. By comparing the experimental fatigue hysteresis dissipated energy with theoretical values, the interface shear stress corresponding to different applied cycles can be estimated, as shown in Under fatigue peak stress of σmax = 105 MPa at room temperature and 800 °C in air, 2D woven C/SiC composite under σmax = 335 and 375 MPa at room temperature, the experimental fatigue hysteresis dissipated energy decreases with increasing cycle number, and lies in the right and left part of the fatigue hysteresis dissipated energy versus interface shear stress curve, as shown in σmax = 350 MPa at 550 °C in air, the experimental fatigue hysteresis dissipated energy increases with increasing cycle number, and lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve, as shown in σmax = 57 MPa at room temperature, and σmax = 300 MPa at 1200 °C in vacuum, the experimental fatigue hysteresis dissipated energy increases first, and then decreases with increasing cycle number, and lies in the right and left part of the fatigue hysteresis dissipated energy versus interface shear stress curve, as shown in The experimental fatigue hysteresis dissipated energy versus cycle number curves of cross-ply and 2D woven C/SiC composites at room temperature, 550 °C, and 800 °C in air, and 1200 °C in vacuum are illustrated in σmax = 350 MPa at 550 °C in air, i.e., 7.4 × 10−2 MPa/cycle, due to high fatigue peak stress and low loading frequency of 0.1 Hz, and is the lowest for 2D woven C/SiC composite under σmax = 375 MPa at room temperature, i.e., 1.7 × 10−6 MPa/cycle, due to high loading frequency of 40 Hz. For cross-ply C/SiC composite under the same fatigue peak stress of σmax = 105 MPa at room temperature and 800 °C in air, the interface shear stress degradation rate increases at elevated temperature in air, i.e., 7.7 × 10−4 MPa/cycle at elevated temperature and 6.3 × 10−6 MPa/cycle at room temperature, due to interface oxidation and interface wear. For 2D woven C/SiC composite at room temperature, the interface shear stress degradation rate increases with increasing fatigue peak stress, i.e., 2.1 × 10−5 MPa/cycle under σmax = 335 MPa, and 1.6 × 10−4 MPa/cycle under σmax = 375 MPa, and decreases with increasing loading frequency, i.e., 1.6 × 10−4 MPa/cycle at the loading frequency of 10 Hz, and 1.7 × 10−6 MPa/cycle at the loading frequency of 40 Hz under the same fatigue peak stress of σmax = 375 MPa.The interface shear stress degradation rate of cross-ply and 2D woven C/SiC composites at room and elevated temperatures are illustrated in σmax = 150 MPa at room temperature and 800 °C in air, and under σmax = 120, 140, 170, and 210 MPa at 1100 °C in air, the experimental fatigue hysteresis dissipated energy increases with increasing cycle number, and lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve, as shown in σmax = 250 MPa at room temperature, as shown in The experimental fatigue hysteresis dissipated energy versus cycle number curves of 2D SiC/SiC composite at room temperature, 800 °C, and 1100 °C in air are illustrated in σmax = 210 MPa at 1100 °C in air, i.e., 2.6 × 10−2 MPa/cycle, and the lowest for 2D woven SiC/SiC composite under σmax = 150 MPa at room temperature, i.e., 5.8 × 10−6 MPa/cycle. For 2D SiC/SiC composite under the same fatigue peak stress of σmax = 150 MPa at room temperature and 800 °C in air, the interface shear stress degradation rate increases at elevated temperature in air, i.e., 7.0 × 10−4 MPa/cycle at elevated temperature, and 5.8 × 10−6 MPa/cycle at room temperature, due to interface oxidation and interface wear. For 2D woven SiC/SiC composite at room temperature, the interface shear stress degradation rate increases with increasing fatigue peak stress, i.e., 5.8 × 10−6 MPa/cycle under σmax = 150 MPa, and 2.2 × 10−5 MPa/cycle under σmax = 250 MPa. For 2D woven SiC/SiC composite at 1100 °C in air, the interface shear stress degradation rate increases with increasing fatigue peak stress, i.e., 4.8 × 10−4 MPa/cycle under σmax = 120 MPa, and 2.6 × 10−2 MPa/cycle under σmax = 210 MPa.The interface shear stress degradation rate of 2D woven SiC/SiC composite at room and elevated temperatures are illustrated in σmax = 150 MPa is lower, i.e., 