Previous studies have demonstrated effectiveness of chimeric PCV1-2 vaccines against PCV2b challenge. In this study, the efficacy of inactivated and live-attenuated  using PCV2b.Both inactivated and live-attenuated chimeric PCV1-2b vaccines induced a robust antibody responses, and significantly decreased microscopic lesion and lower viral loads in serum or superficial inguinal lymph nodes (SILN) compared with that in the nonvaccinated challenged group. PCV2 antibody titers decreased after 7 days post challenge (DPC) in pigs administered the inactivated PCV1-2b vaccine and they were lower than those in pigs inoculated with live-attenuated PCV1-2b on the day of necropsy. Moreover, no viremia was present in pigs inoculated with live-attenuated PCV1-2b vaccine at 21 DPC regardless of the dose difference.The results demonstrated that both inactivated and live-attenuated chimeric PCV1-2b vaccines were effective to induce protective immunity against PCV2b infection. Porcine circovirus type 2 (PCV2) is the primary causative agent of the postweaning multisystemic wasting syndrome (PMWS). This disease was first identified and reported in western Canada in 1991 –5. PMWS Circoviridae [Porcine circovirus (PCV) is a small, nonenveloped virus with a single-strand circular DNA genome of 1.7 kb in the family oviridae . Two genoviridae . PCV2 geoviridae , 10, ORFoviridae , 11, ORFoviridae . Currentoviridae , 14; andoviridae . PCV2a aoviridae , 17. PCVoviridae , 19.3.5 or 104.0 50 % tissue culture infective dose (TCID50)/ml dose live-attenuated chimeric PCV1-2 vaccine developed high levels of antibody and the vaccinated pigs were fully protected against challenge with PCV2 [3.5 or 2 × 104.0 TCID50 dose) in growing pigs. In this study we demonstrated that pigs can be effectively protected against PCV2b challenge by vaccination with inactivated or live-attenuated PCV1-2b vaccines.It has been shown that vaccinations are a major tool for reducing PCVAD losses in swine populations. There are currently several types of commercial vaccine products available worldwide and they differ in antigen. One vaccine is based on the inactivated PCV2a virus . Two subith PCV2 –27. HoweClinical signs and pathological lesions were not found in the vaccinated and mock groups, whereas two pigs in the challenged group were markedly depressed and had fever symptoms for 1 day (40.2 °C-41.4 °C). One pig in the inactivated PCV2b vaccine group died after routine blood collection on 7 days post vaccination (DPV) because of massive hemorrhages in the neck area. The average daily weight gain (ADWG) ranged from 0.30 to 0.40 kg/day during the growing period (3–7 weeks of age), with no significant differences among groups. The ADWG ranged from 0.51 to 0.69 kg/DPC (7–10 weeks of age), with no significant differences among groups  of the pigs in the inactivated and 2 × 104.0 TCID50 dose live-attenuated vaccine groups were seropositive, the IFA antibody titers were 0, 1:10, 1:10, 1:100, and 1:400 and 0, 1:10, 1:10, 1:10, and 1:400, respectively, whereas only 40 % (2 out of 5) of the pigs vaccinated with 2 × 103.5 TCID50 dose live-attenuated vaccine had seroconversion to PCV2; the IFA antibody titers were 0, 0, 0, 1:10, and 1: 400. The IFA antibody titers peaked at 35 DPV or 42 DPV and remained high on the day of necropsy in the pigs vaccinated with the 2 × 104.0 TCID50 dose and the 2 × 103.5 TCID50 dose; however, PCV2 antibody titers decreased from 35 DPV in pigs vaccinated with inactivated PCV1-2b. Regarding commercial inactivated PCV2b vaccine X, the PCV2 antibody levels were highest at 28 DPV (the mean IFA antibody titer was 1:5600) and then declined rapidly at 35 DPV. For the nonvaccinated challenged group, all five pigs seroconverted to PCV2-specific antibodies at 14 DPC (the IFA antibody titers ranged from 1:400 to 1:1600). There were no significant differences in the mean PCV2-specific antibodies between any of the vaccinated groups , and all five pigs in the vaccinated groups were seropositive against PCV2 by 21 DPV (the IFA antibody titers ranged from 1:400 to 1:6400). Pigs vaccinated with the inactivated or 2 × 104.0 TCID50 dose live-attenuated vaccines induced a higher level of NA titers than 2 × 103.5 TCID50 dose live-attenuated and commercial inactivated PCV2b vaccines during the course of the study, which corresponded to the PCV2 IFA antibody titers. The NA titers were not detected in pigs in the mock group throughout the experiment  when they were 25.60 ± 3.92, 28.80 ± 3.20, 28.80 ± 3.20, and 20.0 ± 4.00, respectively. In general, the inactivated and 2 × 10PCV2 DNA was not detected in any pigs before challenge or in pigs in the mock group at any sampling time point. The loads of PCV2 DNA amounts per milliliter of serum are summarized in Table 3.5 TCID50 dose live-attenuated PCV1-2b vaccine group, PCV2 virus DNA was detected in one of five pigs with a load of 101.56 PCV2 genomic copy numbers at 7 DPC. At 14 DPC, two of five pigs were found to have approximately 103.01 PCV2 genomic copy numbers, and no PCV2 genomic copy numbers were detected in any of five pigs at 21 DPC.In the 2 × 104.0 TCID50 dose live-attenuated PCV1-2b vaccine group, no pig had PCV2 genomic copy numbers after challenge, except one pig that harbored 101.33 PCV2 genomic copy numbers at 14 DPC.In the 2 × 101.11 PCV2 genomic copy numbers at 21 DPC.In the inactivated PCV1-2b vaccine group, no PCV2 genomic copy numbers were detected in the pigs after challenge, except for one pig that had approximately 101.42 to 101.74 at 7 to 21 DPC.For the commercial inactivated PCV2b vaccine control group, one out of four pigs had PCV2 genomic copy numbers that varied from 10P < 0.05).Figure 8.62 in group 1, 108.70 in group 2, 107.89 in group 3, 109.08 in group 4, and 1011.50 in group 5 , in groups 2 and 5 (P = 0.0019), in groups 3 and 5 (P = 0.0012), and in groups 4 and 5 (P = 0.0154).At necropsy (DPC 21), PCV2 genomic DNA was detected in the SILN in four of five pigs in group 1, in two of five pigs in group 3, and in all pigs in groups 2, 4, and 5. The mean PCV2 viral genomic copies/g loads in homogenized SILN in samples that tested positive were 104.0 TCID50 dose live-attenuated vaccine group had mild enlargement, whereas two pigs  in the commercial inactivated PCV2b vaccine X group and one pig in 2 × 103.5 TCID50 dose live-attenuated vaccine group had moderate swelling. The lymph nodes of all five pigs in the nonvaccinated challenged group were moderately to severely enlarged.No gross or microscopic lesions were observed in pigs in the mock group Fig. , g. At nIn vaccinated pigs, two pigs in the commercial inactivated PCV2b vaccine X group had lung lesions and only one pig in the inactivated or live-attenuated PCV1-2b vaccine group presented interstitial pneumonia; this was significant differences (one or two pigs versus four pigs) compared with nonvaccinated challenged pigs Table .Table 4D3.5 TCID50 dose live-attenuated vaccine group, commercial inactivated PCV2b vaccine X group, and in no pigs in the 2 × 104.0 TCID50 dose live-attenuated vaccine group, compared with five pigs that displayed LD and four of five pigs that displayed HR in the nonvaccinated challenged group (Table Lymphoid depletion (LD) of lymph node follicles was detected in one pig in the inactivated or live-attenuated PCV1-2b vaccine groups and in two pigs in the commercial inactivated PCV2b vaccine X group. Histiocytic replacement (HR) of lymph node follicles was found in one pig in the inactivated or 2 × 10up Table -l.3.5 TCID50 dose live-attenuated vaccine group, commercial inactivated PCV2b vaccine X group, and in no pigs in the 2 × 104.0 TCID50 dose live-attenuated PCV1-2b vaccine group, whereas LD or HR was detected in the tonsil follicles of three pigs in the nonvaccinated challenged group.LD of the tonsil follicles was observed in one pig in the inactivated or 2 × 103.5 TCID50 dose live-attenuated PCV1-2b vaccine group, commercial inactivated PCV2b vaccine X group, and in no pigs in the inactivated or 2 × 104.0 TCID50 dose live-attenuated vaccine group, but three of five pigs exhibited LD and two of five pigs exhibited HR effects.LD of the spleen follicles was found in one pig in the 2 × 10In general, the characteristic LD and HR effects were lower in vaccinated pigs compared with the nonvaccinated challenged pigs. Results regarding the presence of lesions in all tissues and organs tested are summarized in Table 4.0 TCID50 dose live-attenuated PCV1-2b  PCV1-2b vaccines in a wild-type PCV2b challenge model.Currently, all commercially available PCV2 vaccines are inactivated or subunit vaccines based on the PCV2a subtype , 20, 21.4.0, 2 × 105.0 and 2 × 106.0 TCID50 of inactivated PCV1-2b virus. The trial test results showed that vaccination with 2 × 105.0 TCID50 of inactivated PCV1-2b virus was more effective for PCV2 challenge, and this was confirmed by the data related to the level of antibodies against PCV2, neutralizing antibodies, viral loads in serum or lymph nodes and lesion in tissues. Corresponding to our previous study [The optimal vaccination protocol of the inactivated PCV1-2b vaccine was determined previously . Experimus study , all vacus study .In the current study, live-attenuated chimeric vaccine-induced clinical signs were not observed and the average daily weight gain was comparable with that of pigs in the mock group, which confirmed that the live vaccine was attenuated. All vaccinated pigs had seroconversion to PCV2 by 21 DPV and peaked at 42 DPV in group 1 (average at 1:9600), at 35 DPV in group 2 (average at 1:13120), at 35 DPV in group 3 (average at 1:15680), and at 28 DPV in group 4 (average at 1:5600) . The distribution of PCV2 antigens was described by IHC, PCV2 antigen was undetected in the 2 × 104.0 TCID50 dose live-attenuated vaccine group and detected only in one pig in the inactivated or 2 × 103.5 TCID50 dose live-attenuated PCV1-2b vaccine group, and the commercial inactivated PCV2b vaccine X group was positive for PCV2 antigen in lymphoid tissues. In contrast, all pigs tested were positive in the nonvaccinated challenged group. These findings are in agreement with a previous study [As expected, vaccination reduced PCV2 genomic copy number in serum, SILN samples and PCV2 antigen in tissues sections compared with nonvaccinated challenged pigs. PCV2 DNA levels peaked at 14 DPC and then declined. Vaccination with the 2 × 10lication , 36. Furlication –39. The us study  and implus study , 41.In this study, the observed PCV2-associated microscopic lesions were milder in the lung and lymphoid tissues in all vaccinated pigs compared with the nonvaccinated challenged pigs. In addition, the clinical signs of disease were not observed in the