It is rapidly absorbed and extensively metabolized. AR-C124910XX, the major active metabolite, antagonizes the P2Y12 receptor at approximately equal potency. The metabolism of ticagrelor to AR-C124910XX involves CYP3A4 and CYP3A5. CYP3A polymorphisms have been well documented, and ∗1GCYP3A4  and ∗3CYP3A5  are the most important single nucleotide polymorphisms in Chinese. Genetic differences in CYP3A4 and CYP3A5 expression in human volunteers and patients might affect the clearance of ticagrelor or AR-C124910XX in vivo resulting in subsequent variable patient response. Thus, this study is designed to explore the effects of ∗1GCYP3A4 and ∗3CYP3A5 polymorphisms on the pharmacokinetics and pharmcodynamics of ticagrelor in healthy Chinese subjects. The results indicated that the ∗1GCYP3A4 polymorphism significantly influenced the pharmacokinetics of AR-C124910XX, and it may be more important than ∗3CYP3A5 with respect to influencing ticagrelor pharmacokinetics by increasing CYP3A4 activity. However, the significant effect of ∗1GCYP3A4 polymorphism on AR-C124910XX plasma levels did not translate into detectable effect on inhibition of platelet aggregation. Therefore, it seems not necessary to adjust the dosage of ticagrelor according to the CYP3A4 or 3A5 genotype.Ticagrelor is the first reversible, direct-acting, potent P2Y The frequencies of ∗1GCYP3A4 and ∗3CYP3A5 alleles were 22.7–38.99%  or ∗3CYP3A5  variant or both were recruited from 136 male healthy Chinese volunteers. Each subject was physically healthy with no prior history of significant medical illness according to a medical history, physical examinations, vital signs, routine clinical laboratory tests  and ECGs. Subjects were required to have the following laboratory values within normal ranges: body mass index (BMI) of 19–24 kg⋅m-2, baseline maximal platelet aggregation (MPA) response to 5 μM ADP of ≥70%. Volunteers who had donated blood within the past 2 months, who had a history of conditions affecting drug disposition ; who had a personal or family history of bleeding disorders/events; who smoked ≥5 cigarettes per week ; who consumed grapefruit products 1 week before the study, or who had taken any prescription medication within 2 weeks of the start of the study were excluded.Fourteen healthy male Chinese volunteers who had the All subjects provided written informed consent before participation and the Ethics Committee of the First Affiliated Hospital of Soochow University approved the study protocol. Studies were conducted in accordance with the International Conference on Harmonisation Guideline for Good Clinical Practice and the Declaration of Helsinki.∗1GCYP3A4 or ∗3CYP3A5 alleles by direct sequencing. Briefly, the genomic DNA was amplified by use of two primers for each genotype. The sequences of PCR primers are shown in Table 1. PCR was performed by using Taq DNA polymerase (1.25 U/50 μL), 10× Taq Buffer, 25 mM MgCl2, dNTP and genomic DNA (50–100 ng/50 μL) with a pair of primers (0.3 μM) on T100TM Thermal Cycler. The PCR conditions for ∗1GCYP3A4 were 95°C for 5 min, followed by 35 cycles of denaturizing at 95°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR conditions for ∗3CYP3A5 is almost the same as ∗1GCYP3A4 except for the annealing temperature that was 56°C. Then, the genotyping was carried out by direct sequencing on an ABI 3730 DNA Analyzer.Genomic DNA from EDTA-treated blood samples was extracted using a Wizard Genomic DNA Purification kit  according to the manufacturer’s instructions. Genomic DNA extracted from subjects’ blood was genotyped for AM on the subsequent morning, each subject received one dose  with 200 mL of water after an overnight fast. Blood samples for pharmacokinetics (4 mL each) were collected via an indwelling catheter or direct venipuncture into tubes containing sodium heparin immediately before and 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 16, 24, 36, and 48 h after drug administration. Blood samples were processed by centrifugation , and plasma was harvested and stored at -80°C until analysis. Pharmacodynamics samples were collected via direct venipuncture into tubes containing 0.109 mmol⋅L-1 sodium citrate before and 1, 2, 4, 12, 24, and 48 h after drug administration.All subjects were admitted to the clinical trial center the night before ticagrelor dosing. At 7:00 -1. The linear calibration ranges were 0.5–2,000 ng⋅mL-1 for ticagrelor and AR-C124910XX (r2 ≥ 0.99) and the lower limits of quantification for ticagrelor and AR-C124910XX were both 0.5 ng⋅mL-1. Intraday precision values [relative standard deviations (RSD)] ranged from 6.0 to 13.2% for ticagrelor and 4.2–11.5% for AR-C124910XX at three quality control levels. Interday precision values ranged from 8.7 to 11.4% for ticagrelor and 6.5 to 10.2% for AR-C124910XX.Plasma concentrations of ticagrelor and the metabolite, AR-C124910XX, were analyzed by validated UPLC-MS-MS methods as previously described . ProteinThe inhibition of ADP-induced platelet aggregation of platelet-rich plasma (PRP) was measured in response to 5 μM ADP as described elsewhere . The meat represents the IPA at time t, MPAt represents the MPA at time t, and MPAbaseline represents the MPA value at baseline. In essence, a higher IPA indicated greater antiplatelet effect.where IPAmax was estimated directly from observed plasma concentration–time data. AUC0-t was calculated using the linear trapezoidal rule, AUC0-∞ was calculated as AUC0-∞ = AUC0-48 + Ct/ke, where Ct was the last measured concentration and ke was calculated using linear regression analysis of the log-linear part of the plasma concentration–time curve. t1/2 was calculated as ln2/ke.Pharmacokinetics was analyzed using non-compartmental methods and WinNonlin Pro 5.2 . Concentration–time curves were generated for plasma ticagrelor and AR-C124910XX, and CThe pharmacodynamics effect (IPA) of ticagrelor was expressed as the percentage change from baseline platelet aggregation to 48 h after initial administration of the study drugs. The area under the time-effect curve (AUEC) for the IPA of ticagrelor was calculated from the time vs. IPA value curve, using the linear trapezoidal rule.post hoc test for multiple comparisons and an unpaired Student’s t-test for two groups as appropriate. Comparison of data not normally distributed was performed with a Mann–Whitney U test for comparison of two groups, while a Kruskal–Wallis H test was used for comparison of multiple groups. Two-sided P values ≤ 0.05 were considered statistically significant.Statistical analyses were performed using SPSS 16.0. Continuous variables were presented as means ± SD, and a Kolmogorov–Smirnov test was used to confirm normal distribution of continuous data. Data normally distributed were analyzed using a one-way analysis of variance (ANOVA) with the least significant difference (LSD) All participants given the medication evaluated here were observed closely to preserve their safety. All adverse events (AEs), such as symptoms and their severity, duration, time of onset and disappearance, and relationships to the study drug, were recorded. Physical examinations were performed and vital signs were evaluated, including systolic and diastolic blood pressures. Laboratory tests included urinalysis and hematologic, coagulation, and chemical analysis were performed.∗1GCYP3A4 and ∗3CYP3A5. All enrolled subjects completed the study with no major protocol violations. Subjects’ demographics and SNP characteristics appear in Table 2, and there were no significant differences in age, weight, height, or BMI among the six genotypes.Fourteen healthy Chinese male subjects were recruited from 136 individuals genotyped for CYP3A4 and CYP3A5 genotypes are presented in Figures 2. Pharmacokinetic comparisons for both genotypes are summarized in Tables 4. Because no significant differences exist between the CYP3A4: g.20230GA heterozygotes and CYP3A4: g.20230AA homozygotes, both were combined and compared to CYP3A4: g.20230GG homozygotes.Mean ± SD plasma concentration–time profiles for ticagrelor and AR-C124910XX for 0-last, AUC0-∞, t1/2, tmax, or Cmax between the two CYP3A4 genotypes (P > 0.05). No statistically significant differences were found in pharmacokinetics for ticagrelor among the three CYP3A5 genotypes.No statistically significant differences were observed in the AUC0-last, AUC0-∞, and Cmax of AR-C124910XX in ∗1GCYP3A4 carriers were 1.38 (P = 0.001), 1.43 (P = 0.001), and 1.42 (P = 0.003)-fold higher than in those with the CYP3A4: g.20230GG homozygotes, respectively. The t1/2 of AR-C124910XX in the ∗1GCYP3A4 group was also significantly longer than the CYP3A4: g.20230GG homozygotes (P = 0.015). The CYP3A4 genotype had no statistically significant effect on the tmax of AR-C124910XX. The CYP3A5 genotype had no statistically significant effect on pharmacokinetics parameters of AR-C124910XX.The mean AUCCYP3A4 and CYP3A5 genotypes are presented in Figures 4. Pharmacodynamics comparisons for both genotypes are summarized in Table 5. Strong inhibitory effects on platelet aggregation were observed. A single oral dose of ticagrelor (180 mg) induced a maximum inhibition of 75% at an average of 4 h after dosing. No statistically significant difference in IPA was observed for either CYP3A4 or CYP3A5 genotype.Pharmacodynamics properties of ticagrelor for Ticagrelor was well tolerated, and no study volunteers withdrew from the protocol, and no clinically significant bleeding event or thrombocytopenia was documented.in vivo resulting in subsequent variable patient response. However, influence of ∗1GCYP3A4 and ∗3CYP3A5 on ticagrelor pharmacokinetics and pharmacodynamics is still not quite clear. The present study was thus designed to extensively investigate the effects of ∗1GCYP3A4 and ∗3CYP3A5 genetic polymorphisms on the pharmacokinetics and pharmacodynamics of ticagrelor and AR-C124910XX in Chinese healthy subjects.Effective antiplatelet treatment is particularly important in patients with a high risk of cardiovascular events. As an orally active antiplatelet agent, ticagrelor has been demonstrated to exert a faster and more powerful IPA in comparison to clopidogrel in coronary artery disease patients. Although ticagrelor does not require metabolic activation for antiplatelet activity, it is still extensively metabolized to an active metabolite, AR-C124910XX . Signifi∗1GCYP3A4 allele had greater AR-C124910XX AUC0-last, AUC0-∞, and Cmax and longer t1/2 compared with those subjects with the ∗1/∗1CYP3A4 genotypes. Thus, the ∗1GCYP3A4 allele may enhance CYP3A4 expression and increase biotransformation of ticagrelor. This finding is consistent with previously published studies  polymorphism decreased CYP3A4 activity and modified fentanyl pharmacokinetics. So, patients with the ∗1G/∗1GCYP3A4 genotype required significantly less fentanyl for postoperative pain control compared to those with wild type or the ∗1/∗1GCYP3A4 genotype.Indeed, our results suggest that subjects with the  studies  which shs groups  found th∗1GCYP3A4 on plasma AR-C124910XX levels. Considering the fact that CYP3A4 and CYP3A5 convert ticagrelor into its active metabolite, AR-C124910XX, whose potency is almost the same as ticagrelor in antagonizing P2Y12 receptor, it became not so surprised. However, it’s important to note that a strong linkage disequilibrium exists between the ∗1GCYP3A4 and ∗1CYP3A5 polymorphisms, the presence of ∗1G might be only a marker of yet unstudied allele of greater influence of the variability on ticagrelor therapy. Therefore, to further examine the associations of the ∗3CYP3A5 and ∗1GCYP3A4 polymorphisms on CYP3A activity and to assess the combined influence of these two polymorphisms, a larger study should be on the way.However, statistically significant difference in IPA was not observed as well as the effects of SLCO1B1, CYP3A4, and UGT2B7) with plasma ticagrelor. Because the CYP3A4: is infrequent in the Chinese, we selected ∗1GCYP3A4 for the study, and our data showed that ∗1GCYP3A4 significantly affected AR-C124910XX pharmacokinetics but not the parent drug or platelet reactivity, which is in agreement with data published by Christophe’s group. Meanwhile, considering the fact that, there was no direct evidence whether SLCO1B1 or UGT2B7 participated in the metabolism of ticagrelor, and the effect of genetic polymorphisms of the two proteins on ticagrelor weren’t assessed in our study.∗1GCYP3A4 polymorphism significantly influenced the pharmacokinetics of AR-C124910XX, and it may be more important than ∗3CYP3A5 with respect to influencing ticagrelor pharmacokinetics by increasing CYP3A4 activity. Dosage adjustment according to the CYP3A4 or 3A5 genotype seems unnecessary considering their minor impact on platelet reactivity.In summary, the SL, LM, and ZS contributed to the study design and protocol. SL and XS conducted the study. SL, XZ, and XT performed the pharmacokinetic and pharmacodynamics measurements. All authors reviewed, interpreted the data and agreed on the content. LM and ZS were the lead authors, directed the manuscript content of each draft, supervising the medical writer. All authors approved the final version for submission.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.