5.8 × 10−6 MPa/cycle at room temperature, and 7.0 × 10−4 MPa/cycle at 800 °C in air, than that of cross-ply C/SiC composite under σmax = 105 MPa, i.e., 6.3 × 10−6 MPa/cycle at room temperature, and 7.7 × 10−4 MPa/cycle at 800 °C in air.The experimental and theoretical fatigue hysteresis dissipated energy versus interface shear stress curves of cross-ply C/SiC and 2D woven SiC/SiC composites at 800 °C in air are illustrated in The interface shear stress degradation rate increases at elevated temperature in air compared with that at room temperature due to interface and fiber oxidation, decreases with increasing loading frequency at room temperature due to the increasing interface wear rate between fibers and the matrix, and increases with increasing fatigue peak stress at room and elevated temperatures due to the increasing interface debonding and slipping extent between fibers and the matrix.σmax = 105 MPa lies in the right and left part of the fatigue hysteresis dissipated energy versus interface shear stress curve, corresponding to interface slip Case 2 and Case 4, which indicates that the interface debonding changes from partially debonding to completely debonding with increasing cycle number; however, for 2D woven SiC/SiC composite under σmax = 150 MPa, the experimental fatigue hysteresis dissipated energy just lies in the right part of the fatigue hysteresis dissipated energy versus interface shear stress curve, corresponding to interface slip Case 2, which indicates that the interface debonding remains to be partially debonding with increasing cycle number.At 800 °C in air, the experimental fatigue hysteresis dissipated energy of cross-ply C/SiC composite under σmax = 150 MPa is lower than that of cross-ply C/SiC composite under σmax = 105 MPa.At room temperature and 800 °C in air, the interface shear stress degradation rate of 2D woven SiC/SiC composite under The comparisons of damage evolution between 2D C/SiC and SiC/SiC composites under tension–tension cyclic fatigue loading at room and elevated temperatures have been investigated. The fatigue hysteresis loops models considering multiple matrix cracking modes in 2D CMCs have been developed based on the damage mechanism of fiber slipping relative to matrix in the interface debonded region. The relationships between fatigue hysteresis loops, fatigue hysteresis dissipated energy, fatigue peak stress, matrix multiple cracking modes, and interface shear stress have been established. The effects of fiber volume fraction, fatigue peak stress and matrix cracking mode proportion on fatigue hysteresis dissipated energy and interface debonding and slipping have been analyzed. The experimental fatigue hysteresis dissipated energy of 2D C/SiC and SiC/SiC composites at room temperature, 550 °C, 800 °C, and 1100 °C in air, 1200 °C in vacuum corresponding to different fatigue peak stresses and cycle numbers have been analyzed. The interface shear stress degradation rate has been obtained through comparing experimental fatigue hysteresis dissipated energy with theoretical values. The damage evolution in C/SiC and SiC/SiC composites has been compared using damage parameters of fatigue hysteresis dissipated energy and interface shear stress degradation rate.In the present analysis, the effect of mechanical load waveform, i.e., sinusoidal waveform and triangular waveform, on fatigue damage in C/SiC and SiC/SiC composites has not been considered , which w


Many fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). The application of LPMOs is expected to contribute to ecologically friendly conversion of biomass into fuels and chemicals. Moreover, applications of LPMO-modified cellulose-based products may be envisaged within the food or material industry.MtPPO7 from Myceliophthora thermophila C1 and the subsequent oxidation of cellulose by MtLPMO9B. Interestingly, MtPPO7 shows preference towards lignin-derived methoxylated monophenols. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota reveals a high correlation between MtPPO7 and AA9 LPMO genes.Here, we show an up to 75-fold improvement in LPMO-driven cellulose degradation using polyphenol oxidase-activated lignin building blocks. This concerted enzymatic process involves the initial conversion of monophenols into diphenols by the polyphenol oxidase MtPPO7 from well-known PPOs, such as tyrosinases, and ensures that MtPPO7 is an excellent redox partner of LPMOs. The correlation between MtPPO7 and AA9 LPMO genes is indicative for the importance of the coupled action of different monooxygenases in the concerted degradation of lignocellulosic biomass. These results will contribute to a better understanding in both lignin deconstruction and enzymatic lignocellulose oxidation and potentially improve the exploration of eco-friendly routes for biomass utilization in a circular economy.The activity towards methoxylated phenolic compounds distinguishes The online version of this article (doi:10.1186/s13068-017-0810-4) contains supplementary material, which is available to authorized users. Fungal carbohydrate converting enzymes are considered important for eco-friendly application in plant biomass degradation. The resulting carbohydrates are sources for the production of biochemicals or biofuels and new enzymatically modified cellulose-based products are envisaged for the future.para-coumaryl alcohol. Cellulose consists of β-(1→4)-linked glucosyl chains that interact with each other via hydrogen bonds and van der Waals forces, which results in the formation of crystalline cellulose fibrils. Unlike cellulose, hemicellulose is a heteropolymer varying in its monosaccharide composition and linkages between the monosaccharides. Examples are xylan, mannan, or β--linked β-glucan. Hemicellulose interacts with lignin through ester and ether linkages, thereby forming a network that embeds the cellulose microfibrils , ten AA9 LPMO sequences ; G2QCJ3, Myceliophthora thermophila (strain ATCC 42464); F7W1P4, Sordaria macrospora (strain ATCC MYA-333); G2RB73, Thielavia terrestris (strain ATCC 38088); Q2H8N9, Chaetomium globosum (strain ATCC 6205); G0S408, Chaetomium thermophilum (strain DSM 1495); F8MLY8, Neurospora tetrasperma (strain FGSC 2508); T0L448, Colletotrichum gloeosporioides (strain Cg-14); A0A0H4K9X4 and A0A1C9CXI0, Myceliophthora thermophila C1); and four AbPPOs  as k et al. . Previouk et al.  were usek et al. .Additional file 1: Figure S1. Activity of MtLPMO9B towards amorphous cellulose in the presence and absence of MtPPO7 or AbPPO. HPAEC elution pattern of regenerated amorphous cellulose  incubated with MtLPMO9B  only, or with either AbPPO  or MtPPO7  in the presence of (a) para-coumaric acid (no. 3 specified in Table b) 3-hydroxy-4-methoxycinnamic acid (no. 5 specified in Table MtLPMO9B results in the formation of non-oxidized gluco-oligosaccharides (GlcOSn) and C1-oxidized gluco-oligosaccharides (GlcOSn#). See “Additional file 2: Figure S2. Release of oligosaccharides from RAC incubated with MtLPMO9B in the presence and absence of MtPPO7 throughout 24 h. Samples were incubated in the presence of ferulic acid (no. 8 specified in Table −1) with MtLPMO9B only  and MtLPMO9B together with MtPPO7  based on HPAEC. All incubations were performed in duplicate, and the standard deviations are presented as error bars. See “Additional file 3: Figure S3. Concentration of phenolic compounds incubated in the presence and absence of MtPPO7. (a) guaiacol (no. 9 specified in Table c) 3-methylcatechol  or without (red bar). Samples were incubated in a 50 mM potassium phosphate buffer (pH = 6.0) containing 2.5 µM copper(II)-chloride for 24 h at 50 °C. The conversion of guaiacol and 3-methylcatechol by MtPPO7 was calculated by subtracting the determined concentration of the incubation of guaiacol or 3-methylcatechol in the presence of MtPPO7 from the concentration that was determined by the incubation of guaiacol and 3-methylcatechol alone. This conversion was expressed as the relative decrease of the guaiacol and 3-methylcatechol concentration and is shown in (b) and (d), respectively. See “Additional file 4: Table S1. Compounds detected after incubation of 21 reducing agents without or with MtPPO7 by UHPLC/UV-MS1.Additional file 5: Figure S4. UHPLC-UV-MSn elution profile of guaiacol incubated (a) in the presence and (b) absence of MtPPO7. Guaiacol (no. 9 specified in Table −1) or without