vaccinated pigs. We found that in nonvaccinated challenged pigs, LD and HR of follicles were associated with high PCV2 viral copy numbers and more serious histological lesions in lymph node, spleen, and tonsil which may eventually progress to clinical PMWS , 42. In In summary, the data from this study demonstrate that pigs vaccinated with inactivated or live-attenuated chimeric PCV1-2b vaccines were equally protected against PCV2 challenge, as evidenced by the lack of PCV2 viremia, significant reduction in viral loads in lymphoid tissues and significantly less lymphoid lesions in vaccinated pigs. This chimeric PCV1-2b virus may be a good candidate not only as an inactivated vaccine but also as a live-attenuated vaccine against PCV2 infection.PCV-free PK-15 cells used in this study were purchased from the China Institute of Veterinary Drug Control and grown in Dulbecco’s modified Eagle’s medium , supplemented with 4 % fetal bovine serum . The construction of the recombinant pBSK(+)-dPCV1-2b plasmid was reported previously . PCV2b/12 for 48 h. Cells were then harvested.Chimeric PCV1-2b live viruses were generated by electroporation of PK-15 cells as previously reported . BrieflyTo determine the infectivity titers of the chimeric PCV1-2b viruses, PK-15 cells were cultivated on 96-well plates, and then the cells were infected with a 10-fold serial dilutions of PCV1-2b. After 72 h of incubation, the virus titers were determined by an indirect immunofluorescence assay (IFA) , 43. Bri3.5 TCID50/ml and 104.0 TCID50/ml and were regarded as live-attenuated PCV1-2b vaccines. The inactivated chimeric PCV1-2b vaccine was developed using the following steps. Briefly, live chimeric viruses with a titer of 105.5 TCID50/ml were incubated with formalin at a final concentration of 0.1 %, kept at 4 °C for 12 h, and spun at 120 rpm at 37 °C for 12 h. Virus inactivation was confirmed by the inoculation of the formalin-treated samples into PK-15 cells and serially passaged for two generations, and then tested by IFA. Next, the inactivated PCV1-2b viruses were mixed with ISA 206 adjuvant  at a ratio of 46:54 (V/V), and the inactivated chimeric PCV1-2b viruses in the prepared vaccine reached 105.0 TCID50/ml.Then, the chimeric PCV1-2b viruses were diluted to 103.5 TCID50 and as 2 ml × 104.0 TCID50 (groups 1 and 2), respectively; inactivated PCV1-2b vaccine was given as 2 ml × 105.0 TCID50 (group 3). A commercial inactivated PCV2b vaccine named X, which is available on Chinese market, was given as a 2.0-ml dose as recommended by the manufacturer (group 4). Phosphate-buffered saline  was given as a 2.0-ml dose (groups 5 and 6). At 28 days post vaccination (DPV), pigs in all groups, except for the mock group, were challenged with 2 ml of wild-type PCV2b/1B/Jiangsu/Jingjiang/2012/11/08 at a titer of 104.8 TCID50/ml. After challenge, all pigs were monitored daily for clinical signs of diseases. Blood samples were collected at −1, 7, 14, 21, and 28 DPV and at 7, 14, and 21 DPC until necropsy.The experimental protocol was previously approved by the Animal Care and Use Committee of Yangzhou University . Twenty-seven 3-week-old conventional pigs were randomly divided into six groups, including four vaccination groups and two control groups Table . All pigPigs were weighed at 0 DPV and weekly thereafter until the day of necropsy. Rectal temperatures and clinical observations, including evidence of central nervous system disease, icterus, musculoskeletal disease, and changes in body condition, were recorded daily .50 PCV2 virus was added to PK-15 cells that were grown in 96-well plates. After 72 h incubation, the infected cells were fixed with methanol for 15 min and then incubated with the serum specimen diluted in PBS at 1:10, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12,800, and 1:25,600. After incubation at 37 °C for 1 h, the cells were washed with PBS and incubated with FITC-labeled rabbit anti-pig immunoglobulin G (Southern Biotech) at 37 °C for 45 min. The antibody titer was defined as the highest dilution time of serum under a fluorescence microscope .The serum samples were tested for the presence of PCV2 antibodies by IFA . Briefly50 PCV2 virus at an equal volume ratio and incubated for 1 h at 37 °C. The serum-virus mixture was added to confluent PK-15 cells growing in 96-well plates. After incubation at 37 °C for 72 h, a similar IFA protocol was performed. Serum NA titer was determined as the highest dilution at which there was 90 % or greater reduction in virus replication compared with the virus control.The titer of neutralizing antibody (NA) against PCV2 was assessed according to the fluorescence focus neutralization (FFN) assay , 44, 45,All serum samples at 7, 14, and 21 DPC and superficial inguinal lymph nodes (SILN) were tested for amount of PCV2 DNA. A pair of primers (5′-TGGCCCGCAGTATTCTGATT-3′ and 5′-CAGCTGGGACAGCAGTTGAG-3′), and a probe (5′-6FAM-CCAGCAATCAGACCCCGTTGGAATG-TAMRA-3′) were designed to target a highly conserved region in PCV2 ORF1 according to a previously reported method . Viral DAll pigs underwent necropsy and were evaluated by veterinary pathologists in a blinded fashion at 21 DPC. Sections of lung (five sections), heart, tonsil, thymus, liver, spleen, small intestine, colon, kidney, and lymph nodes  were collected, fixed in 10 % neutral buffered formalin, and routinely processed for histological examination.Immunohistochemistry (IHC) for detection of PCV2-specific antigen was performed on selected formalin-fixed and paraffin-embedded sections of lymph nodes, tonsil, spleen, and thymus tissues using a pig polyclonal antiserum (VMRD) . The amo10 viral copy with Graphpad Prism v5.0 software package  and expressed as means with standard deviations. The t tests (and nonparametric tests) were used to draw the graphs to display PCV2 viral copy loads in serum samples or SILN tissue and the dynamics of seroconversion to PCV2-specific IFA antibody.Statistical analysis was performed using SPSS software (version 13.0). Viral copy loads in serum samples or SILN tissue were transformed to values of log


During the last few decades more and more attention has been paid to triterpenes—a group of compounds with five- or four-ring skeleton and carboxyl, hydroxyl or oxo groups. Triterpenes with unsubstituted C-3 hydroxyl group can be easily transformed into appropriate ketones and then into oximes. The carbonyl group can be created not only from the hydroxyl group at C-3 position, but also at C-2, C-12 or C-28 positions. Several methods of creation of two = NOH groups within one molecule of triterpene are known. There are also known triterpenes with two carbonyl groups, e.g. at C-3 and C-11 positions, which differ in reactivity: among them only C-3 group can be transformed into oxime. A reactive hydroxyimine group can undergo the action of acylating agents, such as carboxylic acids or their derivatives, also the ones with significant pharmacological activity. Acyl derivatives of triterpenic oximes exhibit important pharmacological activity. The biological tests performed with the use of cell cultures inoculated with viruses showed inhibitory activity of some triterpenic acyloximes against type 1 HSV (H7N1), ECHO-6 and HIV-1 viruses. Another acylated oximes derived from triterpenes shown cytotoxic or antiproliferative activity against many lines of cancer cells. In many cases the pharmacological effects of the tested acyloxyiminotriterpenes were comparable to those of appropriate standard drugs. One of the newest application of acyl derivatives of triterpenic oximes is their ability to form organogels. Natural resources have been used for thousands of years in order to combat human diseases. Over the last decade the interest in natural products and their mechanisms of action has been intensified and now naturally occurring substances play an increasing role in drug discovery and development. Natural compounds are subjected to miscellaneous chemical transformations with the use of different chemicals in order to obtain numerous new derivatives. The semisynthetic compounds obtained as a result of such transformations often exhibit more advantageous pharmacological activity than mother compounds.Ganoderma lucidum  can be subjected to the reaction of esters synthesis (Scheme Hydroxyl group at C-3 position of oleanolic acid  can be ss Scheme . The acy1) or its methyl ester (2) can be oxidizied with Jones reagent . The above products are known as oleanonic acid (3) and methyl oleanonate (4), respectively. Formation of reactive C-3 carbonyl function activates the neighbouring methylene group (at C-2 position) so it is possible to obtain many new compounds.Oleanolic acid  as well as its methyl ester 4 form products with indole system: 7 and 8, respectively  or its methyl ester (4) refluxed with 2,4-dinitrophenylhydrazine in acetic acid afford the appropriate phenylhydrazones . Heating of oleanonic acid (3) or methyl oleanonate (4) with hydroxylamine hydrochloride affords the appropriate oximes . Oxime function can be next subjected e.g. to the reaction of reduction, acylation, cyclization to oxazole ring, Beckmann rearrangement.Oleanonic acid Zaprutko  to give Similar transformations can be also performed for lupanes  and glycyrrhetinic acid.O-acyloxyimino compounds are known only within lupanes and oleananes. Such type of compounds (with the acyloxyimino function) presents perspective pharmacological activities, such as anticancer, anti-HIV or anti-inflammatory.According to our knowledge newly obtained 13), one of the most often occured triterpene with lupane skeleton, was oxidizied with pyridinium chlorochromate (PCC) to betulonic aldehyde 14 , combined with a counterscreen for Huh-7 cell viability, was run in order to determine the tentative inhibitory potential of each compound. The results of these tests were expressed as surviving fractions  after exposure to each compound. Betulin (13) yielded 7 % virus SFV replication  was transformed into betulonic acid (17), methyl betulonate (18) and 28-oxo-allobetulon (19) with the use of known methods  with high yields. The compounds 20, 21 and 22 acylated in anhydrous benzene with carboxylic acid anhydrides  were tested for their antiviral activity with the use of cell cultures inoculated with type 1 HSV virus, A/FPV/Rostock/34 (H7N1) influenza virus and ECHO-6 enterovirus. The antiviral effect was judged by reduction of the viral titer in the presence of test compounds in comparison to control sample and characterized by the effective concentration causing a 50 % inhibition of the virus growth (EC50) and by the ratio of a maximum tolerable drug concentration (MTC) to EC50. The MTC values were determined upon 72-h