Staphylococcus aureus (MRSA), are the most frequent cause of sepsis, which urgently demanding new drugs for treating infection. Two homologous insect CSαβ peptides-DLP2 and DLP4 from Hermetia illucens were firstly expressed in Pichia pastoris, with the yields of 873.5 and 801.3 mg/l, respectively. DLP2 and DLP4 displayed potent antimicrobial activity against Gram-positive bacteria especially MRSA and had greater potency, faster killing, and a longer postantibiotic effect than vancomycin. A 30-d serial passage of MRSA in the presence of DLP2/DLP4 failed to produce resistant mutants. Macromolecular synthesis showed that DLP2/DLP4 inhibited multi-macromolecular synthesis especially for RNA. Flow cytometry and electron microscopy results showed that the cell cycle was arrested at R-phase; the cytoplasmic membrane and cell wall were broken by DLP2/DLP4; mesosome-like structures were observed in MRSA. At the doses of 3‒7.5 mg/kg DLP2 or DLP4, the survival of mice challenged with MRSA were 80‒100%. DLP2 and DLP4 reduced the bacterial translocation burden over 95% in spleen and kidneys; reduced serum pro-inflammatory cytokines levels; promoted anti-inflammatory cytokines levels; and ameliorated lung and spleen injury. These data suggest that DLP2 and DLP4 may be excellent candidates for novel antimicrobial peptides against staphylococcal infections.Methicillin-resistant  Multidrug resistant organisms, especially methicillin-resistant Staphylococcus aureus (MRSA), which is the most common organism with higher mortality than that of methicillin-sensitive S. aureus (MSSA)4, are expected to become more frequent causes of death than cancer in the coming decades5. Despite the urgent need for new antibiotics that are effective against resistant bacteria, new compound sources, such as the large inventory of antimicrobial host defense peptides from different organisms, which have the advantages of the broad spectrum activity, rapid action and low target-based resistance, can open up new avenues for the development of new anti-infectives6.In recent years, there has been a rapid emergence of highly antibiotic-resistant bacterial infections in both nosocomial and community settings, which is a major cause of morbidity and mortality worldwide7. Insect AMPs, which are essential components of insect innate immune system, may have potential applications in the medicine field. Most insect AMPs are small cationic molecules that are active against bacteria, virus and fungi8. According to structural similarity or unique sequences, insect AMPs can be categorized into four classes: α-helix (cecropin and moricin), Cys-rich (insect defensin and drosomycin), Pro-rich , and Gly-rich peptides (attacin)7. Defensins, which are composed of 34‒51 residues, are the most widespread AMPs characterized in insects8. At present, more than 70 insect defensins have been identified in various arthropods, including Sarcophaga, Sapecin, and Aedes11. They commonly contain an N-terminal loop, an α-helix, and an antiparallel β-sheet, forming a “cysteine-stabilized alpha beta (CSαβ)” or “loop-helix-sheet” structure12. These insect CSαβ defensins are active mainly against Gram-positive bacteria, including S. aureus, Micrococcus luteus, and Aerococcus viridians7.Although insects make up 90% of the total number of animals on earth, insect-derived AMPs only account for approximately 10% of more than 2,830 antimicrobial peptides (AMPs) listed in the Antimicrobial Peptide Database, thus there are more AMPs with activity just waiting to be discoveredHermetia illucens13. The 40-residue DLP4 peptide shares high sequence similarity (60‒67.5%) to other insect defensins, such as sapecin14, defensin A12, and lucifensin15; it exhibited potent antibacterial activity against Gram-positive bacteria including MRSA with the minimal inhibitory concentrations (MICs) of 0.59‒2.34 μM, but not Gram-negative bacteria such as Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa13. However, little is known about the clinical pharmacology and antibacterial mechanisms of DLP4 and its analogue DLP2.Recently, a novel insect defensin-like peptide DLP4 is isolated and identified in immunized hemolymph and various tissues of black soldier fly DLP2 and DLP4 genes were firstly expressed in Pichia pastoris X-33. The structure, antimicrobial activity, toxicity, resistance and pharmacodynamics of DLP2 and DLP4 against MRSA were systematically evaluated in vitro. A possible mechanism of DLP2 and DLP4 against MRSA were further investigated. Additionally, the antibacterial and inflammatory properties of DLP2 and DLP4 were examined in a mouse peritonitis model of MRSA infection.In this study, the synthesized 16, insect defensin A12, and lucifensin8, which are composed of 40 amino acid residues  to insect defensins: sapecinues Fig. . DLP2 anues Fig. , which fues Fig. , indicatXhoI and XbaI was inserted into pPICZαA and transformed into E. coli DH5α , and approximately 175 mg/l of target peptides was obtained from the culture supernatant in the shaking flask after 120 h of induction. To enhance the yield of peptides, high-density cultivation was performed in a 5-l fermentor. As shown in Fig. S. aureus ATCC25923 and ATCC43300 (MRSA) displayed time-dependent relationship  of S. aureus significantly (p < 0.001) decreased by 0.98, 1.68‒1.89 and 1.06‒1.34, respectively after treatment with 20 × MIC vancomycin, 10 × MIC and 20 × MIC DLP2 or DLP4.As shown in Table S. aureus was 0.67, 3.67, and 10.67 h, respectively at 1, 2, and 4 × MIC. The PAEs at the same concentrations of DLP4 on S. aureus were 3.33, 6.33, and 13.33 h, respectively, whereas the PAE of vancomycin at 2 × MIC was 2 h  of DLP2 and DLP4 against MRSA ATCC43300 were shown in Fig. 2 h Fig. , indicat17. However, the combination interactions of vancomycin with DLP2 and DLP4 demonstrated an indifference effect with FICI values of 1.188  ranging from 0.291 to 0.5, and detected between DLP4 and ciprofloxacin (FICI = 0.333) Fig. . However188 Fig. .S. aureus ATCC25923 of greater than 94.9% in a temperature range from 20 °C to 60 °C; 85% and 93% of the activity of DLP2 and DLP4 were remained after a 1-h incubation at 80 °C, and 60‒73.7% of the activity of DLP2 and DLP4 were observed to remain at 100 °C  Fig. . MeanwhiAfter 30 serial passages in the presence of peptides or antibiotics, the MICs of DLP2 and DLP4 against MRSA ATCC43300 did not change, which indicated that no mutants of bacteria resistant to peptides were produced Fig. . In cont18. As shown in Fig. S. aureus stained with PI was 0.06%, indicating the integrated cell membranes. The percentages of PI-positive MRSA ATCC43300 cells treated with 2 × MIC DLP2 for 0.5 h, 1 h and 2 h were 2.53%, 21.70% and 27.10%, respectively , which can penetrate the damaged cell membrane and intercalate into nucleic acid, was used as an indicator to evaluate effects of DLP2 and DLP4 on the plasma membrane of MRSA ATCC43300 cells by flow cytometryely Fig. . After tely Fig. . Comparaely Fig. . This suS. aureus cells exhibited the smooth surface and intact cell morphology with a prominent septal midline. After exposure of S. aureus to 2 × MIC DLP2 or DLP4 for 2 h, some filiferous adhesions were observed outside of cells  and transmission electron microscopy (TEM) were used to directly observe the effects of DLP2 or DLP4 treatment on the cell morphology and integrity of MRSA ATCC 43300. As shown in Fig. lls Fig. . The TEMlls Fig. . Approxi20. In contrast, DLP2 and DLP4 weakly bound to the genomic DNA from E. coli CVCC1515 and S. typhimurium ATCC14028  and D-[6-3H(N)] glucosamine hydrochloride  incorporation was observed at 0.5 h of exposure of S. aureus to 2 × MIC DLP2 or DLP4. Moreover, both DLP2 and DLP4 also induced a weak decrease in DNA synthesis  in a time-dependent manner and protein synthesis   Fig. , which wS. aureus (1 × 1010 CFU) and followed by treatment with DLP2 and DLP4. As shown in Fig. S. aureus, and all were dead within 36 h. Mice injected with PBS in the control group survived throughout the experimental period. After treatment with 3, 5 and 7.5 mg/kg DLP2 or DLP4, the survival rates of mice were 80%, 100% and 100%, respectively. In the positive control, the survival rates of mice treated with 5 and 10 mg/kg vancomycin were 60% and 83.3% respectively, which was lower than DLP2 and DLP4. This result indicated that both DLP2 and DLP4 can protect mice from a lethal S. aureus challenge in vivo.To evaluate the therapeutic activity of peptides in the peritonitis model, mice were challenged with 9 CFU) leads to the translocation of S. aureus into deep organs, infected mice were treated with DLP2, DLP4 or vancomycin at 2 h post-inoculation, and spleen and two kidneys were harvested at 3 h post-treatment and analyzed by the plate count method. As shown in Fig. S. aureus cells in spleen and two kidneys in mice treated with 7.5 mg/kg DLP2 or DLP4 decreased by 95‒99% and 98‒99%, respectively. Treatment with 10 mg/kg vancomycin resulted in a reduced bacterial burden in the spleen and two kidneys by 26% and 73%, respectively.To test whether intraperitoneal inoculation , interleukin-6I (IL-10), tumor-necrosis factor alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were determined at 3 h and 16 h post-treatment using an enzyme-linked immunosorbent assay (ELISA) kit. As shown in Fig. S. aureus, the organ damage degree was examined at 1 d and 5 d after treatment. As shown in Fig. To explore if DLP2 and DLP4 can reduce lung and spleen injury from a lethal challenge with S. aureus challenge in vivo.These data indicated that, superior to vancomycin, both DLP2 and DLP4 could protect mice from lethal 23, so more attentions have been concerned on the newly effective AMPs which have been considered as promising antibiotic alternatives in the medical field24. In the current study, DLP2 and DLP4, the novel insect AMPs from H. illucens, were firstly expressed in P. pastoris and purified by cation-exchange columns; their antibacterial, pharmacological activities and the mechanism of action against MRSA ATCC43300 were systematically investigated for the first time.The continuous and selective antibiotic pressure in humans and in farm animals have promoted the emergence of MRSAE. coli, Klebsiella pneumoniae, and P. aeruginosa, insect CSαβ AMPs such as sapecin and lucifensin can inhibit the growth of Gram-positive bacteria (i.e. S. aureus)27. In the present study, however, CSαβ DLP2 and DLP4 exhibited a more potent antibacterial activity against Gram-positive bacteria than that found in the previous study13. DLP4 exhibited 2-‒4-fold higher antimicrobial activity compared to DLP2  is more effective at reducing the S. aureus burden than treatment with DLP2 (7.5 mg/kg) and vancomycin (10 mg/kg)  Fig. , which mkg) Fig. 31.31. Results of the present study showed that both DLP2 and DLP4 obtained a significant concentration-dependent PAE, and shorter PAE was observed for MRSA ATCC43300 after exposure to DLP2 (0.67‒10.67 h) than DLP4 (3.33‒13.33 