MtPPO7. Samples were incubated in a 50 mM potassium phosphate buffer (pH = 6.0) containing 2.5 µM copper(II)-chloride for 24 h at 50 °C. Annotation of the peaks based on UV was done by using mass spectrometry . These ortho-quinones are expected to polymerize and form insoluble complexes. Guaiacol itself forms insoluble complexes via auto-oxidation, which results from the presence of copper during the incubation for 24 h at 50 °C. The decrease in guaiacol concentration during the incubation of guaiacol without MtPPO7 is also shown in Additional file n after incubation of guaiacol (2 mM) with MtPPO7 (5.0 μg mL−1). Samples were incubated for 24 h at 50 °C in 50 mM potassium phosphate, pH 6.0, containing 2.5 µM copper(II)-chloride.Additional file 7: Table S2. Selected cellulase-rich Ascomycota from the JGI database1.Additional file 8: Table S3. Selected cellulase-rich Basidiomycota from the JGI database1.Additional file 9: Figure S6. Correlation of AA9 LPMOs, AbPPOs and MtPPO7s encoding genes in Ascomycota and Basidiomycota. Correlation between the three gene families encoding AA9 LPMOs, AbPPOs and MtPPO7s of (a) 336 Ascomycota and (b) 208 Basidiomycota. Correlation between genes encoding AA9 LPMOs, AbPPOs and MtPPO7s of (c) 27 Ascomycota and (d) 23 Basidiomycota, which have at least ten annotated genes encoding cellulose-degrading enzymes. Species of selected fungal classes are listed in Additional file


Background. The aim of this study was to assess the effects of low concentrations of H2O2 on angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro and explore the underlying mechanisms. Methods. HUVECs were cultured and stimulated with different concentrations of H2O2. Flow cytometric analysis was used to select an optimal concentration of H2O2 for the following experiments. Cell proliferation, migration, and tubule formation were evaluated by Cell Counting Kit-8 (CCK-8) assays, scratch wound assays, and Matrigel tubule formation assays, respectively. For gain and loss of function studies, constitutively active MEK5 (CA-MEK5) and ERK5 shRNA lentiviruses were used to activate or knock down extracellular signal-regulated kinase 5 (ERK5). Results. We found that low concentrations of H2O2 promoted HUVECs proliferation, migration, and tubule formation. ERK5 in HUVECs was significantly activated by H2O2. Enhanced ERK5 activity significantly amplified the proangiogenic effects of H2O2; in contrast, ERK5 knock-down abrogated the effects of H2O2. Conclusions. Our results confirmed that low concentrations of H2O2 promoted HUVECs angiogenesis in vitro, and ERK5 is an essential mediator of this process. Therefore, ERK5 may be a potential therapeutic target for promoting angiogenesis and improving graft survival.  Tissue transplantation is commonly used in plastic and reconstructive surgery. Timely and effective revascularization is important in ensuring graft survival, and angiogenesis plays an essential role in this process. Angiogenesis, the formation of new blood vessels from existing vasculature, involves many cellular components and signaling pathways, including reactive oxygen species (ROS), which play an important role in this process .2O2), superoxide radicals (O2−), and hydroxyl radicals (OH−), are important signaling molecules that regulate multiple biological responses, including angiogenesis [2O2 in particular, have biphasic effects on angiogenesis; ROS at physiological levels mediate endothelial cell proliferation, migration, tubular formation, and enhanced angiogenesis, but ROS at pathological levels result in detrimental effects, including cell apoptosis, death, and impaired angiogenesis [ROS, including hydrogen peroxide HO2, superogenesis –5. Low c2O2, suggesting that, compared with other MAPK family members, ERK5 is a redox-sensitive kinase [Among various ROS downstream signaling molecules, mitogen-activated protein kinases (MAPKs) have a crucial role . The MAPe kinase . Targetee kinase .2O2 and ERK5 play important roles in angiogenesis and ERK5 is redox-sensitive, we hypothesized that low-concentration H2O2 promotes endothelial cell angiogenesis through the ERK5 pathway. To verify this hypothesis, we investigated the role