incubation of noninfected cell cultures. As the result showed, the most active compound turned to be the oxime of betulonic acid (20). The EC50 values with respect to viruses: H7N1, HSV-1 and ECHO-6 for this compound were, respectively: 2.17, 81.56 and 4.19 μM  upon the action of Jones reagent (Scheme 17) with hydroxylamine hydrochloride in the presence of sodium acetate afforded the appropriate oxime 20. The obtained hydroxyimino derivative 20 was subjected to the action of acetyl chloride or p-toluenesulfonyl chloride in DMF to obtain, respectively, compound 23a known from the earlier presented publication and new product 23d.The further study concerning acylated oximes of betulinates was published in the same year by Mukherjee and et al. . Betulint Scheme  and then20, 23a and 23d as well as another compounds obtained in this work, were subjected to biological tests to determine their cytotoxic activity against 9 lines of cancer cells: MOLT-4, Jurkat E6.1, CEM.CM3 (human lymphoblastic leukemias), BRISTOL8 (human B cell lymphoma), U937 (human histiocytic lymphoma), DU145 (human prostate cancer), PA-1 (human ovary cancer), A549 and L132 (human lung cancers). The ED50 values (50 % effective dose) for oxime 20 varied from 0.7 μg/ml for PA-1 cell line to 2.6 μg/ml for CEM.CM3 (Table 23a were from 2.2 μg/ml (MOLT-4) to more that 10 μg/ml (A549) and for derivative 23d varied from 1.5 μg/ml (CEM.CM3) to >10 μg/ml (for A549 and L132 cell lines). The compounds 20 and 23d were more active towards the tested cancer cell lines than betulinic acid only for PA-1 cell line (ED50 for betulinic acid for PA-1 was >10 μg/ml); ED50 values for derivatives 20 and 23d and for PA-1 cell line were, respectively, 3.7 and 1.8 μg/ml.The obtained compounds 20 upon the action of carboxylic acid anhydrides in refluxed pyridine , with an EC50 of 1.04 μM and an efficacy of 83 %.The obtained compounds were evaluated for their TGR5 agonist potency expressed as EC25 obtained from betulin 13 and refluxed for 30 min in anhydrous toluene with equimolar amount of 5-phenyl-2,3-dihydrofuran-2,3-dione gave unexpected product 26  subjected to the action of 5-phenyl-2,3-dihydrofuran-2,3-dione after decarboxylation gave O-benzoylacetic acid derivative 26  or its methyl ester (2) were oxidizied with pyridinium chlorochromate and the resulted products (3 and 4) were subjected to the action of hydroxylamine hydrochloride in pyridine  were tested towards their activity as inhibitors of HIV-1 dimerization protease (HIV-1 PR). It was stated that the introduction of acyl function with six-membered chain into hydroxyimino group intensifies the inhibitory effect of the tested compound against the above mentioned enzyme  and CC0 value: 15.6 μg/ml (CC0—the minimum concentration for appearance of MT-4 cell toxicity by microscopic observation), but at the same time it is not active towards HIV-1 dimerization protease  were 5.5 and 4.0 μM, respectively, for compounds with AZT system (27a and 28a) were 1.9 and 16.0 μM, respectively, and for acylated oxime with FK-3000 system (28c) the IC50 value also exceeded 100 μM; it means that this compound in fact was inactive as HIV-1 PR inhibitor.The resulted triterpenic derivatives were tested towards their activity as HIV-1 PR inhibitors and HIV-1 RT inhibitors. The inhibitory activity against HIV-1 dimerization protease was evaluated by HPLC analysis of the cleavage products of a synthetic substrate. The ICμM Table , for oxi100 and CC0 values.The inhibitory activity against HIV-1 reverse transcriptase of the obtained conjugated compounds was tested with the use of MT-4 cells. The results were shown as IC27a and 28a turned to be practically inactive, with CC0 values 209.0 and 40.9 μM, respectively  were: 1.84 and 4.53 μM, respectively, the CC0 values were 145.0 and 120.0 μM, respectively. The triterpenic derivative with FK-3000 system (28c) was inactive, with CC0 value 31.0 μM.The derivatives 28c, both, against reverse transcriptase of HIV-1 and dimerization protease of HIV-1   only in the presence of an additional α-hydroxyl group at C-2 position (Scheme 33), after blocking of C-2 hydroxyl group, the appropriate oximes  were obtained. Next, the above compounds (34a and 34b) were transformed into acetyloxyimino derivatives  in the same conditions and by means of using of the same reagents as for methyl oleanolate (2)  or its methyl ester (2) were oxidizied with Jones reagent in acetone to the appropriate ketones 3 and 4, respectively. Next the oxoderivatives 3 and 4 were subjected to the action of n-butyl nitrite and as a result α-ketooximes 37 and 38, respectively, were obtained  and its methyl ester (2) into appropriate oximes  were obtained with the yields of 86–96 %.The received oximes 3 Scheme a were su11, 12, 42 and 43 as well as their derivatives obtained by acylation of the above oximes with heterocyclic carboxylic acids  were subjected to the biological tests in order to estimate their cytotoxic acitivity against selected cancer cell lines. The tests were conducted with the use of doxorubicin eluting beads (DEB) and the procedure according to NCI methods  to 2.02 × 10−6 for HL-60 TB (Table −6 (UO-31 cell line) and 5.36 × 10−6 (HL-60 TB) and the LC50 values were found from 2.62 (HL-60 TB) to 11.5 (HOP-92). The obtained results of the cytotoxicity of the oleanolic acid acylated oximes expressed in a form of the above parameters, pointed at potent antileukemic activity of the triterpenic oximes acylated with some carboxylic acids containing thiazolidone moiety. In molecules of the most potent antileukemic products thiazolidone skeleton was joined with carboxyl function via short carbon chain.The GI1) was transformed into its methyl ester oxime (12) in three-step set of reactions (Scheme 12) was acylated with octanoic acid in the presence of DCC in THF  in the dose of 2.0 g/kg. It was found that the acute administration of 48 did not cause any changes in skin and mucous appearance, cardiovascular, respiratory and central nervous system function and mortality during the next 14 days proving non toxic effects upon mice. In locomotor activity tests, horizontal and vertical locomotor activities of mice were evaluated using an activity meter which recorded spontaneous activity of animals in the time of 30 and 60 min after triterpene 48 administration in the dose of 3.0, 30.0, 100.0 and 300.0 mg/kg intragastrically (p.o.). It was found that compound 48 did not affect horizontal activity of mice in doses of 3.0, 30.0 and 100.0 mg/kg in both periods, but after administration of 300.0 mg/kg m.c. an increase in activity was observed. Moreover, similar effects in vertical activity were observed.For toxicity studies, the group of mice were administered triterpene 48 in the time of 30, 60, 90 and 120 min following the application. For comparative purposes, morphine in a dose of 5.0 mg/kg (s.c.) was administered to mice. It was observed that the compound 48 showed analgesic activity in the dose of 300.0 mg/kg and 60 min after administration. The combined treatment of triterpene 48 and morphine showed stronger analgesic activity in the doses of 30.0 and 300.0 mg/kg in comparison to morphine effect after 60 min.In analgesic activity test pain reflexes of mice in response to a thermal stimulus were measured in hot plate test after the same way of application and the same doses of triterpene 48 in range of 0.3, 3.0, 30.0 and 300.0 mg/kg were given intragastrically (p.o.) 30 min after carrageenan injection. For comparison, one group of rats was treated with the acute diclofenac  injected 60 min after carrageenan administration. The interaction between methyl 3-octanoyloxyiminoolean-12-en-28-oate (48)  and diclofenac was also tested. The rate of oedema of the two paws was measured at 1.5, 3.0, 6.0, and 10.0 h after carrageenan injection using a plethysmometer. The triterpene 48 caused a significant antioedemic effect in all doses after 1.5 and 3 h after carrageenan application in comparison to the control. The effect was also observed in three smallest doses after 6 h and in the dose of 3.0 mg/kg after 10 h from carrageenan application.In the next test, skin inflammation was induced in the right hindpaw of rats by the topical application of 2 mg/paw of carrageenan dissolved in 0.2 ml of 0.9 % saline solution. To the control, the rear left paw of the rats received the same volume of 0.9 % saline solution. Single doses of the compound 1) was transformed into its methyl or benzyl esters and next into the appropriate oximes 11 and 49, respectively  and exhibited the inhibition rate at 0.35 mM, respectively 37.2, 40.8, 34.2, 330 and 33.1 % (Table 1) applied in the same concentration.Among the compounds o et al.  only the50a–50j and 51a–51o Zhao et al. evaluated against Sclerotinia sclerotiorum (Lib.) de Bary, Phytophthora parasitica Dast, Botrytis cinerea Pers, Rhizoctonia solani Kuhn, Rice blast and Fusarium wilt using the mycelium growth rate test  to 74.4 % (for 51i), for P. parasitica was from 1.8 % (50a) to 67.6 % (50g) (Table B. cinerea ranged from 1.4 % (50i) to 69.1 % (51i), for R. solani from 5.5 % (50j) to 93.6 % (51c), for Rice blast waried from 21.0 %  to 85.5 % (51o) and for Fusarium wilt ranged from 5.1 % (50j) to 36.7 % (51c). In the same conditions oleanolic acid (1) exhibited the inhibition rate for this 6 fungi from 1.0 % (for B. cinerea) to 25.3 % (for R. solani).The inhibition rate for 2) was obtained and described also by Bednarczyk-Cwynar et al.  was acetylated with acetic anhydride in anhydrous pyridine according to literature protocol  in methylene chloride, based on literature data  as a reference compound and 12-ketone 52, 12-oxime 53 as well as acyloxyiminoderivatives 54a–54e were tested as to their cytotoxic activity against KB, MCF-7 and HeLa cell lines. Oleanolic acid (1) turned to be moderately active, with IC50 value for 14.93, 13.95 and 11.82 μM/l, respectively (Table 54d) was about 21-fold more active against the above mentioned cell line (IC50 of 0.72 μM/l) than oleanolic acid (1), and about 6.5-fold more active against MCF-7 and HeLa cells .Oleanolic acid  was oxidizied at C-3 position with the use of Jones reagent and next subjected to the action of hydroxylamine hydrochloride to give oxime 56  is a well known compound that inhibits growth and induces apoptosis of cancer cells. The antiproliferative and antiapoptotic effects for the above acid and its derivatives, e.g. acyloxyimino ones were determined with the use of human leukemia HL-60 cells.Glycyrrhetinic acid  used as a reference substance  with a high level of purity. This simple and quick manner involves small amounts of a solvent excellently soluble in water, that provides simple workup.