h) , tigecycline (3.2 h), and arbekacin (3.0‒3.2 h) against S. aureus reported in previous studies33. Long PAEs might lead to longer interval of administration, which thus potentially reducing treatment cost and inhibiting development of drug resistance34.PAE is an important pharmacodynamic parameter that may be considered in choosing the antibiotic dosing regiments in clinical use h) Fig. , which w16. Furthermore, both Asp4 and Arg23 residues are also conserved in lucifensin and insect defensins   Fig.  suggestsion Fig. . Secondlion Fig. . Moreoveays Fig. . Comparaays Fig. , which mS. aureus-evoked expression of pro-inflammatory cytokines of TNF-α, IL-6 and MCP-1, and promote production of anti-inflammatory ones of IL-10 and GM-CSF than DLP4 and vancomycin  to activate the downstream signaling . The three-dimensional structures of DLP2/DLP4 were modeled using SWISS-MODEL workspace (http://swissmodel.expasy.org/workspace/) and PyMol 1.8.Multiple sequences alignment and the sequence conservation analysis were performed by ClustalX 1.8 and the WebLogo    using a broth microdilution technique as previously described5) were incubated with mid-exponential-phase MRSA ATCC43300 for 2 h at 37 °C in a 5% CO2 incubator. Gentamicin (100 μg/ml) was added into the cultures and incubated for 1 h and then thoroughly washed with PBS for three times to remove extracellular bacteria57. Fresh DMEM containing different concentrations of DLP2, DLP4 or vancomycin  were added into the cultures and incubated at 37 °C for 18 h. Monolayers cells were then washed three times with PBS, lysed with lysis buffer . The internal S. aureus cells were estimated by colony counting on MHA  plates.Murine macrophage RAW264.7 cells  by the viable plate count method as described previously59.The interaction of combinations of peptides and traditional antibiotics was evaluated in MHB by a checkerboard microtiter assay as described in detail in the supplemental material and expressed as FICI for each agentS. aureus strain ATCC25923 was tested using an inhibition zone assay16.The thermal stability of DLP2 and DLP4 was determined after 1-h incubation of 25 μl (32 μg/ml) of peptides at 4, 20, 40, 60, 80, and 100 °C in deionized water, respectively. The antimicrobial activity of DLP2 and DLP4 against 60. The method was described in detail in the supplemental material.The hemolytic activity of DLP2 and DLP4 was evaluated against fresh mRBCs by determining the amount of the released hemoglobin at 540 nmThe cytotoxicity of DLP2 and DLP4 on the viability of mouse peritoneal macrophages RAW264.7 cells was measured by a colorimetric 3--2,5-diphenyl-2H-tetrazolium bromide assay (MTT) according to the method in the supplemental material.The resistance experiment for DLP2 and DLP4 was performed in MH by sequential passaging. These methods are described in detail in the supplemental material.The membrane permeabilization activity of DLP2 and DLP4 was investigated by a PI uptake assay as described in the supplemental material.2 dried. The samples were sputter-coated with platinum and observed using a QUANTA200 SEM  as described in detail in the supplemental material.The exponential-phase MRSA ATCC43300 were treated with 2 × MIC DLP2 or DLP4 for 2 h at 37 °C, fixed with osmium tetroxide, dehydrated by ethanol, and COAfter treatment with peptides for 2 h, the MRSA ATCC43300 cells were fixed twice, dehydrated with ethanol, embedded in Spur resin, and sectioned. Then, samples were stained with uranyl acetate and lead citrate and observed using a TEM  as described in the supplemental material.To examine whether binding of DLP2 and DLP4 to bacterial DNA, the gel retardation experiment was performed by mixing peptides and bacterial DNA, which was extracted from MRSA ATCC43300 using a TIANamp Bacteria DNA Kit  as described in detail in the supplemental material.CD measurements were also carried out on a MOS-450 spectropolarimeter using a quartz cuvette with 1.0-mm path length to examine further DLP2 and DLP4 binding to DNA at the ratios of 1, 5 and 10, respectively. The spectra were the average of 10 scans collected from 220 to 320 nm at 25 °C with a 20 s bandwidth.S. aureus treated with DLP2 and DLP4 was analyzed by PI staining using flow cytometry according to a previous method in the supplemental material60.The cell cycle of S. aureus was measured by isotope labeling experiment as described in detail in the supplemental material.Effect of DLP2 and DLP4 on the macromolecular synthesis of All mouse experiments were performed in accordance with the Animal Care and Use Committee of the Feed Research Institute of Chinese Academy of Agricultural Sciences (CAAS) and protocols were approved by the Laboratory Animal Ethical Committee and its Inspection of the Feed Research Institute of CAAS (AEC-CAAS-20090609).10 CUF/ml, 0.5 ml) at the lower left side of abdomen, and followed by intraperitoneal injection at other side with two doses of DLP2, DLP4  or vancomycin at 2 h and 12 h post-infection, respectively. Mice injected with only bacteria or PBS were used as negative or blank controls. Survival of mice was recorded daily for seven days.To establish a peritonitis or sepsis mouse model, six-week-old female BALB/c mice (five mice/group) were intraperitoneally injected with MRSA ATCC43300  and treated with a single dose of DLP2, DLP4 (7.5 mg/kg) or vancomycin (10 mg/kg) at 2 h post infection. Sera was collected from the mice sacrificed at 3, and 16 h post-treatment. The levels of TNF-α, IL-6, IL-10, GM-CSF, and MCP-1 in sera were detected at Jiaxuan Biotech. Co. Ltd.  using an ELISA kit62. Moreover, kidneys and spleens were removed at 3 h post-treatment and homogenized in sterile PBS for a CFU assay for MRSA ATCC43300 to evaluate bacterial translocation.Similarly, mice (30 mice/group) were intraperitoneally injected with MRSA ATCC43300 . Lungs and spleens were harvested from mice which were sacrificed at 1 d, 3 d and 5 d after infection, washed with PBS, and fixed in 4% paraformaldehyde at 4 °C for 24 h. After washing with PBS and dehydrating with a graded series of ethanol (75‒95%), the organs were infiltrated with xylene, embedded in paraffin wax and sectioned. After staining with hematoxylin and eosin, the tissue samples were examined by a light microscope. Mice treated with vancomycin or PBS served as a positive or negative control, respectively.Mice were intraperitoneally injected with DLP2, DLP4 (7.5 mg/kg) or vancomycin (10 mg/kg) at 2 h post-intraperitoneal infection of MRSA ATCC43300 (8 × 10All data are presented as means ± standard deviation (SD). GraphPad Prism software v6.0  was used for all statistical analyses.Supplementary Information


In contrast, tumor and organ uptakes of 89Zr-amatuximab were dose-dependent, whereby a high dose (60 μg) was needed to achieve tumor targeting comparable to the low dose (2 μg) of 89Zr-B3, suggesting that shed mesothelin may affect amatuximab tumor targeting as well as serum half-life. The autoradiography analysis showed that the distribution of 89Zr-B3 was nonuniform with the radioactivity primarily localized at the tumor periphery independent of the B3 dose. However, the autoradiography analysis for 89Zr-amatuximab showed dose-dependent distribution profiles of the radiolabel; at 10 μg dose, the radiolabel penetrated toward the tumor core with its activity comparable to that at the tumor periphery, whereas at 60 μg dose, the distribution profile became similar to those of 89Zr-B3. These results suggest that shed antigen in blood may act as a decoy requiring higher doses of mAb to improve serum half-life as well as tumor targeting. Systemic mAb concentration should be at a severalfold molar excess to the shed Ag in blood to overcome the hepatic processing of mAb-Ag complexes. On the other hand, mAb concentration should remain lower than the shed Ag concentration in the tumor ECS to maximize tumor penetration by passing binding site barriers.We investigated the effect of shed antigen mesothelin on the tumor uptake of amatuximab, a therapeutic anti-mesothelin mAb clinically tested in mesothelioma patients. The B3 mAb targeting a nonshed antigen was also analyzed for comparison. The mouse model implanted with A431/H9 tumor, which expresses both shed mesothelin and nonshed Lewis-Y antigen, provided an ideal system to compare the biodistribution and PET imaging profiles of the two mAbs. Our study demonstrated that the tumor and organ uptakes of  Some parameters are related to tumor environment factors, including vascular and stroma density, interstitial pressure, and tumor binding site barriers –3. In adculation , 6. In cculation .κ and 10−9 M KD binding affinity), a therapeutic mAb currently investigated in mesothelioma patients, and control anti-Lewis-Y mAb B3 (murine IgG1κ with 10−8 M KD binding affinity). Comparative studies using this system enabled us to define the effects of shed Ag on the tumor uptake and penetration of mAb apart from the effects of other factors such as vascular density, high interstitial fluid pressure (IFP), and extracellular protein contents.In this study, we investigated the effects of shed Ag on the tumor targeting and penetration of mAb. To achieve this goal, we used a nude mouse model implanted with A431/H9 tumor that overexpresses both shed MSLN (5 million Ag molecules/cell) and nonshed Lewis-Y (4 million Ag molecules/cell). MSLN is a membrane glycoprotein of 40 kDa that is actively internalized into the cell's cytosol as well as shed from the tumor cell surface, generating soluble MSLN in the tumor's interstitial space and blood circulation with its concentrations proportional to the size of tumor , 9. Lewi89Zr , which decays with a low positron emission energy of 395.5 keV, allowing for PET imaging with higher resolution [89Zr can enhance PET radioimmune detection of labeled mAbs and extend autoradiography preparation time due to the longer half-life and higher imaging resolution compared with 111In and 64Cu. In addition to the biodistribution (BD), PET imaging, and autoradiography studies for the two 89Zr-labeled mAbs, here we also report a new autoradiography analysis method to define the tumor uptake profile of the two 89Zr mAbs irrespective of tumor size and shape.The two mAbs were labeled with solution –16. 89Zr89Zr) was produced at the National Institute of Health  cyclotron facility using a 16.5 MeV proton cyclotron  by proton irradiation   reaction (2~5 h) on 89Y-metal mesh . 