of ERK5 in H2O2-induced human umbilical vein endothelial cell (HUVECs) proliferation, migration, and tubule formation.Since both H2O2 was critical for its proangiogenic function; downregulation of p-ERK5 by shERK5 inhibited H2O2 proangiogenic activity in vitro, whereas upregulation of p-ERK5 by CA-MEK5 facilitated H2O2 proangiogenic action. The concentration of H2O2 (50 μmol/L) used in our study was in the range of that reached under pathophysiological conditions in vivo (from 0.2 nmol/L in red blood cells to 200 μmol/L in wound fluid) [In this study, we found that ERK5 activation by Hd fluid) , 12; the2. Cells were used for experiments at 80–90% confluency after 2-3 days of culture. In some cases, 5 U/ml catalase  was added 5 min before H2O2 was administered.HUVECs were acquired from China Infrastructure of Cell Line Resources . Cells were grown in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  and 1% penicillin-streptomycin . The cell cultures were incubated at 37°C in a humidified atmosphere with 5% COFor gain of function studies, a constitutively active MEK5 (CA-MEK5) lentivirus was used to activate ERK5. A nonspecific green fluorescent protein (GFP) lentivirus with the same multiplicity of infection  was used as a negative control. For loss of function studies, ERK5 shRNA (shERK5) lentivirus was used to reduce ERK5 expression in endothelial cells. A nonsilencing shRNA lentivirus with the same MOI was used as a negative control (control siRNA). Cells were seeded in 6-well plates, cultured overnight, and then incubated with lentivirus for 48 h for infection. All the plasmids and lentiviruses used were constructed by the Shanghai Obio Technology Company .2O2, cells were labeled with phycoerythrin- (PE-) conjugated annexin V and 7-amino-actinomycin (7-AAD)  following the kit instructions. Approximately 5,000 cells were counted from each sample using flow cytometry , and early apoptotic cells were defined as annexin V positive/7-AAD negative.After a 30 min incubation with different concentrations of Hμg of protein per sample was used for polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked with fat-free milk or 5% BSA for 1 h at room temperature and then incubated with various antibodies: ERK5 , phosphorylated-ERK5 , MEF2C , phosphorylated-MEF2C , ERK1/2 , p-ERK1/2 , and β-actin  overnight at 4°C. After the membranes were washed with TBST, they were incubated with HRP-linked secondary antibodies: goat anti-rabbit IgG  or goat anti-mouse IgG  for 1 h at room temperature. The density of the protein bands was determined by ImageJ software. Densitometric values of the ERK5, MEF2C, and ERK1/2 bands were normalized to that of β-actin. Phosphorylated protein levels were normalized to the respective total protein levels.Cells were lysed in RIPA buffer  containing a protease inhibitor cocktail . The protein concentration was measured using a BCA kit, and 30 3 cells per well. After the cells were cultured in a standard environment for 24 h, they were analyzed using CCK-8 assays. Following treatment with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min, the culture media were changed to normal medium. Then, 10 μl CCK-8 was added, and cells were further cultured for 2.5 h. The optical density (OD) at 450 nm was measured using a microplate reader .Cell proliferation was assessed using a Cell Counting Kit-8  assay. Following the manufacturer's instructions, HUVECs were seeded in 96-well plates at a density of 1 × 106 cells per well (confluence) and cultured overnight. Following treatment with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min, a scratch was made by a sterile 200 μl pipette tip in the center of each well and washed twice with PBS. The wounded HUVECs monolayers were then incubated with medium containing 2% FBS for 12 h or 24 h. Photographs were taken at 0 h and 12 h or 24 h at fixed locations along the scratch with an Olympus microscope, and the closure of the wound area was quantified using ImageJ software.Cell migration was assessed using scratch wound assays. HUVECs were seeded in 6-well plates at a density of 1 × 10μl per well) was coated on 6-well plates and incubated at 37°C for 30 min to form a solid