Escherichia coli and showed thermal instability. In this study, rational design was employed to enhance the solubility and the thermostablity of the bacterial laccase Lac15-His6 based on the crystal structure obtained previously. After deletion of His-tag and residues323–332, the obtained Lac15D was completely expressed in soluble form even at a higher temperature of 28°C, compared to only 50% of Lac15-His6 expressed solubly at 16°C. It showed a two-time higher activity at temperatures lower than 35°C and a half-life increasing from 72 min to 150 min at 45°C. When used in chromogenic reactions, Lac15D showed constant activity toward dye precursors and their combinations under alkaline conditions, demonstrating its application potential in hair coloring biotechnology.Bacterial laccases are ideal alternatives of fungal laccases for specific industrial applications due to specific characteristics such as alkalescence dependence and high chloride tolerance. However, some bacterial laccases presented as inclusion bodies when expressing in  However, a number of bottlenecks on the production of bacterial laccases exist. As E. coli6 in this study) showed an excellent tolerance to halogen ion and had the ability to decolorize several industrial dyes of reactive azo class under alkaline conditions. However, the soluble expression temperature of Lac15-His6 must be kept at 16°C, and only about 50% of the expressed protein was soluble under this condition. Moreover, the half-life of Lac15-His6 was only 72 min at 45°C Lac15 is a bacterial laccase screened from a marine metagenomic library and was successfully expressed in E. coli BL21 (DE3) was purchased from TransGen . The strain was cultured in Luria–Bertani (LB) liquid medium. Syringaldazine was purchased from Sigma-Aldrich . Isopropyl-β-D-thiogalactoside (IPTG) and Taq DNA polymerase were purchased from TaKaRa . All other chemicals and reagents were of analytical grade.6 and Lac15, respectively; lac15 complete sequence as the template (GenBank accession No. HM623889). A two-step PCR method was used to construct a deletion mutant of lac15 (lac15(-), the expression product is called Lac15D, lac15(-). Compared with Lac15, residues 323–332 were deleted from Lac15D. Three expression plasmids were constructed as described using the expression plasmid pET22b(+) E. coli BL21(DE3) to express Lac15 derivatives.Genes coding for two Lac15 derivatives (with or without histidine tag (His-Tag) at the C-terminal and called Lac15-HisE. coli BL21 (DE3) cells carrying recombinant plasmids were grown at 37°C in 200 mL LB medium containing 100 µg/mL ampicillin. IPTG at a final concentration of 0.2 mM was added into the culture of OD600  = 0.6. Immediately after adding of IPTG, the cultures were cultured at 16°C, 28°C, and 37°C for 10 h, 5 h, and 3 h, respectively. The cells were then collected by centrifugation. The pellets were resuspended in cold 20 mM Tris–HCl buffer (pH 8.0), disrupted by sonication, and then centrifuged at 30,000×g for 30 min.When inducing the expression of proteins, ???1). The purified protein was dialyzed into a 50 mM Na2HPO4–KH2PO4 buffer (pH 7.5) and stored at 4°C.The supernatants with soluble target proteins were applied to Hitrap Capto Q affinity chromatography  to purify laccase proteins according to the manufacturer's instructions. Briefly, the column was eluted with a linear gradient of NaCl  after PAGE using syringaldazine as the substrate. Protein concentration was determined using the BCA method .The supernatants and precipitations of each induction group, and the purified laccase proteins were applied to SDS-PAGE to monitor the soluble expression of each protein and the purity of the purified proteins following the common procedure. Proteins were stained with Coomassie brilliant blue R-250. The purified laccase proteins were also applied to native-PAGE to determine the purity of the purified proteins. Activity staining of laccases was performed in 50 mM Na2HPO4–KH2PO4 buffer (pH 7.5) containing 100 µM syringaldazine  and 100 µM CuSO4. The reaction was initiated by adding enzyme to the solution. After incubation at 45°C for 5 min, the mixture was transferred into an ice-water bath for 30 s to stop the reaction, and the absorbance was measured at 525 nm The assay mixture consisted of 10 µL of appropriately diluted laccase protein and 990 µL of 50 mM Na2HPO4–KH2PO4 buffer (5.5–8.0), and Tris–HCl buffer (8.0–9.0). The temperature effect on the activity was measured by incubating laccase proteins within the temperature range of 20°C to 55°C at the optimum pH for the substrate. The enzyme stabilities against temperature were determined by incubating laccase at 45°C in a 50 mM Na2HPO4-KH2PO4 buffer (pH 7.5). The residual activities were then determined as mentioned above.The pH effect on laccase activity was examined from pH 4.5 to 9.0 at 45°C in 50 mM sodium citrate buffer (4.5–5.5), Nap-phenylenediamine (PPD), o-phenylenediamine (OPD), o-aminophenol (OAP), 2,6-dimethylphenylalanine (DMP), and p-aminophenol (PAP) were dissolved in 10% DMSO solution (v/v). When inducing the reactions, the 100 mM Na2HPO4-KH2PO4 buffer ; 20 U/L Lac15D, and substrates were mixed in the 96 well plates. The total volume of the reaction mixture was 100 µL, whereas the final concentration of the substrate, buffer, CuSO4, and DMSO was 5 mM, 0.1 M, 100 µM, and 1% (v/v), respectively. Reaction mixtures were incubated at 40°C for 3 min, and then the oxidation was monitored spectrophotometrically according to Saito When conducting the chromogenic reactions, Azospirillum lipoferum, a few bacterial laccases have been cloned and recombinantly expressed in E. coli in recent years 6 was rather unstable, with only 50% of the initial activity retained after 72 min of incubation at 45°C Since the first report on bacterial laccase from 6 was resolved using X-ray crystallography method 6 (PDB No. 4F7K) adopts a typical fold of a bacterial laccase, which comprises three cupredoxin like domains formed by multiple-stranded Greek key β-barrel .Highly flexible regions represent an important part of the protein structure and have been found to be important in protein function, stability, and folding  at 45°C . The enh6 (data not shown). Notably, the fragment invisible in the crystal structure of Lac15 is missed in Lac15-D as well, which means the shortened loop is still significantly flexible. In addition, using the maximum emission wavelength of intrinsic Trp fluorescence as probe, we measured the thermodynamic stability of non-tagged Lac15 and Lac15-D against denaturant GdnCl, and the result showed that the two proteins are indistinguishably stable (data not shown). Thus, there must be some other mechanisms through which the flexible region contributes to the thermostability of Lac15. Its elucidation needs more in-depth investigation.However, our further investigation indicated that enhanced thermostability of Lac15-D might not be due to decreased flexibility in the shortened loop. We have resolved the crystal structures of non-tagged Lac15 and Lac15-D, both of which display little difference when compared with that of Lac15-HisLac15 also showed improved thermostability compared to Lac15-His6, showing that His-tag also affects the thermostability of Lac15-His6. As indicated above, the attachment of the His-tag at the end of C-terminus would interfere with the protein conformation. This scenario may decrease the interaction forces such as the hydrogen bond and hydrophobic effect between the residues in Lac15-His6, cause its sensitivity to temperature compared with Lac15, and finally cause physical inactivation at a shorter time.6 share similar catalytic properties such as the same optimal pH values  plasmid and the proteins expressed by these constructs. Lac15-His6, single His-tag fusion at the C-terminus. Lac15, protein without any His-Tag. Lac15D, residues of 323–332 were deleted from Lac15.(TIF)Click here for additional data file.Table S1Primers used in plasmids construction steps.(DOCX)Click here for additional data file.