89Zr was separated as 89Zr-oxalate from irradiated 89Y-metal mesh using 0.1 M oxalic acid solution [Amatuximab was obtained from Morphotek, Inc. , and B3 was provided by Dr. Ira Pastan . p-Isothiocyanatobenzyl-desferrioxamine (p-SCN-Df) was purchased from Macrocyclics, Inc. . Zirconium-89 (89Zr using desferrioxamine (Df) with an isothiocyanate linker as a chelating agent following a method of Vosjan et al. [µmol DTPA to block nonspecific protein binding sites and remove potential metal contaminants and then washed with metal-free sodium acetate . The mAb concentrations were measured according to the method of Bradford [89Zr distribution between the peaks corresponding to Df-mAb and free DF on the size exclusion HPLC when the product mixture was radiolabeled as described below.The mAbs were radiolabeled with n et al. . BrieflyBradford . The levµM) was labeled with 89Zr , which was neutralized with a solution containing sodium carbonate (0.135 M)/HEPES buffer  and d-mannitol (5.5 mg/mL) at 25°C for 1 h. One mL of 0.25 M sodium acetate containing 5 mg/mL gentisic acid (pH 5.5) was then added to the reaction solution. The labeled product was purified with PD 10 columns eluted with metal-free elution buffer . Each PD 10 column was pretreated with 25 mg BSA containing 1 µmol DTPA to block nonspecific protein binding sites and remove potential metal contaminants and then washed with metal-free elution buffer. The radiolabeling yield and the radiochemical purity were assessed by analytical size exclusion HPLC  before and after the purification  and unbound 89Zr (retention time: 9.45 min) on the HPLC profiles.Purified Df-amatuximab or Df-B3  of the conjugate samples were incubated side-by-side with an increasing number of A431/H9 cells  in 100 µL of PBS with 1% BSA at 4°C for 3 hours. Nonspecific binding to the cells was determined by performing the cell-binding assays under a condition of excess amount of antibodies (50 µg amatuximab and 50 µg B3).The immunoreactivity of and Bunn , as prevand Bunn . Aliquot2 in media supplemented with 10% FBS, 2 mmol/L L-glutamine, 100 U penicillin, and 100 mg streptomycin (Invitrogen Corporation). The cell line was authenticated at the source and grown from frozen stocks prepared from an early passage of the original line [A431/H9 is a derivative of the A431 epidermoid carcinoma cell line that has been stably transfected with vectors for human mesothelin and grown in media supplemented with 750 mg/mL G-418 (Geneticin) for selection . This cenal line .89Zr-labeled amatuximab or B3 conjugate with Df molecules, groups (n = 4-5 mice/group) of mice were injected (i.v.) with 89Zr-labeled mAb conjugates  mixed with corresponding unlabeled intact antibodies  in 0.2 mL PBS containing 1% BSA. The tumor sizes at the time of the BD studies were as follows: 245.5 ± 6.5, 190.8 ± 5.0, and 226.4 ± 7.1 mm3 for 2, 10, and 60 µg amatuximab, respectively, and 205.9 ± 3.4, 321.2 ± 9.0, and 372.5 ± 8.2 mm3 for 2, 15, and 60 µg B3, respectively. The animals were euthanized at 24 hours by CO2 inhalation and exsanguination by cardiac puncture. We performed the BD studies as described previously  with A431/H9 tumor were injected (i.v.) with 89Zr-amatuximab  or 89Zr-B3  in 0.2 ml of normal saline through the tail vein and then 15 min static PET scans were performed at 3, 24, and 48 h p.i. The tumor sizes at the time of the PET imaging were as follows: 429 ± 141 mm3 (range: 253–599 mm3) for 10 µg amatuximab and 406 ± 23 mm3 (range: 385–440 mm3) for 60 µg amatuximab; 700 ± 220 mm3 (range: 436–1042 mm3) for 15 µg B3 and 441 ± 126 mm3 (range: 304–630 mm3) for 60 µg B3.PET imaging studies were performed as described in the previous study of us paper ). The mi3  and 388 ± 5 mm3  for 10 and 60 µg amatuximab, respectively, and 574 ± 121 mm3  and 364 ± 60 mm3  for 15 and 60 µg B3, respectively. The tumors were embedded and frozen in Tissue-Tek® CRYO-OCT compound  at −20°C for 3 h. Serial 20 µm thick short axis sections were cut in 400 µm intervals covering the entire tumor. Two or three consecutive tumor slices were selected at 3 tumor regions  as representative sections throughout the tumor and exposed on the phosphor screen for 16 h. Signals were obtained by the use of the Typhoon FLA 7000  with 25 µm pixel resolution and analyzed with Image Quant TL8.1 software. Values were grouped together from the 3 tumor regions to represent a tumor. Each tumor was treated as an independent sample. To analyze the microdistribution of the radioactivity in the tumor sections, we introduced a normalized length analysis method as described below. The first line was drawn along the longest axis, and the second line was drawn along a short axis perpendicularly at the center of the first longest line  of less than 0.05 was considered significant.Statistical analysis was performed using ANOVA for comparing multiple groups, and Student's n = 3) for B3 and 0.9 ± 0.2 (n = 3) for amatuximab. The 89Zr-labeled mAbs were purified on PD-10 columns eluted with acetate buffer (pH 5.5) containing gentisic acid at 5 mg/ml. The purified products were >95% radiochemically pure based on the size exclusion HPLC profiles. The specific activities of the purified product were 296 kBq/µg for 89Zr-amatuximab and 148 kBq/µg for 89Zr-B3. The immunoreactivities of  89Zr-amatuximab and 89Zr-B3 were 84.0 ± 2.2% (n = 2) and 70.0 ± 1.0% (n = 2), respectively.The level of Df conjugation was 1.6 ± 0.3  at both dose levels, while the majority of radioactivity was still localized in heart (blood pool) and liver  at 24 h and 48 h  was 20.43 ± 2.49, 5.09 ± 1.78, and 2.52 ± 0.78 at 3, 24, and 48 h, respectively, for 10 µg and 27.37 ± 4.03, 12.44 ± 3.66, and 6.80 ± 2.04 at 3, 24, and 48, respectively, for 60 µg. Liver uptake (%ID/g) was 24.17 ± 3.11, 25.64 ± 2.43, and 23.03 ± 3.51 at 3, 24, and 48 h, respectively, for 10 µg and 22.33 ± 3.89, 21.14 ± 2.21, and 20.78 ± 3.67 at 3, 24, and 48 h, respectively, for 60 µg. Thus, these data indicate that a higher injection dose of amatuximab is advantageous for the tumor visualization by PET. The tumor-to-organ and the tumor-to-blood ratios from PET imaging had similar values observed in 64Cu-NOTA-amatuximab study [µg but a value <1 for 10 µg  of 21.28 ± 1.54, 12.99 ± 2.83, and 10.21 ± 1.92 at 3, 24, and 48 h, respectively, for 15 µg and 21.05 ± 1.85, 12.40 ± 0.56, and 9.92 ± 1.33 at 3, 24, and 48 h, respectively, for 60 µg; liver uptake (%ID/g) of 25.04 ± 1.03, 20.37 ± 3.4, and 16.55 ± 3.98 at 3, 24, and 48 h, respectively, for 15 µg and 25.21 ± 1.90%ID/g, 18.90 ± 1.06, and 16.72 ± 1.83 at 3, 24, and 48 h, respectively, for 60 µg. The tumor-to-organ and the tumor-to-blood ratios from PET imaging did not change as the dose increased  and sequestered into the reticular endothelial system of liver and spleen, thereby lowering both blood retention and tumor uptake. Based on this knowledge, we hypothesized that a dose of 60 µg could be sufficiently high to saturate shed MSLN as well as create an excess of 89Zr-amatuximab available for a more effective tumor targeting. This effect could be explained by correlating the concentration of shed MSLN in the blood and in the ECS of A431/H9 tumor, as estimated in previous studies [64Cu-NOTA-amatuximab. 89Zr-amatuximab injected at 10 µg dose results in an estimated blood concentration of 43.3 nM (assuming blood volume of 1.6 mL for a 20 g mouse) immediately after injection and 1.75 nM at 48 h  would be mostly bound to shed MSLN in blood during a 48 h period and this complex sequestered into liver and spleen. At 48 h, unbound 89Zr-amatuximab would have crossed the tumor vasculature and diffused into tumor ECS . Because the concentration of shed MSLN is ~300 nM in the ECS of a tumor of 300 mm3, 89Zr-amatuximab (53.6 nM) in the ECS would mostly exist as an antibody-Ag complex distributed throughout the entire tumor, presumably bypassing the binding sites on the surface of tumor cells nearest to the vasculature. On the other hand, 89Zr-amatuximab injected at 60 µg dose results in an estimated blood concentration of 260 nM immediately after injection and 28.3 nM at 48 h (6.8%ID/g blood), that is, at a molar excess compared to the average concentration of shed MSLN in the blood (~6 nM). Consequently, 89Zr-amatuximab would remain mostly unbound, overcoming the sequestration into the hepatic reticuloendothelial system during a 48 h period. Therefore, a larger portion of the injected dose (95.2 nM based on 22.90%ID/g tumor at 48 h and 952 nM in the ECS) would have crossed the tumor vasculature and diffused into the tumor ECS. This concentration (952 nM) is larger than the shed MSLN concentration in the tumor ECS. The excess of 89Zr-amatuximab not bound to shed MSLN would bind to MSLN on tumor cells in the periphery rather than in the tumor core, as observed for 89Zr-B3 as well as Alexa-labeled B3 [These findings could be explained by a threshold effect whereby  studies , 23, simbeled B3 . Accordibeled B3 . Therefoµg) at 24 h and thereafter because the tumor-to-blood and tumor-to-muscle ratios of 89Zr-amatuximab were higher than 2. However, it would be necessary to inject the higher dose (60 µg) to visualize the tumor in the abdominal area. The PET study also suggests that 89Zr-amatuximab could be useful in a clinical setting because 89Zr with a long half-life (78.4 h) would be more suitable for the detection of tumor and the tumor-to-background ratio increased over time with the injection of the higher dose (60 µg).It is noteworthy that our PET study demonstrated that the tumor localized outside of the abdomen could be visualized even with the lower dose (10 In this study, we demonstrated the effect of shed mesothelin on the tumor targeting and tumor microdistribution of anti-mesothelin mAb amatuximab in A431/H9 tumor using anti-Lewis-Y B3 as a negative control. However, it is possible that other shed antigens with a different binding epitope or a different affinity for the targeting mAb might show a different degree of shed antigen effects. Thus, careful investigation of other shed antigen systems would be needed to determine whether our findings could be generalized.89Zr with a 3.4-day half-life to label the mAbs for PET imaging and autoradiography studies provided the advantages of high image sensitivity and resolution which enabled us to quantify the tumor and organ uptakes as well as tumor penetration of the mAbs.The use of A431/H9 tumor which overexpresses both the shed Ag mesothelin and the nonshed Ag Lewis-Y as a tumor model and the use of anti-mesothelin mAb amatuximab and anti-Lewis-Y mAb B3 as model mAbs for the BD, PET, and autoradiography studies allowed us to make a direct assessment on the effect of the shed Ag on the tumor and organ uptakes and tumor penetration, apart from the effects of other factors related to the tumor microenvironment. In addition, the use of The findings of this study imply that the systemic mAb concentration should be at a severalfold molar excess to the shed Ag concentration in the blood to increase the concentration of free mAb available for the tumor uptake and reduce the fraction of the mAb bound to the shed Ag in the blood and the subsequent hepatic processing of the mAb-Ag complexes. However, the mAb concentration in the tumor ECS should remain lower than the shed Ag concentration in the tumor ECS to maximize the tumor penetration of the mAb-Ag complexes by bypassing the binding site barrier. It will be the future challenge for both antibody engineers and clinical investigators to find this balance.