gel. Cells were pretreated with PBS, H2O2 (50 μmol/L), or catalase (5 U/ml) and H2O2 (50 μmol/L) for 30 min and then trypsinized, collected, and seeded onto Matrigel-coated wells (1 × 104 cells per well) in complete medium (DMEM containing 10% FBS and 1% penicillin-streptomycin) and cultured for 4 to 8 h. Tubule formation was observed with an Olympus microscope and analyzed by measuring branch length and counting tubule number with ImageJ software.In vitro tubule formation of HUVECs was assayed on Matrigel . Briefly, Matrigel  protein was determined by a human VEGF ELISA kit  according to the manufacturer's instructions.Cells were seeded in 60 mm tissue culture dishes at a density of 1 × 10p value was less than 0.05 (p < 0.05). Each experiment included triplicate measurements for each condition tested, and the experiment was repeated at least three times.All data are presented as the mean ± standard deviation (SD). Comparison among multiple groups was performed by one-way analysis of variance (ANOVA) followed by the SNK post hoc test. The results were considered statistically significant when the 2O2, we assessed the effects of five different concentrations. The results showed that 0–50 μmol/L H2O2 did not induce significant apoptosis of HUVECs. However, 100 μmol/L H2O2 increased the apoptotic rate of the cells, and 200 μmol/L H2O2 induced significant cell apoptosis (2O2 (versus control group) than with 25 umol/L H2O2, we used 50 μmol/L H2O2 in the following experiments.To determine the optimal concentration of Hpoptosis . Based oμmol/L H2O2 significantly increased HUVECs proliferation, and this effect was completely abrogated by catalase, an enzyme that eliminates H2O2. Additionally, cell migration is an important step in angiogenesis. Scratch wound assays were performed; the area recovered represents endothelial cell migration  did not differ from the control cells [(70 ± 1.9)%, 2O2 on endothelial cell angiogenesis, we performed a Matrigel tubule formation assay . As observed previously, cells treated with 50 μmol/L H2O2 and 5 U/ml catalase did not differ from those of the control. Overall, low-concentration H2O2 promoted HUVECs angiogenesis; however, catalase eliminated all H2O2 proangiogenic effects.Stimulation of endothelial cell proliferation is essential for angiogenesis. As shown in igration . Controlon assay . After t2O2 in HUVECs. Endogenous ERK5 activity was measured by assessing phosphorylation of ERK5 and its substrate, MEF2C. H2O2 treatment of the cells resulted in activation of ERK5 without changes in total ERK5. Pretreatment of the cells with 5 U/ml catalase inhibited ERK5 activation by H2O2. ERK1/2 and p-ERK1/2 protein levels did not change significantly among the experimental groups  promoted HUVEC proliferation, migration, and tubule formation  levels rationally may be an angiogenic strategy.Low concentrations of H2O2-induced angiogenesis would help develop novel treatment strategies for promoting revascularization of grafts and improving their survival. The finding that ERK5 activity regulates low-concentration H2O2-induced angiogenesis suggests that ERK5 is a potential target for therapies that modulate graft survival. Many drugs have been developed to promote angiogenesis and improve graft survival after tissue transplantation. However, most of these drugs target growth factors , endothelial-specific receptor tyrosine kinases, and other extracellular molecules [2O2 in endothelial cells.Furthermore, elucidating the in-depth mechanisms underlying Holecules . Drugs tolecules . Therefoolecules . Additio2O2-induced angiogenesis in HUVECs. We showed that low concentrations of H2O2 activated ERK5 in the HUVECs. Enhanced activity of ERK5 amplified the proangiogenic effects of H2O2. In contrast, reduced activity of ERK5 abolished the effects of H2O2. However, further experiments are needed to clarify the mechanisms involved. Nevertheless, we believe that the present study provides helpful insights into the role of ERK5 in angiogenesis and thus provides a theoretical basis for developing new treatment strategies to promote revascularization of grafts and improve their survival.Here, we demonstrated a novel mechanism of H