The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis.c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6–7  and was also higher in non-survivors compared to survivors on day 3–4 and day 6–7 (p = 0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1–2 by 24±5% (p = 0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week.Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post-translational enhancement of mitochondrial respiration rather than augmented mitochondrial mass. Despite extensive research, sepsis continues to be a devastating disease with high mortality, primarily due to unresolving multiple organ failure (MOF) e.g. the regeneration of mitochondria within the cell. Mitochondrial biogenesis and increased mitochondrial respiratory capacity have been demonstrated in animal models and in humans in the later stages of the septic process Mitochondrial dysfunction has been implicated in the pathogenesis of sepsis Only 13 of the essential respiratory complex subunits are encoded by the mitochondrial DNA (mtDNA). Hence, mitochondrial biogenesis is dependent on protein synthesis derived from transcription and translation of both mitochondrial and nuclear DNA In sepsis, production of cytokines is greatly upregulated and released by cells from the innate and adaptive immune system to modulate the inflammatory response Cytokines, such as IL-1β and TNFα, and NO have been shown to activate PGC-1α by phosphorylation and a cGMP signaling pathway, respectively, and as such serve as a physiological stimuli of mitochondrial biogenesis Adult (>18 years) patients with severe sepsis or septic shock, as previously defined 2EDTA tubes . Patients were sampled three times during the first week at the ICU; within the first 48 h (day 1–2), on day 3–4 and day 6–7. Platelet preparation was commenced immediately after sampling. The vials were centrifuged at 300×g 15 min. The supernatant, platelet rich plasma, was collected and centrifuged at 4600×g for 5 min. The ensuing pellet was suspended in a small amount of plasma with a final platelet concentration of approximately 2000×109/L. Respirometric measurements were performed directly after isolation and within 5 hours of sampling. The analyzed contents from the respirometry chamber were stored frozen until further use.Blood were sampled from an arterial line in patients and by venous puncture in controls and collected in K2EDTA vials and platelets were isolated as described above. The platelets were then resuspended in either healthy donor plasma or plasma from septic patients.For incubation experiments, platelets were isolated from buffy coat collected from healthy donors the same day. The buffy coat was divided into Ki.e. the negative time derivative of oxygen concentration . Calibration with air-saturated Millipore water was performed daily. The oxygen concentration was calculated from the digitally recorded barometric pressure and the oxygen solubility at 37°C. The oxygen solubility factor relative to pure water was set to 0.92 for MiR05. Oxygen consumption was expressed as pmol/s/106 cells.Respiration was measured at 37°C in 2 ml glass chambers using a high-resolution oxygraph with online display of the calibrated oxygen concentration and oxygen flux, 6/ml were suspend in respiration medium consisting of 110 mM sucrose, 0.5 mM EGTA, 3.0 mM MgCl2, 80 mM KCl, 60 mM K-lactobionate, 10 mM H2PO4, 20 mM taurine, 20 mM HEPES and 1.0 g/l BSA, pH 7.1 (MiR05) 6 platelets). For complex I-dependent respiration (OXPHOSCI), substrates were pyruvate (5 mM) plus malate (5 mM) and glutamate (5 mM) which provide nicotinamide adenine dinucleotide (NADH) to the respiratory chain. Maximal OXPHOS is obtained by convergent electron input through both complex I and complex II (OXPHOSCI+II) and was determined by sequentially adding succinate (10 mM). State 4  was evaluated by adding oligomycin (1 µg/ml) and maximal capacity of the electron transport system (ETSCI+II) was further obtained by careful titration of the protonophore, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). For measurement of complex II-dependent respiration (ETSCII) complex I was subsequently inhibited by rotenone (2 µM). Electron flow through complex I to III was inhibited by addition of antimycin-A, and the residual oxygen consumption was subtracted from prior oxidative values. Complex IV-dependent respiration (CIV) was measured by adding N,N,N′,N′-tetramethyl-p-phenylendiamine . As TMPD exhibited a wide range of auto-oxidation in the sample preparation, respiration was finally inhibited with sodium azide (10 mM) and the difference between the oxygen consumption before and after the addition of sodium azide was determined as complex IV respiration. The coupling of phosphorylation to oxidation was determined by calculating control ratios for both maximal capacity of OXPHOS and ETS by dividing the respective rate with state 4 respiration. For experiments on intact cells, platelets were incubated in their own plasma and allowed to stabilize at routine level. Subsequent addition of oligomycin induced State 4 (LEAK) and FCCP was thereafter titrated until maximal respiration was achieved. Addition of rotenone and antimycin-A ended the experiment and residual oxygen consumption was subtracted from prior respiration values.Platelets at a concentration of 50–200×10Cytokines were analyzed with a multiplex sandwich immunoassay format with electrochemiluminescence according to manufacturer's instructions . In short, 96-well plates pre-coated with capture antibodies for TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 were incubated with plasma samples for 2 hours. Subsequently, detection antibodies were added and the plate incubated for another 2 hours. After washing, the plate was read with MSD Sector Imager. Since the variation of cytokine levels in the healthy controls was low the sampling was restricted to 12 randomly selected subjects.3) and nitrite (NO2) using the Griess reaction. A commercial kit was used with modifications adapted for plasma samples NO levels in plasma are short lived and were thus evaluated by its stable metabolites nitrate . Samples were analyzed in duplicates.Mitochondrial DNA (mtDNA) was measured as previously described c (Cyt c) content was quantified using an immunoassay kit . Frozen samples where thawed, sonicated and subsequently processed according to the manufacturer's instructions.Human cytochrome A commercially available kit , was used according to manufacturer's instructions, to determine citrate synthase (CS) activity in the frozen samples.Platelets were incubated in 1 ml of plasma from septic patients or healthy volunteers for 1 hour in room temperature before experiments. Mitochondrial respiration was then assessed with the protocol for intact cells described above.2EDTA tubes. IL-8 was added to a final concentration of 10 ng/ml and tubes were placed on a tilting board in room temperature for 24 h. Platelets were then isolated and analysed as described above.Blood from healthy volunteers was collected in Kt-test. Correlations were evaluated using linear regression. A p-value of <0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 5.04 software .Data were tested for normal distribution with D'Agostino and Pearson omnibus normality test. Parametric data are presented as mean ± SD and non-parametric data as median ± interquartile range (IQR). Differences between controls and patients at different time points and plasma incubation experiments were analyzed with ANOVA with post-analysis using Dunnett's Multiple Comparison Test for parametric data and Kruskal-Wallis test with post-analysis using Dunn's Multiple Comparison Test for non-parametric data. Differences between first and last time points in the patient cohort were analysed with paired Student n = 31 and n = 27 at day 6–7.Demographics of the 38 patients and 38 controls recruited are presented in CI 30±9.5 vs. 21±5.6, OXPHOSCI+II 46±11 vs. 33±7.7, LEAK 6.2±2.2 vs. 4.9±1. ETSCI+II 54±16 vs. 32±7.4 and ETSCII 20±4.0 vs. 15±3.3, CIV 65±19 vs. 37±11 and intact cells 24±9.6 vs. 16±5.1 pmol O2×s−1×10−8 platelets, p<0.001. Also, the respiratory control ratio (ETSCI+II/LEAK) was significantly increased in the septic patients compared to controls .In Of the 10 cytokines measured, plasma concentrations of all, except for INFγ, were significantly increased at the first sampled time point as compared to controls (IL1β 1.9 (0.8–3.7) vs. 0.45 (0.2–0.6), IL-4 1.5 (0.8–2.7) vs. 0.6 (0.3–1.5), IL-5 2.6 (0.9–6.2) vs. 0.9 (0.7–1.3), IL-6 228 (115–1351) vs. 1.7 (1.0–2.3), IL-8 40 (20–104) vs. 2.4 (1.9–3.5), IL-10 26 (11–69) vs. 1.5 (0.9–1.9), MCP-1 1457 (720–2954) vs. 256 (181–382) TNFα 23 (9.0–35) vs. 3.7 (2.7–4.3) pg/ml, p<0.001 except IL-5 p = 0.03). At the subsequent time points IL-6, IL-8, IL-10, IL-17, MCP-1 and TNFα remained significantly elevated compared to controls (30 (18–67), 23 (13–35), 7.7 (4.0–13), 16 (8.8–31), 688 (473–1217) and 7.8 (5.7–12) pg/ml respectively, p<0.01 except IL-17 and TNFα p<0.05) whereas IL-1β, IL-4, IL-5 returned to values similar to controls. The relative changes in cytokine levels as compared to controls are displayed in 3+NO2 levels were 14.5±3.2 µM in controls. In the septic patients NO2+NO3 levels were not significantly increased  within 48 h of admission to the ICU, and returned toward control values at the subsequent time points (24.5±6.0 µM at day 3–4 and 15.0±6.0 µM at day 6–7).Mean plasma NOCI+II and ETSCI+II . Of the analyzed cytokines TNFα, sampled at day 1–2, displayed a weak positive correlation with the patients' severity of disease as expressed by SAPS II scores  and APACHE II .Of the analyzed cytokines, IL-8 levels at day 6–7 correlated positively with both maximal ATP generating as well as maximal non-ATP generating rates of respiration i.e. OXPHOSCI+II and OXPHOSCI+II respiration at day 6–7 as compared to survivors . Among the different cytokines IL-8 demonstrated the same profile with higher values in non-survivors in samples taken at day 6–7 compared to survivors (35 (22–47) vs. 18 (13–27) pg/ml respectively, p = 0.025). None of the other cytokines differed between survivors and non-survivors.In agreement with our previous findings 2 = 0.38, p<0.001). Cyt c was reduced by 24±5% (p = 0.03) at day 1–2 as compared to controls and then returned to similar values as controls at the subsequent time points. Cyt c levels also correlated to CS activity, although weaker than mtDNA .MtDNA copy number and CS activity were similar as compared to controls at the different time points and did not change between the different time points analyzed and also correlated to each other . Routine respiratory rate and LEAK state were similar regardless of which plasma they were exposed to.As shown in Platelet mitochondrial respiration remained similar as compared to controls after 24 hours of incubating platelets with IL-8 at 10 ng/ml (data not