III and EuIII depletion from the culture medium by the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV. The assay can also be modified to screen the uptake of other REEs, such as PrIII, or to monitor the depletion of LaIII from growth media in neutrophilic methylotrophs such as Methylobacterium extorquens strain AM1. The AS III assay presents a convenient and fast detection method for REE levels in culture media and is a sensitive alternative to inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS).Recently, methanotrophic and methylotrophic bacteria were found to utilize rare earth elements (REEs). To monitor the REE content in culture media of these bacteria, we have developed a rapid screening method using the Arsenazo III (AS III) dye for spectrophotometric REE detection in the low μM (0.1 to 10 μM) range. We designed this assay to follow LaIMPORTANCE REE-dependent bacterial metabolism is a quickly emerging field, and while the importance of REEs for both methanotrophic and methylotrophic bacteria is now firmly established, many important questions, such as how these insoluble elements are taken up into cells, are still unanswered. Here, an Arsenazo III dye-based assay has been developed for fast, specific, and sensitive determination of REE content in different culture media. This assay presents a useful tool for optimizing cultivation protocols, as well as for routine REE monitoring during bacterial growth without the need for specialized analytical instrumentation. Furthermore, this assay has the potential to promote the discovery of other REE-dependent microorganisms and can help to elucidate the mechanisms for acquisition of REEs by methanotrophic and methylotrophic bacteria. Methylobacterium radiotolerans, Methylobacterium extorquens, and Bradyrhizobium sp. strain MAFF211645, which are dependent on LaIII and CeIII  and copper(II), will significantly interfere with the assay. In addition, the sensitivity of this dye is generally lower (Rare earth elements (REEs) are relevant for Gram-negative methylotrophic bacteria, which utilize these strong Lewis acids in the active sites of alcohol dehydrogenases, including methanol dehydrogenases (MDHs), such as XoxF, and ethanol dehydrogenases, such as ExaF . The biod CeIII 24. In 201um SolV 518. Addit as PedH . Due to  as PedH , 16. How strains . Furthereobacter . While ieobacter . The meteobacter . Chromogeobacter , 27. Of eobacter  has beeneobacter , 27–29. acter 27–. REEs reacter 27–. While nacter 27–, 29. In ly lower , 32.III absorbance at 650 nm will increase with increasing pH, as demonstrated in III complex exhibits a maximum absorbance at 650 nm. For concentrations higher than 2 μM, an additional peak can be detected at 600 nm under certain conditions. This observation has also been made by Fernandez-Gavarron et al., who detected an additional peak at 597 nm when using a mixture of 25 μM AS III and 10 μM La at pH 3.1  this correlation is strongly dependent on the conditions of the assay . III to a solution of 10 μM AS III in citrate/phosphate buffer (pH 2.8) in the presence of bulk medium and trace elements of M. fumariolicum SolV. In III to a solution of 10 μM AS III in citrate/phosphate buffer (pH 2.8) in the presence of minimal PIPES  (MP) medium, as used for the cultivation of methylotrophs, is shown. Different REE ions can be screened using this assay. However, individual calibration curves have to be recorded for each derivative, as the intensity of the absorption at 650 nm varies within the series , which reflected a depletion of the AS III-La complex, was observed at different time points during microbial growth (600]) and the LaIII content of the medium was also analyzed by ICP-MS . III by Mb. extorquens AM1.In order to evaluate the precision of the AS III assay in monitoring Lae medium . DepletiP medium  during bM. fumariolicum SolV depletes most REEs before growth starts , while Mb. extorquens AM1 shows a linear decrease in REE content over the whole growth period. We present here a quick and simple assay for direct determination of REE concentrations in the growth media of methanotrophic and methylotrophic bacteria using the Arsenazo III azo dye. The assay can be used for different REEs, although it was observed that the larger the REE, the more sensitive the assay. REE depletion can be monitored for both acidophilic and neutrophilic bacterial strains. Control of the pH in the calibration curve samples and test solutions is essential for the accuracy of the REE content determination.Overall the ICP-MS and AS III measurements show a good correlation. It is interesting to note that the uptake mechanisms of the two bacterial species investigated here seem to be different. 3·7H2O was of 99.999% purity. EuCl3·6H2O and PrCl3·6H2O were obtained in 99.99% purity. Buffer, metal stock solutions, and dye stock solutions were made up in deionized Millipore water. Additional treatment of the Millipore water with Chelex was not necessary. The assay was tested in Hellma quartz Suprasil cuvettes, as well as with disposable cuvettes (polymethyl methacrylate [PMMA]) and 96-well plates, and all were found to be suitable for this assay. The assay was tested on conventional diode array and xenon flash lamp UV-visible (UV-Vis) spectrometers, which can both be used, since AS III does not show significant decomposition during the short duration time of the assay when subjected to UV light. The assay can also be conducted using a microplate reader .Arsenazo III (AS III) and the REE salts were purchased from Sigma-Aldrich and used in the experiments without further purification. Initially, the dye (85% purity) was further purified using a published protocol . HoweverM. fumariolicum SolV, the final element solution contained the bulk elements (bulk medium solution) 0.2 mM MgCl2·6H2O, 1 mM Na2SO4, 2 mM K2SO4, 4 mM (NH4)2SO4, 1 mM NaH2PO4·H2O, and 0.2 mM CaCl2·2H2O and the trace elements 2 μM FeSO4·7H2O, 0.1 μM ZnSO4·7H2O, 0.1 μM CoCl2·6H2O, 2 μM MnCl2·4H2O, 3 μM CuSO4·5H2O, 0.1 μM NiCl2·6H2O, and 0.1 μM Na2MoO4·2H2O. The stock solution of the trace elements was dissolved in 1 to 2% sulfuric acid (vol/vol) before addition to the bulk medium solution. Usually, 10× and 1,000× concentrated solutions of bulk and trace elements were prepared and diluted together as needed.In the case of strain 3 was performed in a 10-liter fermenter . The liquid volume was 5 liters and the temperature was set to 55°C. The pH was kept at 2.8 using 0.2 M NaOH. A total of 8 to 100 ml/min methane was supplied to the reactor. The air supply was 50 to 1,200 ml/min, and the agitation speed was between 400 and 1,000 rpm, in order to maintain a dissolved oxygen concentration of up to 10% air saturation. Samples were taken approximately every 12 h and were centrifuged at 2,770 × g for 20 min at room temperature. The clear supernatant was transferred to clean 15-ml centrifuge tubes stored at 4°C prior to analysis. One part of the supernatant was transferred to a clean 1.5-ml Eppendorf tube and used for the Arsenazo dye III assay. The remaining supernatant was used for ICP-MS analysis. All analyses were performed at the end of the batch experiments. Batch cultivation with 5 μM LaCl3 was performed in a 500-ml fermenter with a working volume of 400 ml. The bioreactor was operated at 55°C with a stirring rate of 700 rpm, using a stirrer bar. The pH was kept at 2.8 using 0.2 M NaOH. Ten milliliters per minute of gas containing 10% CH4 and 3% CO2, and 8% O2 was supplied to the reactor. Samples for the Arsenazo III dye assay were taken approximately every 2 h for the first 8 h of the experiment, and for the remaining time period, samples were taken every 12 h. Samples for the Arsenazo III dye assay were centrifuged at 20,238 × g for 10 min at room temperature. The supernatant was transferred to a clean Eppendorf tube and stored at −20°C prior to analysis. Samples for ICP-MS were taken at the start, after 8 h, and then approximately every 12 h and were centrifuged at 2,770 × g for 20 min at room temperature, and the clear supernatant was stored at 4°C prior to analysis. All analyses were performed at the end of the batch experiments. To analyze europium or lanthanum content in the SolV culture medium with ICP-MS, 10 ml of the clear supernatant was collected and passed through by 0.2-μm filter and acidified with 65% nitric acid to a final concentration of 1%. After sample preparation, metal analysis was performed using an inductively coupled plasma mass spectrometer (ICP-MS)  at the Faculty of Science, Radboud University (Nijmegen). Batch cultivation with 2 μM EuCl2HPO4·3H2O, 1.88 mM NaH2PO4·H2O, 0.5 mM MgCl2·6H2O, 8 mM (NH4)2SO4, 45.6 μM Na3C6H5O7·2H2O, 1.2 μM ZnSO4·7H2O, 1 μM MnCl2·4 H2O, 18 μM FeSO4·7H2O, 2 μM (NH4)6Mo7O24·4H2O, 1 μM CuSO4·5H2O, 2 μM CoCl2·6H2O, 0.33 μM Na2WO4·2H2O, 20 μM CaCl2, and 125 mM methanol  (3 in a flask at 30°C on a rotary shaker at 150 rpm for 48 h. Samples (5 ml each) were collected at time points of 0, 1, 4, 6, 8, 12, 24, 28, 32, and 48 h and centrifuged at 4,696 × g for 10 min at 4°C. Supernatants were filtered using a 0.2-μm syringe filter and acidified with 5 M HCl. Precipitated PIPES buffer was removed by centrifuging for 15 min at 4,696 × g at room temperature. Collected supernatants were then used either for the Arsenazo III assay or for ICP-MS analysis. For ICP-MS analysis, samples were diluted 1,000-fold with nitric acid to the final concentration of 2% nitric acid, and the samples were analyzed with a ICAP Q quadrupole ICP-MS (Thermo Scientific) at the Department of Earth and Environmental Sciences at Michigan State University.One-hundred-milliliter  strain)  were gro strain)  containiThe general protocol for the AS III assay is described in


In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70.  Heat shock factor 1 (HSF1) orchestrates an elaborate transcriptional program that enhances adaptation and survival under conditions of stress. It is activated in response to stresses such as heat shock, hypoxia, heavy metals, reactive oxygen species, and changes in pH. In an unstressed system, monomeric HSF1 is bound to its negative regulators, heat shock protein 40 (Hsp40), Hsp70, and Hsp90 13. DuringIncreased nuclear HSF1 levels correlate with poor prognosis in breast, colon, and lung cancer , 6. Furt303SPPQ307SPRV) after obligatory priming phosphorylation at S307 by the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1), is carried out by glycogen synthase kinase 3, inhibiting the function of the transcription factor (–326SPTAL) activates HSF1, and the mutation of S326 to alanine (S326A) reduces its transcriptional activity by >80% , a vegetable from this family, is a rich source of the glucosinolate gluconasturtiin. Phenethyl isothiocyanate (PEITC)   is an is (PEITC) , 23. PEI (PEITC) , but how (PEITC) . We showAll general chemicals and reagents were of analytical grade and obtained from Sigma-Aldrich . PEITC was prepared as a stock solution in acetonitrile and diluted 1:1,000 in the cell culture medium before treatment. The concentration of the solvent was maintained at 0.1% (vol/vol) in all wells. The p38α/β MAPK inhibitor SB202190 was purchased from SYNkinase. The c-Jun N-terminal kinase (JNK) inhibitor JNK-In-8 was kindly provided by Dario Alessi (University of Dundee).2 in air at 37°C and were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum. The medium in which HeLa-HSE-luc cells were grown also contained 100 μg/ml G418 (Invitrogen), whereas the medium for MEF cells was additionally supplemented with nonessential amino acids and 50 U/ml penicillin-streptomycin.MDA-MB-231 cells were from ATCC. HeLa-HSE-luc cells  were a gg for 10 min at 4°C, and the supernatant was transferred to a new tube. The lysates derived from the SDS lysis buffer were subjected to sonication at 20% amplitude for 20 s. A BCA assay (Thermo) was used to determine protein concentrations. Proteins were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and probed with specific antibodies against Hsp70 , Hsp90 , HER2 , RAF1 , HSF1 , pS326-HSF1 , p38 MAPK , pp38 MAPK , JNK1/2 , pJNK1/2 , pERK1/2 , pT334-MK2 , and pS235/6 S6 . Isoform-specific p38γ and p38δ MAPK antibodies were from the Division of Signal Transduction Therapy and were used at a concentration of 1 μg/ml. Equal loading was confirmed by probing the blots with antibodies against GAPDH  or β-actin , both from Sigma  or lamin A . The Western blots shown are representative of at least three independent experiments.Cells grown in six-well plates were washed twice with phosphate-buffered saline (PBS) and lysed in 150 μl of either radioimmunoprecipitation assay (RIPA) buffer  or SDS lysis buffer . The lysates derived from RIPA buffer were transferred into 1.5-ml Eppendorf tubes, which were placed on a rotator at 4°C for 30 min. The cell debris was then removed by centrifugation at 16,300 × 6 per dish) were plated in 6-cm dishes and treated for the indicated periods of time with 0.1% (vol/vol) acetonitrile or PEITC. The REAP method described by Suzuki et al.  in PBS. The lysates were then subjected to a further centrifugation at 10,000 × g for 30 s at room temperature. The supernatant was collected as the cytoplasmic fraction. One volume of 5× sample SDS loading buffer  glycerol, and 0.025% [wt/vol] bromophenol blue) was added to 4 volumes of the cytoplasmic fraction, and the samples were heated for 5 min at 100°C and subjected to SDS-PAGE. The remaining pellet containing the nuclear fraction was washed twice with ice-cold 0.1% NP-40 (vol/vol) in PBS and dissolved in 1× sample loading buffer  and heated for 5 min at 100°C. The nuclear fractions were sonicated before subjecting them to SDS-PAGE.MDA-MB-231 cells . Cells (2 × 105 per well) were seeded in six-well plates. After 24 h, the cells were exposed to vehicle (0.1% acetonitrile) or PEITC for a further 8 h (MEFs) or 16 h (MEFs and MDA-MB-231 cells). After cell lysis, total RNA was extracted using RNeasy kit , and 500 ng of total RNA was reverse transcribed into cDNA with an Omniscript reverse transcription kit . Real-time PCR was performed on an Applied Biosystems 7900HT Fast real-time PCR system. The data were normalized using β-actin  as an internal control.The primers and probes for quantifying the levels of the mRNA species were from Applied Biosystems (5 per well) were seeded in each well of a 24-well plate and 24 h later treated with PEITC or 0.1% (vol/vol) acetonitrile vehicle for 8, 16, 24, or 48 h. The cells were washed twice with 0.1% PBS, and 100 μl of 1× reporter lysis buffer (Promega) was added to each well. The plate was placed at −20°C for a minimum for 2 h and then transferred to thaw on a shaker at room temperature for 30 min. Cell lysates were collected into Eppendorf tubes and subjected to centrifugation at 15,000 × g for 2 min at 4°C. The luciferase activity was measured in 10 μl of cell lysate in opaque 96-well plates (Corning) using a microplate-reader based luminometer  and normalized for protein concentration determined by a Bradford assay (Bio-Rad).HeLa-HSE-luc cells (106 per dish) were seeded in 6-cm dishes. After 24 h, the cells were treated for a further 24 h with 0.1% acetonitrile as the vehicle control for sulfoxythiocarbamate alkyne  and PEITC (20 μM) treatments or with 0.1% dimethyl sulfoxide (DMSO) as the vehicle control for the geldanamycin  and celastrol  treatments. The cells were harvested by scraping into 300 μl of lysis buffer , frozen, thawed, and lysed for 30 min at 4°C. ATP-agarose beads (Jena Bioscience) were washed with the incubation buffer . Cell lysates  were added to a suspension of 30 μl of beads in 1.25 ml of buffer, and the samples were incubated with rotation overnight at 4°C. The beads were collected by centrifugation and washed three times with the incubation buffer. SDS loading buffer (10 μl) and incubation buffer (40 μl) were added to the beads, and the samples were incubated for 5 min at 100°C. The beads were pelleted by centrifugation, and the supernatants were collected and subjected to Western blot analysis.MDA-MB-231 cells (0.5 × 106 per dish) cells were grown on 10-cm dishes for 24 h and treated with 0.1% acetonitrile or 20 μM PEITC for a further 3 h. The cells were then washed twice with PBS. Next, 10 ml of 0.4% (wt/vol) paraformaldehyde in PBS (0.4% PFA-PBS) was added to the dishes over 10 min, where fresh 0.4% PFA-PBS was added every 5 min. Next, the PFA-PBS was removed, and the reaction was quenched with the addition of 3 ml of ice-cold 1.25 M Glycine-PBS. After washing twice with PBS, nuclear and cytoplasmic fractions were obtained. The cells were lysed in buffer A . The lysates were subjected to centrifugation at 1,000 × g for 5 min at 4°C. The supernatant containing the cytoplasmic fraction was transferred to a fresh Eppendorf tube where one volume of 5× SDS sample loading buffer was added to four volumes of the cytoplasmic fraction. The pellet containing the nuclear fraction was washed there times with the buffer A before dissolving it in 1× sample loading buffer . The nuclear fractions were subjected to sonication. Both the cytoplasmic and the nuclear fractions were subjected to SDS-PAGE before immunoblotting.MDA-MB-231 (2 × 106 per dish) cells were grown on 10-cm dishes for 24 h and then treated with 0.1% DMSO or 20 μM PEITC for 45 min. The dishes with cells were placed on ice and washed twice with ice-cold PBS. Protein G-Dynabeads (30 μl slurry [Invitrogen]) were washed twice for 5 min with PBS and incubated with 1 μg of mouse monoclonal HSF1 antibody (Santa Cruz) for 1 h at room temperature, after which the beads were washed three times every 10 min with PBS. Cells were lysed with 1.0 ml ice-cold CO-IP buffer  supplemented with one EDTA-free protease cocktail inhibitor tablet (Roche) and one phosphatase inhibitor tablet (PhoSTOP Roche). The cell lysates were passed through a 23-gauge needle 10 times before they were clarified by centrifugation at 4°C for 30 min at 16,000 × g. Then, 50 μl of the clarified lysate (IP sample) was transferred to a fresh Eppendorf tube to serve as an input sample. To preclear the IP sample, 30 μl of protein G-Dynabead slurry was washed twice for 5 min with PBS, and the beads were added to each of the IP sample (containing 0.8 to 1.0 mg of protein) and then incubated for 1 h at 4°C on a tube rotator. Subsequently, the protein G-Dynabead-antibody conjugate was added to the precleared IP sample, followed by incubation for 16 h at 4°C on a tube rotator. The immunoprecipitated complexes were washed three times with ice-cold CO-IP buffer every 10 min and then eluted from the beads by adding 70 μl of 1× LDS buffer (Invitrogen) and heating the sample at 70°C for 10 min. After cooling, 7 μl of sample reducing agent (Invitrogen) was added to the sample, followed by incubation for 15 min at room temperature. Immunoprecipitated proteins (35 μl) were resolved by electrophoresis. Antibodies against Hsp90  and HSF1  were used for detection of the respective proteins.MDA-MB-231 . A lentivirus containing the control pLKO.1 or the shRNA plasmids was used to infect MDA-MB-231 cells. To produce the virus, HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) with empty pLKO.1-puro vector or the shRNA constructs against p38γ or p38δ, together with the packaging vectors (psPAX2 and pMD2.G) in serum-reduced medium. On the following day, the medium was replaced with complete DMEM and, after 24 h, the lentivirus-containing supernatant was collected, filtered, and used to transduce MDA-MB-231 cells. Cells containing the shRNA plasmid were selected, expanded, and maintained with supplementation of puromycin (2 mg/ml) for approximately 3 weeks, during which time cell lysates were collected every 3 to 4 days to ensure the respective p38 expression levels were reduced throughout the selection period.E. coli [BL21(DE3)pLysS], the cells were grown to an optical density at 600 nm of 0.6, and induced for a further 3.5 h at 37°C with 400 μM IPTG . The induced cells were harvested by centrifugation and resuspended in extraction buffer . After freezing and thawing, the cells were disrupted by sonication for 5 min on ice. Cell debris were then cleared by centrifugation at 10,000 × g for 15 min at 4°C. The resultant supernatant was left on ice for 30 min before it was applied to nickel agarose resin . The resin was washed with 20 mM Tris-Cl (pH 7.9), 150 mM NaCl, and 5 mM imidazole. The supernatant (20 ml) was then incubated for 1 h at 4°C with 1 ml of resin. After three washes with buffer, the protein was eluted with 2 ml of 20 mM Tris-Cl (pH 7.9), 150 mM NaCl, and 250 mM imidazole. To remove the imidazole, the preparation was dialyzed in 50 mM Tris-Cl (pH 7.4)–150 mM NaCl. Mutant S326A HSF1 was generated by site-directed mutagenesis of the plasmid vector pet15b containing the HSF1 cDNA by using the primers 5′-GTGGACACCCTCTTGGCCCCGACCGCCCTCATTG-3′ and 5′-CAATGAGGGCGGTCGGGGCCAAGAGGGTGTCCAC-3′ and a QuikChange II mutagenesis kit (Stratagene). The hexahistidine-tagged mutant S326A HSF1 recombinant protein was generated using the method described above.Full-length HSF1 cDNA was amplified by PCR from a plasmid obtained from Addgene  and subcloned into the bacterial expression vector pet15b using NdeI and XhoI. After transformation into 3 cells/well and transfected with 50 ng/well green fluorescent protein (GFP)-tagged wild-type or S326A or S326E mutants of HSF1 using Lipofectamine LTX reagent (Invitrogen), using eight replicate wells per condition. At 24 h after transfection, the cells were fixed using 4% paraformaldehyde in PBS for 10 min at room temperature and permeabilized using methanol at −20°C for 5 min. The cells were blocked using 2.5% normal goat serum in PBS–0.1% sodium azide and counterstained using rabbit anti-ERK1/2 monoclonal antibody  and Alexa Fluor 546-labeled highly cross-adsorbed goat anti-rabbit secondary antibody (Invitrogen). DNA was labeled with 300 nM DAPI (Sigma) in PBS, and the images were acquired using an IN Cell Analyzer 2000 robotic fluorescence microscope  using a 20× lens to capture four fields per well for each fluorophore  using 2×2 pixel binning to maximize the signal/noise ratio. The images were analyzed using a custom algorithm constructed within IN Cell Developer software , using DAPI and ERK1/2 images to identify nuclear and cytoplasmic regions, respectively, in order to assess fluorescence distribution within the GFP channel.HSF1-knockout MEFs were seeded in black-walled 96-well plates (Corning Costar 3904) at 4 × 102, 0.1 mM [γ-32P]ATP (approximately 0.5 × 106 cpm/nmol), and kinase buffer  in a total volume of 50 μl. The kinase assays were performed at 30°C. At the times indicated, a 15-μl aliquot of each incubation mixture was removed, the reaction was terminated by the addition of SDS gel loading buffer, the sample was loaded on SDS-PAGE, and the excess [γ-32P]ATP was removed by electrophoresis. The gels were dried and subjected to autoradiography. Protein-containing gel pieces  were then excised, and phosphate incorporation into HSF1 was quantified by scintillation counting.The incubation mixtures contained purified recombinant kinase (at a specific activity of either 6 or 0.06 mU/μl), recombinant HSF1 (1 μg) substrate, 10 mM MgCl2 (10 mM), and ATP (0.1 mM) instead of [γ-32P]ATP. For identification of the phosphorylated sites, the gel bands were excised, reduced with DTT (10 mM), alkylated with iodoacetamide (50 mM), and digested overnight (16 h) with trypsin  at 30°C. The resulting peptides were extracted from the gel, dried in a SpeedVac concentrator (Thermo Scientific), resuspended in 10 μl of 5% formic acid, and diluted five times. Any residual particles were removed by centrifugation, the samples were then transferred to high-pressure liquid chromatography vials, and analyzed by liquid chromatography-tandem mass spectrometry on an Ultimate3000 RSLCnano System (Thermo Scientific) coupled to a LTQ OrbiTrap VelosPro (Thermo Scientific) with an EasySpray source. The data files were analyzed with Proteome Discoverer (v1.4.1) using Mascot (v2.4.1) as the search engine using a protein-specific database (HisTag-HSF1) and the IPI-Human (ipi.HUMAN.v3.87) database.Cold assays were performed in an analogous manner using purified recombinant kinase (at a specific activity of 0.06 mU/μl), recombinant HSF1 (1 μg) substrate, MgClt test. Analyses were performed using Excel (Microsoft Corp.).Values are expressed as ±1 standard deviation (SD). The differences between groups were determined by using a Student hspa1a were upregulated by 4.1- and 3.5-fold after the exposure of wild-type MEF cells to 10 μM PEITC for 8 or 16 h, respectively  45% decrease in the mRNA levels for HER2, although the mRNA levels for RAF1 were not affected (not shown). The reason for this decrease in HER2 expression and its potential contribution to the decreased HER2 protein levels at the high concentrations of PEITC are presently unknown. In contrast to geldanamycin (GA), an Hsp90 inhibitor that competes with ATP, but similarly to celastrol (CL) and sulfoxythiocarbamate alkyne (STCA), which inhibit Hsp90 by modifying its cysteine residues without interfering with ATP binding  phosphorylate and activate HSF1 . We