shown).High-resolution respirometry performed in the O2k Oxygraph is a sensitive method of analyzing mitochondrial respiration. With the present experimental protocol we could assess whole cell mitochondrial function as well as the contribution of the individual complexes I, II, IV, V and the maximal function of the ETS with convergent electron input through both complex I+II. Accordingly this generates a comprehensive picture of the respiratory capacity of the mitochondria analysed. The evaluated mitochondrial respiratory function is also independent from the levels of markers of platelet activation since thrombin receptor agonist peptide-6 (TRAP) stimulation was not associated with an increase in respiration in our experimental setup (data not shown).The respiratory data on the 20 patients newly recruited for the present study were in agreement with those previously presented Increased plasma cytokine levels have been demonstrated in several studies of human sepsis and both individual cytokines as well as different combinations have been used as predictors for severity of disease and outcome 2+NO3 levels displayed a similar, however non-significant, trend being 171% higher compared to controls upon admission to the ICU with a subsequent decrease towards control values at later time points. The levels of NO2+NO3 measured in the present study were in the same range as those previously reported Plasma NOOf the analyzed cytokines, IL-8 differed significantly between survivors and non-survivors in the last two time points studied. In accordance with this, IL-8 has previously been analyzed in septic patients where the levels were found to be higher in non-survivors compared to survivors c levels compared to controls. Apart from a net increase in mitochondrial density, post-translational regulation of the OXPHOS system is increasingly recognized as an important modulator of respiratory capacity. Complexes of the ETS as well as a majority of the metabolic pathways in the mitochondria have been shown to be subjected to regulation by phosphorylation and dephosphorylation which, at least in part, seems to be controlled by intracellular signaling cascades triggered by extracellular receptor interactions in vivo effect . Secondly, platelets are anucleated cells and any translational change would have to take place in the megakaryocytes in the bone marrow. This together with a likely tissue diversity in mitochondrial function and regulation precludes drawing any firm conclusions regarding other tissues than the one examined. Peripheral blood immune cells have previously been investigated in septic patients In conclusion, we have demonstrated that in patients with severe sepsis and septic shock, plasma concentration of ten analyzed cytokines and NO, only IL-8 correlated with mitochondrial respiratory levels in the later phase of the disease. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied with an increase in markers of mitochondrial content, arguing for a post-translational regulation of mitochondrial respiratory capacity rather than augmented mitochondrial mass in this cell type.


In this study, we investigated the effects of H2 on human sperm motility in vitro.Sperm motility is a critical factor in male fertility. Low motility can be caused by a variety factors including abnormal spermatogenesis, oxidative damage, or depletion of intracellular ATP. Recent findings indicate that hydrogen molecule (H2, their forward motility was measured with a counting chamber. A time-lapse movie was recorded to analyze sperm swimming speed. Mitochondria were stained with a membrane potential-sensitive dye.Experimentally damaged sperm suspensions from patients left at room temperature for > 5 days or frozen immediately after ejaculation were used. After exposure with H2 treatment significantly improved the rate of forward motility, whereas treatment with nitrogen gas did not. While treatment for 30 min was sufficient to improve motility, it did not affect sperm swimming speed. After 24 h, retreatment with H2 increased the motility again. H2 treatment also increased mitochondrial membrane potential. Forward motility of low motile frozen-thawed sperm from patients significantly improved with cleavage medium containing H2.H2 treatment stimulates low sperm motility. H2 is a new promising tool for male infertility treatments.Our results illustrated that H Several factors are present in infertile males with sperm function defects caused by asthenozoospermia . Gene de10 on semen quality, and their effects in vitro .  10% PPF .2-treated, and N2-treated. During experiments, sperm suspensions were kept in approximately atmospheric O2 concentration to enhance oxidative damage. A sperm suspension of 100 μL on a culture dish was placed into an exposure chamber  with H2-mixed gases  or N2-mixed gases . After closing the exposure chamber tightly, these concentrations of mixed gases (1 L/min flow rate) were reached within approximately 5 min. To confirm saturation of the sperm suspensions with mixed gases, we monitored H2 and O2 concentrations with needle-type sensors . After exposure to mixed gases, each sperm suspension was mixed well by pipetting and a 5–10 μL drop was placed in a counting chamber . We then treated the sperm suspensions with mixed gases containing 50% H2 gas (H2-mixed gases) for 40 min and found that the forward motility rate increased significantly  . At that time, pH was almost equal between suspensions treated with N2- and H2-mixed gases (pH7.3 ± 0.1). Next, we examined the effect of treatment duration using other suspensions with relatively higher motility (n = 12). Both 30 and 60 min treatments with H2-mixed gas significantly improved sperm motility, whereas the 5 min treatment did not . After treatment with H2-mixed gas, we found that the forward motility in 16 suspensions significantly increased  and 3.6% (90% CI: 0.0-17.6%), respectively. Thus, H2 treatment effectively and significantly improved sperm motility in suspensions with a higher survival rate.The survival rate of sperm is different in each patient and affects the mobility . We seleentclass1pt{minima2 treatment on sperm swimming speed, we used time-lapse microscopy and analyzed images with sperm analysis computer software, CASA. Ten sperm suspensions, which were randomly selected from 17 suspensions with a higher survival rate used in Figure 2 treatment does not affect sperm swimming speed.To examine the effect of H2 is an important indicator of IVF and ICSI success. After treatment with H2- or N2-mixed gases for 30 min, 10 sperm suspensions were kept at 25°C in room air. After 2.5 h, we found that forward motility in H2 treated suspension was still higher than that before treatment  and kept it at 25°C in room air for 30 min, and found that sperm motility increased again, reaching a level approximately 67% of that of the first treatment.After the first H2 treatment might be due to changes in mitochondrial function. Therefore, after treatment with H2- or N2-mixed gases for 30 min by the same method described in Figures 2 treatment may enhance mitochondrial function and sperm motility.Since sperm motility is dependent on ATP content, we hypothesized that the improvement of sperm motility by H2, we prepared frozen-thawed sperm suspension from each patient, which could be used for infertility treatment. The average forward motility of total 21 suspensions was 37.1% (95%CI: 28.7-45.6). Asthenozoospermia was defined according to the WHO guideline as samples with < 50% progressive sperm motility [2-treatment were 61.3% (95% CI: 50.8-71.8%) and 27.5% (95% CI: 21.7-33.2%), respectively. All suspensions were treated with sperm-wash media containing different concentrations  of H2. Dunnett's t-test for unpaired values indicated that the motility in suspensions of lower motile, asthenozoospermic sperms significantly increased after treatment with sperm-wash media containing 50% of H2, whereas treatment with 75% and 100% of H2 did not  with higher survival rates in which an increase of forward motility was observed with Hpulation . The sam2 did not enhance the sperm swimming speed  and that of N2 (0.48 to 0.98%) was very low.We found that the higher sperm motility induced by H2 Figure , which iroom air . Neverth2 treatment enhanced mitochondrial membrane potential (Figure 2 treatment enhances mitochondrial function, promotes ATP production, and then stimulates sperm motility. Indeed, we observed previously that H2 prevented a decrease in cellular levels of ATP synthesized in mitochondria [2.A functional relationship among sperm mitochondrial membrane potential, sperm motility, and fertility potential has been proposed . We founl Figure . Since schondria . Precise2 treatment on the motility of damaged sperm. Frozen sperm suspension is used routinely in assisted reproduction treatment. However, freezing has been reported to cause changes in sperm morphology, including damage to mitochondria. Sperm motility is particularly sensitive to freezing damage [2 (Figure 2 treatment is clinically a potential approach to activate low motility sperm. However, treatment with medium containing higher concentration, 100%, of H2 was not effective, indicating the possibility that very low concentration of O2 in the medium might repress the sperm motility [2-treatment.Finally, we used frozen-thawed sperm suspensions to validate the effects of Hg damage ,35. Effe2 Figure b, indicamotility . FurtherThe fertilization rates after IVF and ICSI using normal sperm are approximately 60% and 70%, respectively . In gene2 treatment activates low motility sperms. Importantly, recent studies demonstrated that H2 might have potential for wide use in medical applications as a novel, safe, effective antioxidant with minimal side effects [2 is a new promising agent for male infertility treatment. However, to make practical use of H2 treatment, we further need to examine the effects of H2-treated sperm on fertilizing ability, embryonic development and safety for IVF and ICSI in future studies.The findings of this study strongly indicate that H effects -19. We p


Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein–coupled receptors (S1P1–5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis. Ltd. The S1P3 genotypes were determined by PCR analyses of genomic DNA isolated from tail biopsy specimens Animal care and experimental procedures were approved by the Kyoto Sangyo University Committee on Animal Welfare . S1P3Seven- to ten-week-old mice were anesthetized by ether inhalation and 30 µL of bleomycin hydrochloride  solution containing 2.15 U/kg of bleomycin dissolved in sterile saline Histological analysis was performed on formalin-fixed, paraffin-embedded lung tissue sections stained with H&E or by Masson's trichrome method. The sections were taken at 7 or 28 days after intratracheal administration of bleomycin or saline. The degree of fibrosis was quantified using the Ashcroft scoring method BALF analyses were performed on the seventh day after bleomycin administration. Immediately after the mice were sacrificed, the lungs and trachea were extracted en bloc, and a 20-gauge intravenous catheter was inserted into the trachea. A total of 800 µL of PBS was instilled three times and withdrawn from the lungs via an intratracheal cannula. More than 90% of the fluid was recovered as bronchoalveolar lavage fluid (BALF), which was then centrifuged at 1,000 rpm for 5 minutes at 4°C. The supernatants were collected and stored at −80°C for ELISA. For the analysis of total and differential white blood cell counts, after removing the supernatant, the resultant pellet was washed with PBS and resuspended in 700 µL of PBS. A 200 µL aliquot of the 700 µL BALF solution was diluted with 400 µL of Turk's solution and the total number of BALF cells was counted using a Fuchs-Rosenthal hemocytometer . The rest of the BALF solution was placed in a cytospin , centrifuged at 700 rpm for 10 minutes and stained with Diff-Quick  to obtain the differential white blood cell count. At least 200 cells per slide were evaluated on the basis of morphological criteria using a light microscope. The concentrations of TGF-β1, monocyte chemotactic protein-1 (MCP-1), connective tissue growth factor (CTGF), and S1P were measured with ELISA kits according to the manufacturer's instructions. ELISA kits for TGF-β1 and MCP-1 were provided by eBioscience , for CTGF by Uscn Life Science  and for S1P by Echelon Biosciences Inc. . Collagen content was estimated by collagen assay using a Sircol collagen assay kit .The data are presented as the mean ± SD and all sample sizes were ≥5. All values except S1P levels were analyzed by Welch's t-test. Differences in S1P levels in BALF between WT and S1P3 KO mice were analyzed by Mann-Whitney U test. Differences were considered significant at the P<0.05 level.To determine the biological significance of S1P3 deficiency after acute lung injury, we tracked changes in body weight after exposure to bleomycin. The average body weight at baseline did not differ significantly between WT (n = 9) and S1P3 KO mice (n = 8). After treatment with bleomycin, body weights in both groups decreased, but weight loss was significantly reduced in S1P3 KO mice . The bodTo determine the survival rate after administration of bleomycin, data from four independent experiments were combined. The survival rate of WT mice decreased to 61.8% (21/34) on the eleventh day and that of S1P3 KO mice to 87.1% (27/31). The survival rate of S1P3 KO mice after administration of bleomycin was significantly higher than that of WT mice  .Lung tissues from WT and S1P3 KO mice on the seventh and 28th day after treatment with bleomycin or saline were stained with H&E or Masson's trichrome staining to evaluate histological changes during the acute and chronic phases of lung injury. On the seventh day after treatment, inflammation was apparent with slight progression of fibrosis in lung tissues of WT mice, which showed infiltration of inflammatory cells, hyperplasia of the alveolar/bronchiolar epithelium, and interstitial collagen deposition in affected lesions. Lung tissues from S1P3 KO mice exhibited less inflammation than those from WT mice . On the When fibrosis was examined by Masson's trichrome staining of lung tissues, no significant differences in fibrosis on the seventh day after treatment were detected in either group . Howeverp = 0.12) (p = 0.0006) (No significant differences were recognized in the Ashcroft score of KO mice (2.72±0.30) and WT mice   on the s 0.0006) . In sali 0.0006)  was evidn = 5, (2.33±0.52) ×106 cells vs. (1.03±0.11) ×106 cells in WT vs. KO mice, respectively; p = 0.003) (5 cells (59.3±5.2%) vs (6.13±1.13) ×105 cells (59.1±3.8%), lymphocyte: (3.88±1.13) ×105 cells (16.9±3.8%) vs (1.44±0.76) ×105 cells (17.3±1.1%), neutrophil: (5.56±0.21) ×105 cells (23.8±4.7%) vs (2.41±0.21) ×105 cells (23.6±3.8%), n = 3; (1.74±0.13) ×105 cells vs. (1.31±0.21) ×105 cells in WT vs. KO, respectively; p = 0.02) (5 cells (91.4±4.6%) vs (1.19±0.22) ×105 cells (90.5±4.0%), lymphocyte: (0.05±0.008) ×105 cells (2.8±1.2%)vs (0.05±0.02) ×105 cells (3.6±1.7%), neutrophil: (0.10±0.03) ×105 cells (5.8±3.4%) vs (0.08±0.02) ×105 cells (5.9±0.8%); Figure5B). There were no differences of cell numbers between non-treated WT and KO mice  ×105 cells vs KO: (1.08±0.42) ×105 cells; p = 0.44, Figure5A).Analysis of BALF collected on the seventh day after treatment revealed a 56% reduction in total cell count in S1P3 KO mice compared with WT mice (= 0.003) . However = 0.02) . Like bln = 5; 683.7±187.0 µg/ml vs. 421.0±67.3 µg/ml in WT vs. KO, respectively; p = 0.02)  (S1P3 KO mice had significantly lower collagen levels in BALF compared with WT mice on the seventh day after bleomycin administration ( = 0.02) . The dif0.00015) . In salip<0.05) (p = 0.30) (p = 0.20) (n = 3) and S1P3 KO (n = 3) mice.The CTGF concentration in BALF on the seventh day after intratracheal administration of bleomycin was significantly decreased in S1P3 KO mice compared with WT mice  . However = 0.30)  or MCP-1 = 0.20)  the seveFinally, there were no significant differences between the S1P concentrations in BALF from WT and KO mice at baseline or on the seventh day  after blIn this study, we demonstrated that S1P3 deficiency has a significant effect on the pathogenesis of bleomycin-induced pulmonary inflammation and fibrosis. In general, the murine model of bleomycin-induced injury initially showed an acute inflammatory reaction when there was loss of body weight, followed by a fibrotic reaction. In this study, the loss of body weight in S1P3 KO mice was significantly attenuated, suggesting that the inflammatory response to bleomycin was reduced and that S1P3 deficiency was protective against the decline in physical health and feeding induced by bleomycin. During the acute phase of lung injury, H&E staining revealed less histological evidence of inflammation in the lungs of S1P3 KO mice than in those of WT mice, and analysis of BALF collected on the seventh day showed that S1P3 KO mice had a 56% reduction in total cell count compared with WT mice. These results are consistent with the observed changes in body weight.Sammani et al. showed that compared with control mice, S1P2 KO mice as well as mice with reduced S1P3 expression via silencing S1P3-containing nanocarriers were protected against LPS-induced barrier disruption in vitroKeul et al. reported that S1P is chemotactic for monocytes/macrophages via the S1P3 receptor, and bone marrow-derived S1P3-deficient macrophages produced less MCP-1 in response to LPS stimulation In saline-treated control mice, analysis of BALF collected on the seventh day revealed a 25% reduction in total cell count in S1P3 KO mice compared with WT mice, while there were no differences in cell numbers between non-treated WT and KO mice. It is possible that microaspiration occurred in saline-treated control mice, and that the differences in the total number of cells in BALF occurred due to inflammation caused by aspiration.S)-FTY720-phosphonate) reduced the degree of radiation-induced lung injury Idiopathic pulmonary fibrosis (IPF) is the most common interstitial fibrotic pulmonary disease; however, there is no effective treatment for preventing the development of fibrosis in IPF. Recent studies have implicated S1P, SphK and S1P receptors in human IPF. Huang et al. reported that the expression of SphK1 was increased in lung tissues from patients with IPF and bleomycin-challenged mice. They also showed that knockdown of SphK1 or treatment with an SphK inhibitor attenuated S1P generation and reduced mortality and pulmonary fibrosis in bleomycin-challenged mice The standard animal model for induction of experimental pulmonary fibrosis is the intratracheal bleomycin model. This model does not recapitulate the progressive and irreversible characteristics of human IPF; however, histological hallmarks, such as intra-alveolar buds, mural incorporation of collagen and obliteration of the alveolar space, are present in bleomycin-treated animals Although S1P signaling is implicated in lung fibrosis, its function is not completely understood. It may depend on the pathophysiology or time course of the disease, or it may depend on the concentration of S1P and the expression of receptor subtypes. Our results showed that S1P levels in BALF did not change between baseline and the seventh day after bleomycin challenge, regardless of whether S1P3 was knocked out. Differences between the conditions in the above reports and our study could account for these results, such as variations in SphK activity, different sample sources , or different disease time courses. Although previous reports have not examined the expression or distribution of S1P3 in the lungs of IPF patients or bleomycin-induced fibrosis models directly, our results showed that the degree of pulmonary fibrosis and the concentration of CTGF in BALF were dependent on the expression of S1P3, not differences in S1P levels in WT and S1P3 KO mice.in vitro, and CTGF mRNA expression was up-regulated in bleomycin-induced lung fibrosis in mice in vivoTGF-β is a known profibrotic protein and is considered a key player in the pathogenesis of fibrosis. It is synthesized by different cell types, such as monocytes, lymphocytes, or eosinophils, which are recruited to the site of injury or inflammation. It induces the transformation of fibroblasts into myofibroblasts, which are able to secrete TGF-β and stimulate extracellular matrix (ECM) deposition in vitro reports suggest that cross-talk occurs between S1P and TGF-β signaling. Xin et al. reported that S1P transactivated the TGF-β receptor and triggered activation of Smads followed by CTGF gene transcription in renal mesangial cells Several In this study, we clarified the importance of S1P3 receptor signaling on bleomycin-induced pulmonary inflammation and fibrosis. Our findings suggest that this signaling occurs via CTGF expression, resulting in the onset of pulmonary fibrosis. This pathway could be a therapeutic target for pulmonary diseases such as IPF or interstitial pneumonia, although further investigation is required to elucidate the role of S1P3 signaling in these diseases.Figure S1Survival rate after administration of bleomycin. The survival rate after administration of bleomycin. S1P3 knockout (KO) mice (n = 31) and wild-type (WT) mice (n = 34) received a single intratracheal dose of bleomycin (2.15 U/kg). Data from four independent experiments were combined; n = 4–10 mice/group in total. Survival rate of WT mice decreased to 61.8% (21/34) on the eleventh day and survival of S1P3 KO mice to 87.1% (27/31). The survival rate of S1P3 KO mice after administration of bleomycin was significantly higher than that of WT mice .(TIF)Click here for additional data file.