therRecently, it was reported that HSF1 physically interacts and is phosphorylated at S326 by MAPKK  . We therin vitro. HSF1 was expressed as a His-tagged fusion protein in Escherichia coli. Purified His-HSF1 migrated as a major band during NuPAGE at the expected molecular weight and showed a tendency to form spontaneously dimeric and trimeric species , the stoichiometry of the reaction was 2, 1.3, 0.8, and 2.2 mol/mol for the α, β, γ, and δ isoforms, respectively (data not shown), suggesting that, under these experimental conditions, p38α, p38β, and p38δ were able to phosphorylate at least two sites on HSF1. With 0.06 mU/μl of enzyme and 15 min of incubation, the stoichiometry of the reaction for HSF1 was 0.04, 0.03, 0.03, and 0.28 mol/mol for p38α, p38β, p38γ, and p38δ, respectively , which iTo confirm the unusually high substrate preference of p38δ for HSF1 relative to the more widely studied p38α/β isoforms, we carried out analogous reactions with MK2 as a substrate. In comparison to p38δ, the initial rates of activation of this physiological substrate are ∼20 times faster for the α or β isoforms . Indeed,As mentioned above, we used MBP-MKK6 to obtain active bacterially produced human p38 MAPK. To make absolutely certain that the phosphorylation of HSF1 was not due to any residual MKK6, a new preparation of p38δ was made in parallel: a preparation of a kinase-dead mutant (D168A) version of the enzyme under identical conditions. After incubation and removal of the activating kinase, each protein was used at an equivalent concentration in a reaction with HSF1 as a substrate. Phosphorylation of HSF1 occurred with the active but not the kinase-dead p38δ, as revealed by autoradiography and Western blot analysis , establiin vitro by all p38 MAPK isoforms at S326 and that p38δ is the most efficient catalyst among the isoforms, whereas p38γ is the most specific. However, it is important to note that S326 is not the only site of phosphorylation by these kinases as the S326A mutant HSF1 was also phosphorylated, albeit with the expected reduction in stoichiometry. To identify the phosphorylation sites precisely, we used a protease-mass spectrometric approach. Recombinant HSF1 was incubated with recombinant p38α or p38δ and, after electrophoretic separation and in-gel proteolytic digestion, the resulting tryptic peptides were analyzed by mass spectrometry. Under these conditions, the sequence coverage was ∼50%. We found that the two p38 isoforms phosphorylated identical sites  of the longest peptide was in precise agreement with the calculated molecular weight of a peptide containing phosphorylated S326 . The mass of the shorter peptides  corresponded exactly to the molecular weight of peptides K.EEPPSPPQ307SPR.V  and R.VKEEPP303SPPQ307SPR.V , in which both S303 and S307 were phosphorylated. Notably, S303 was found in both phosphorylated and unphosphorylated forms. The phosphorylation of HSF1 at S303/307 by the same kinase which phosphorylates the transcription factor at S326 was at first glance surprising, because in contrast to the activating S326 phosphorylation, phosphorylation at S303/307 is inhibitory   28). In. In28). in vitro  to D. Trin vitro .in vitro with both WT and S326A mutant HSF1 (Similar results were obtained in MDA-MB-231 cells: shRNA-mediated knockdown (by >90%) of p38γ led to a substantial reduction (by ∼50%) in the phosphorylation of HSF1 at S326 at basal cell culture conditions, whereas the knockdown of p38δ did not have this effect . The knoant HSF1  and C; tThe conclusion that p38α and p38β do not contribute significantly to the phosphorylation of S326 in A431 cells was further supported by studies in PEITC-treated MDA-MB-231 cells, where SB202190 either had no effect (at the high concentration of PEITC) or even enhanced by 2.5-fold (at the low concentration of PEITC) the levels of pS326 HSF1 after exposure to the isothiocyanate . The actC. elegans, ectopic expression of HSF1 produces a gain-of-function phenotype  phosphorylating this site. It was reported that the mechanistic target of rapamycin (mTOR) is able to catalyze the phosphorylation of HSF1 at S326 . HoweverInterestingly and unexpectedly, we found that in addition to S326, p38 MAPK can also catalyze the phosphorylation of HSF1 at S303/307. In contrast to the activating function of the S326 phosphorylation, phosphorylation at S303/307 inhibits the function of HSF1 –16. Alth–Previous investigations have suggested the possible involvement of MAPK signaling in the activation of HSF1. Thus, loss of the tumor suppressor neurofibromatosis type 1 (NF1) leads to activation of MAPK signaling and HSF1 . Chronic


Preeclampsia (PE) is a life-threatening pregnancy disorder characterized by wide-spread endothelial dysfunction. Placental factors circulating in the maternal blood are believed to cause endothelial dysfunction. Our previous study identified HtrA4 as a placenta-specific serine protease that is released into the maternal circulation and significantly increased in early-onset PE. In this study, we examined the impact of HtrA4 on expression of endothelial genes related to vessel biology, using human umbilical vein endothelial cells (HUVECs) as a model.HUVECs were treated with 0 or 3 μg/ml HtrA4 (highest concentration seen in PE circulation) for 24 h and analysed by an endothelial cell biology PCR array containing 84 genes. HtrA4-induced changes were then validated by real-time RT-PCR and ELISA for time and dose dependency.IL6, PTGS2 (COX2) and IL1B were significantly increased by HtrA4. IL6 protein in HUVEC media was also drastically increased. THBD, an anticoagulant factor reported to be increased in PE, was significantly up-regulated by HtrA4. In contrast, THBS1, which is involved in many regulatory processes of endothelial cell biology, was severely down-regulated by HtrA4.High levels of HtrA4 significantly altered the expression of a range of genes related to inflammation, vaso-activity, angiogenesis, cell adhesion, platelet activation and coagulation. In particular, pro-inflammatory genes HtrA4 significantly increased the inflammatory responses of HUVECs, and altered their expression of a number of genes important for vessel biology. These data suggest that placenta-derived HtrA4 that is increased in PE circulation is a potential causal factor of endothelial dysfunction. •HtrA4 altered expression of endothelial genes involved in vessel biology.•HtrA4 significantly induced inflammation in endothelial cells.•Placental-derived HtrA4 is a potential causal factor of endothelial dysfunction. The starting passage of the HUVECs was 13, and the experiments were completed within eight passages. The cells were cultured in 12-well plates (Thermo Fisher Scientific) at 1 × 105 per well density for 24 h (h), then treated with recombinant HtrA4  or vehicle control for 24 or 48 h. The vehicle control contains 150 mM NaCl, 5 mM CaCl2, 50 mM Tris-HCl pH 7.5, 0.05% Brij 35 solution, 50 mM imidazole in ultrapure H2O. The two doses of HtrA4 were chosen to represent the median and highest levels of HtrA4 found in early-onset PE circulation HUVECs  were cultured at 37 °C in a humidified atmosphere of 5% CO2.2RNA was extracted using RNeasy Mini Kit  and contaminating DNA was removed using RNase-free DNase (Qiagen) according to the manufacturers' protocols. The RNA concentration was determined using Nanodrop ND-1000 (Thermo Fisher Scientific).2.32 First Strand Kit (Qiagen). A RT2 Profiler 84 gene PCR array (Qiagen) was screened as per manufacturer's instruction on an ABI 7900 HT Fast real-time machine . The results were analysed using Qiagen RT2 profiler PCR array data analysis software.RNA samples from vehicle control or 3 μg/ml HtrA4 treatment for 24 h were pooled from three independent experiments, and 500 ng of the pooled RNA presenting control and HtrA4 treatment  was reverse transcribed into complementary DNA (cDNA) using RT2.4Genes showing more than 2-fold differences in expression on the array between the vehicle control and HtrA4 treatment were validated by real-time RT-PCR. RNA (300 ng) from three independent experiments of 24 h and 48 h treatment with 0, 1.5 μg/ml and 3.0 μg/ml HtrA4 was reverse transcribed in 20 μl using SuperScript III First-Strand kit  as per manufacturer's protocol. Real-time RT-PCR was performed with primers specified in 2.5The secreted levels of interleukin (IL)6 and monocyte chemoattractant protein (MCP)1 in the above treated cell media were measured by ELISA  according to manufacturer's instruction.2.6A cytokine antibody array for 36 cytokines  was analysed to determine whether HtrA4 alters these cytokines in HUVECs. Conditioned media from cells treated with vehicle control or 3.0 μg/ml HtrA4 for 24 h from four independent experiments were pooled for the array analysis. Dots representing 36 cytokines were analysed by densitometry using ImageJ software .2.7Statistical analyses were conducted using GraphPad Prism . Data are expressed as mean ± SD, comparison was made using two-way ANOVA followed by Tukey's post-hoc tests, and difference was considered significant if p < 0.05.3We used HUVECs as a model and examined the effect of HtrA4 on the expression of 84 genes that are involved in endothelial cell biology . These g3.1CCL2  and ALOX5, CCL2 mRNA was significantly reduced by HtrA4 even at the lower dosage of 1.5 μg/ml; and the reduction was more profound at 48 h than 24 h , IL6 and IL1B were all validated by real-time RT-PCR. PTGS2 mRNA was significantly up-regulated by 3 μg/ml HtrA4 at 48 h time point  and two up-regulated (FGF2 and VEGFA) genes  genes A. Real-tt manner B, but PTby HtrA4 C. The twby HtrA4 D–E. The by HtrA4 D–E.3.3THBS1) and two up-regulated (SERPINE1 and IL11) genes  genes A. THBS1 t manner B. Both Sendently C–D.3.4OCLN, MMP1 and THBD) genes  genes A. BCL2, e RT-PCR B. For th at 48 h C. For th at 48 h D. In cont manner E.In total, thirteen genes were confirmed by real-time RT-PCR to be significantly altered by HtrA4, ten were up-regulated and three down-regulated . Functio3.5IL6, CCL2, CCL2, which encodes MCP1, was significantly reduced at the protein level by HtrA4 in a dose- and time-dependent manner  and IL1B, further suggesting that the circulating HtrA4 of placenta-origin can intensify the inflammatory response of maternal endothelial cells, which is frequently observed in early-onset PE Several studies reported that maternal serum IL6 is significantly increased in PE patients Additional inflammatory factors such as TNF-α are reported to be significantly elevated in preeclamptic serum CCL2, which encodes for MCP-1. The main function of this protein is to recruit monocytes or macrophages to the site of inflammation as a result of tissue injury or infection The only down-regulated gene identified in the inflammation category was THBD, an endothelial cell-specific membrane-bound receptor that functions as an anticoagulant, was highly up-regulated by HtrA4. This is consistent with other studies showing that serum THBD is increased in PE women compared to normal controls Our data also showed that HtrA4 affected several regulatory factors of endothelial cell biology. For Instance, SERPINE1, another gene up-regulated by HtrA4, encodes the inhibitor for fibrinolysis and is also elevated in the circulation of PE women THBS1, which is reported to be lower in women with severe PE THBS1 down-regulation can significantly impair the normal cellular function of endothelial cells Surprisingly, only a small number of genes in the angiogenic and vasoactive group were found to be regulated by HtrA4. This suggest that endothelial genes in this group may not be the major targets of HtrA4 action, and other placental-derived factors may be responsible for their dysregulation and contribute to the endothelial dysfunction.Overall, our study suggests that high levels of placental-derived HtrA4 that is circulating in early-onset PE women is a potential causal factor of endothelial dysfunction. HtrA4 profoundly altered HUVEC expression of several factors essential for normal endothelial cell function and inflammation responses. As endothelial dysfunction is a major aspect of early-onset PE development, our study provides new insight into the underlying causes of this disease.One limitation of this study is that HUVEC was used as a model. HUVEC is a well characterized cell line and commonly used for studying endothelial biology, but it may not reflect all the features of human vascular endothelial cells. Future studies will confirm the impact of HtrA4 on endothelial cells using other cell models.The authors declare no conflict of interests.


For comparison, the LiBax1−PO4:xTm3+ powders sintered at the same sintering condition but in a conventional furnace were also investigated. LiBaPO4 without second phase was formed no matter which furnace was used. More uniform grain size distributions are obtained by microwave sintering. When the concentration of Tm3+ ion was x = 0.015, the luminescence intensity reached a maximum value, and then decreased with the increases of the Tm3+ concentration due to concentration quenching effect. The microwave sintering significantly enhanced the emission intensity of LiBax1−PO4:xTm3+ phosphors. Additionally, the d-d interaction is the key mechanism of concentration quenching for LiBaPO4:Tm3+. The chromaticity  for all LiBax1−PO4:xTm3+ phosphors are located at , which will be classified as a blue region.An investigation of the photoluminescent properties and crystalline morphology of blue emitting LiBa The combination of blue LED with yellow luminescence from Y4, where A is a mono-valent cation and B is a divalent cation, are interesting because of their optical, ferroelectric properties, excellent thermal and hydrolytic stability [4 phosphate by solid-state reaction for WLEDs [3+-doped materials have been widely adopted as blue emitting phosphors because of their intense 1D2 → 3F4 emission. The investigation from Li et al. has shown that Tm3+-doped NaCaPO4 phosphor exhibits relatively strong absorption in near-UV region of 356 nm and intense blue emission of 451 nm with excellent color purity [Phosphates with a general formula like ABPOtability ,4,5,6,7.or WLEDs ,4,5,6,7.2O7:Eu3+ [2ZnO5:Eu3+ [2SiO4:Eu3+ [4:Dy3+ [Regarding the sintering process, it has been reported that, when the phosphors were sintered using the microwave energy as the heating sources, the energy can be absorbed immediately and uniformly compared to results from a conventional solid state sintering process . This te2O7:Eu3+ , BaY2ZnOnO5:Eu3+ , and Sr2iO4:Eu3+  and LiBa [4:Dy3+ .3+ ions doped LiBaPO4 phosphors prepared by microwave-assisted sintering or conventional sintering have not yet been reported. In addition, the luminescent characteristics of Tm3+ ions doped LiBaPO4 and the mechanism of energy transfer of Tm3+-doped LiBaPO4 phosphor has been little-reported until now. Therefore, in this paper, the luminescent characteristics, microstructure properties and mechanism of energy transfer of LiBaPO4:Tm3+ phosphors prepared using different sintering processes are investigated.However, to our knowledge, Tm0.985PO4:0.015Tm3+ sintered at 1200 °C for 3 h in different furnace, respectively. According to the Joint Committee on Powder Diffraction Standards (JCPDS #14-0270), the LiBaPO4 has a hexagonal crystal structure with a space group P63 [0.985PO4:0.015Tm3+ phosphors with different sintering furnace can be indexed and match the reference [0.985PO4:0.015Tm3+ phosphors prepared using microwave sintering is smaller than that prepared using the conventional one, indicating that the crystallinity of LiBa0.985PO4:0.015Tm3+ was improved by microwave sintering. It is known that a long sintering time causes grain growth, resulting in good crystallinity. However, at the same sintering condition  with a different sintering method, the heat through conventional method is indirect, but in a microwave furnace, the material is rapidly heated both internally and externally. The heat generated within the material, and the susceptors provided the heat to the specimen externally by thermal conduction [eference  and no o0.985PO4:0.015Tm3+ sintered in microwave furnace and in conventional furnace at 1200 °C for 3 h, respectively. The microstructures of the LiBa0.985PO4:0.015Tm3+ powders changed significantly through different sintering processes. The shapes of the particles are not very different from one another, but the grain size distribution from microwave sintered powder is uniform. The grain distribution of conventionally sintered powder reveals the agglomeration of particles. Additionally, the particle sizes of LiBa0.985PO4:0.015Tm3+ phosphors are in the range of 9–11 μm.0.985PO4:0.015Tm3+ phosphors with different concentrations of Tm3+ ions using different sintering process, respectively. LiBa0.985PO4:0.015Tm3+ phosphors can be effectively excited by NUV LED because all the LiBa0.985PO4:0.015Tm3+ phosphors are excited by a xenon lamp at a wavelength of 359 nm. The inset of x1−PO4:xTm3+ at 0.005 < x ≤ 0.030 sintered in a microwave furnace. The inset of x1−PO4:xTm3+ at 0.010 < x ≤ 0.030 sintered in conventional furnace. Both of the emission spectra of the LiBax1−PO4:xTm3+ phosphors display four emission bands at 454, 476, 513, and 660 nm, respectively corresponding to the 1D2 → 3F4, 1G4 → 3H6, 1D2 → 3H5 and 1G4 → 3F4 transitions of Tm3+ ions.0.985PO4:0.015Tm3+ phosphors with different sintering processes. It is shown that LiBa0.985PO4:0.015Tm3+ phosphor prepared by microwave sintering has much higher luminescent intensity than that of a conventionally sintered one. Referring to 0.985PO4:0.015Tm3+ phosphors prepared by microwave sintering is better than that using conventionally sintering so that higher luminous intensity is expected. As the concentration of Tm3+ ion increased, the probability of the energy transfer among Tm3+ ions also increased. The luminescence intensity reached a maximum when the concentration of Tm3+ ion was at 0.015, and then decreased with the increases of the Tm3+ concentration due to the concentration quenching effect.Rc) to realize the mechanism of energy transfer in phosphors whereby Rc is about equal to twice the radius of a sphere with the volume as shown in Equation (1) [Blasse proposed the critical transfer distance (tion (1) ,15,16. of LiBax1−PO4:xTm3+ phosphor is larger than 5 Å. Therefore, the energy transfer mechanism between Tm3+ ions in LiBaPO4 phosphor could be suggested by multipole-multipole interaction from Dexter's theory [3+ ions, the multipole-multipole interaction effect can be determined from of the difference of the emission intensity according to the emitting level with multipolar interaction. The emission intensity (I) per Tm3+ ion can be calculated by the Equation (2) [x is the activator concentration; Q = 6, 8 or 10 for dipole–dipole (d-d), dipole-quadrupole (d-q) or quadrupole-quadrupole (q-q) interaction, respectively; and K and β are constants for a given host crystal with the same excitation condition. The doped Tm3+ concentration, which is not less than the critical concentration , is used to determine the dependence of the emission intensity of LiBaPO4:Tm3+ phosphor excited at 359 nm. As shown in I/x) on log (x) in microwave sintering is linear and the slope is −2.231. The value of Q can be calculated as 6.693. In addition, the dependence of log (I/x) on log (x) in conventional sintering is linear and the slope is −2.186. The value of Q can be calculated as 6.558. Both are approximately equal to 6 based on Equation (2). Therefore, the d-d interaction is the major mechanism for concentration quenching of the LiBaPO4:Tm3+ phosphor no matter what sintering method was used.In which nsitions . When ths theory . If the tion (2) ,15,16: coordinates located in the blue region . Therefore, if we choose appropriate green and red phosphors mixed with the LiBaPO4:Tm3+ onto the ultraviolet LED chip, the goal to form WLEDs could be achieved.It is known that color coordinates could be established by Commission International de l’Eclairage (CIE) 1931 according to a two-dimensional graphical representation of any color perceptible by the human eye. The CIE coordinates could be obtained through commercial software by converting the data of photoluminescence emission spectrum. x1−PO4:xTm3+ phosphors used Li2CO3 (99.94%), BaCO3 (99.9%), NH4H2PO4 (98%), and Tm2O3 (99.9%) powders as starting materials and different concentrations of Tm3+ ions (x = 0.005–0.03) were chosen as a variable parameter. The starting materials were mixed using alcohol as a solvent and then ball-milled for 1 h with zirconia balls. After drying, the mixed powders were sintered in a microwave furnace and conventional furnace to form LiBax1−PO4:xTm3+ phosphors. As in the case of microwave sintering, a microwave furnace (Therm Wave Mod. III) with a continuously variable power of 2.45 GHz microwaves up to 1.3 kW was used. Silicon carbide (SiC), having a very strong heating response to 2.45 GHz microwaves, was used as a susceptor to provide indirect heating of the powders. For comparison, the sample was also sintered at 1200 °C for 3 h in conventional furnace under an air atmosphere with the heating rate controlled at 10 °C/min. The average heating rate of microwave furnace was greater than 100 °C/min. After sintering, the phosphor samples were cooled to room temperature and then characterized.The LiBaThe crystalline phases of the phosphors were identified by X-ray diffraction  with CuKα radiation of λ = 1.54 Å using a Ni filter, and a secondary graphite monochromator. A scanning range of 2θ = 10°~60° was used with a step of 0.03° and 0.4 s as a per-step count time. The particle morphology of phosphors was identified by scanning electron microscopy . Additionally, the excitation, emission spectra and the color coordinates and the Commission International de l’Eclairage (CIE) information were obtained using a photoluminescence spectrophotometer  with a 150 W xenon lamp as the light source.0.985PO4:0.015Tm3+ phosphors with various sintering process were successively synthesized at 1200 °C for 3 h. XRD results indicate that pure LiBa0.985PO4:0.015Tm3+ phosphor phase was formed. The major emission peak centered at 454 nm corresponds to the 1D2 → 3F4 transition and the maximum photoluminescence intensity appeared at the Tm3+ concentration of 0.015. Under the microwave sintering, good crystallinity and uniform grain size distributions are obtained because microwave energy provides the material rapidly heated both internally and externally. The d-d interaction plays a major role in the mechanism of concentration quenching of LiBaPO4:Tm3+ phosphor based on the theoretical calculation no matter what sintering method was used. Additionally, WLEDs could be achieved by mixing appropriate green and red phosphors with the LiBaPO4:Tm3+ onto the ultraviolet LED chip.In this paper, LiBa