The lithium ion conductivity and salt diffusion coefficient of PEO18LiTFSI were dramatically enhanced by the addition of TEGDME. The water-stable lithium electrode with PEO18LiTFSI-2TEGDME, as the protective layer, exhibited a low and stable electrode resistance of 85 Ω·cm2 at 60 °C, after 28 days, and low overpotentials of 0.3 V for lithium plating and 0.4 V for lithium stripping at 4.0 mA·cm−2 and 60 °C. A Li/PEO18LiTFSI-2TEGDME/LTAP/saturated LiCl aqueous solution/Pt, air cell showed excellent cyclability up to 100 cycles at 2.0 mAh·cm−2.The electrochemical properties of a composite solid polymer electrolyte, consisting of poly(ethylene oxide) (PEO)-lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) and tetraethylene glycol dimethyl ether (TEGDME) was examined as a protective layer between lithium metal and a water-stable lithium ion-conducting glass ceramic of Li The aqueous lithium-air battery system, proposed by Imanishi and co-workers, is composed of a lithium metal electrode, a lithium ion conducting polymer electrolyte based on poly(ethylene oxide) (PEO)x-lithium bis(trifluoromethanesulfonyl)imide (LiTFSI), a water-stable lithium ion-conducting glass ceramics of Li1x+y+1x−AlxPy3−SiyO12 (LTAP), an aqueous electrolyte with saturated LiCl, and a carbon air electrode [−4 S·cm−1 at ambient temperature, and is stable in saturated LiOH and LiCl aqueous solution, but exhibits poor chemical stability towards lithium metal [Aqueous lithium-air batteries have been considered as a promising electrochemical energy storage system for electric vehicles. The theoretical energy density for the cell reaction of 4Li + 6Hum metal . The two18LiTFSI reduced the interface resistance and suppressed lithium dendrite formation [et al. [et al. reported that the addition of poly(ethylene glycol) dimethyl ether (PEGDME) into propylene carbonate (PC) with LiClO4 suppressed dendrite formation [et al. reported that the oxidative stability of glyme molecules is enhanced by complex formation with LiTFSI in a molar ratio of 1:1 [et al. [3SO3/carbon, O2 cell capable of operating over many cycles with capacity and rate values as high as 5000 mAh·g−1 and 0.5 A·g−1, respectively. We have previously reported an increase in the lithium ion transport number and decreases in the interface resistance of the lithium metal and polymer electrolyte by the addition of PEGDME to PEO18LiTFSI, where the mean molecular weight of PEGDME was 500 [et al. [4 decreased with a decrease in the molecular weight of PEGDME in the range of 90–400. In this study, the interface resistance between lithium and the polymer electrolyte, and the transport properties of the PEO18LiTFSI-xTEGDME (M = 222.28 g·mol−1) composite polymer electrolyte (CPE) are examined as a function of the amount of TEGDME (x). In addition, the electrochemical performance of a Li/PEO18LiTFSI-2TEGDME/LTAP/saturated LiCl aqueous solution/Pt, air cell is evaluated at 60 °C. In our previous studies ,7,8, we ormation . The add [et al.  reportedormation . Moreoveo of 1:1 . Recentl [et al.  demonstr was 500 . Naoi et [et al.  conclude18LiTFSI-xG4 (TEGDME abbreviated as G4 hereafter) were measured as a function of x (the molar ratio of G4 to ethylene oxide). 18LiTFSI-xG4. The conductivity increases significantly with increasing x up to 2 and there is little difference in conductivity between the samples with x = 2 and 3. The electrical conductivity of PEO18LiTFSI-2G4 at 25 °C is almost one order of magnitude higher and that at 60 °C is three times higher than that of PEO18LiTFSI. TEGDME loosens the coordination of lithium ions with EO units in the PEO matrix and thus enhances the mobility of ions, and could also enable lithium ions to decouple from ion pairs, as shown by Kriz et al. [18LiTFSI-xG4 at high temperature decrease with increasing x. That of PEO18LiTFSI-2G4 was calculated to be 25.3 kJ·mol−1, in the range of 55 to 80 °C, which is lower than that for PEO18LiTFSI (38.8 kJ·mol−1) and that for PEO18LiTFSI-2.0 N-methyl-N-propylpiperdinium bis(fluorosulfonyl) imide (PP13FSI) (33.8 kJ·mol−1) [The electrical conductivities of PEOz et al. . The actJ·mol−1) .18LiTFSI/LTAP/Pt, air cell at high current density, it is necessary to enhance the limiting current density of lithium-ion conducting PEO-based polymer electrolytes (Il), which is proportional to the salt diffusion coefficient and lithium ion transference number, as shown by Equation (1):Co is the initial concentration, e is the elemental charge, D is the salt diffusion coefficient, ta is the anion transport number, and L is the thickness of the electrolyte. The salt diffusion coefficient of PEO18LiTFSI-xG4 was estimated using the method proposed by Ma et al. [x. Typical curves for the natural logarithm of potential versus time for the Li/PEO18LiTFSI-xG4/Li cells at 60 °C are shown in D) of PEO18LiTFSI-xG4 was calculated from the slope of the linear curves using Equation (2):D are summarized in −7 cm2·s−1 was determined for x = 2.0, which is almost one order of magnitude higher than that for PEO18LiTFSI (3.60 × 10−8 cm2·s−1) and PEO18LiTFSI-1.44PP13FSI (2.25 × 10−8 cm2 s−1) [18LiTFSI-18 wt % PEGDME (8.38 × 10−8 cm−1 s−1) [The overpotentials between lithium metal and polymer electrolytes for lithium deposition and stripping generally increase with current density and undergo a sudden increase with the polarization period at a current density (limiting current density). When the ionic concentration in the vicinity of cathode drops to zero, the limiting current density is reached and dendrite growth is initiated (Sand time) . To opercm2 s−1) , and foum−1 s−1) . xG4 were measured using the method reported by Evans and Vincent [ta was calculated using the Evans and Vincent equation, and the results are summarized in i.e., decrease of the anion transport number) was observed by addition of G4 into PEO18LiTFSI. The lithium ion transport number for PEO18LiTFSI-2G4 is slightly higher than that for LiTFSI-2G4. Based on these results for D and ta, the limiting current density (Il) for PEO18LiTFSI-xG4 was calculated and the results are summarized in L) was 100 μm. PEO18LiTFSI-2G4 exhibits the highest Il of 15.8 mA·cm−2, which is higher than that for PEO18LiTFSI by a factor of approximately 15. The high limiting current density of PEO18LiTFSI-2G4 suggests that a WSLE with PEO18LiTFSI-2G4 could be operated at a high current density.Lithium ion transference numbers for PEO18LiTFSI- Vincent . Figure 18LiTFSI-xG4/Li cells at 60 °C as a function of storage time. PEO18LiTFSI-G4 and PEO18LiTFSI-1.5G4 show a significant increase of interface resistance with storage time; the initial cell resistance of 69 Ω·cm2 increased to 141 Ω·cm2 for PEO18LiTFSI-G4 after 28 days. However, low interface resistance was obtained for PEO18LiTFSI-2G4 and PEO18LiTFSI-3G4. After 28 days, PEO18LiTFSI-2G4 and PEO18LiTFSI-3G4 showed interface resistances of 34 and 74 Ω·cm2, respectively, which are much lower than 253 Ω·cm2 for PEO18LiTFSI after storage for 28 days [18LiTFSI-2G4 was unusual, where the interfacial resistance increased during the initial seven days and then decreased continuously to a value lower than the original. A similar change in the interface resistance was reported for the Li/PEGDME-LiTFSI/Li cell by Bernhard et al. [18LiTFSI-xG4/Li cells as a function of x. The activation energy for the inverse of the passivation film resistance (Rp) decreased from 76.2 kJ·mol−1 for PEO18LiTFSI to 54.7 kJ·mol−1 for PEO18LiTFSI-2G4. A decrease was also observed for PEO18LiTFSI-G4 and PEO18LiTFSI-3G4, but it was not as significant as that for PEO18LiTFSI-2G4. The activation energies for the inverse of the charge transfer resistance (Rc) decreased with increasing x. The lowest value of 63.4 kJ·mol−1 was calculated for PEO18LiTFSI-3G4, in comparison with 81.7 kJ·mol−1 for PEO18LiTFSI and 68.1 kJ·mol−1 for PEO18LiTFSI-2G4. These results suggest that the low-molecular weight oligomer ether G4 could reduce the resistance of the SEI and facilitate the charge transfer reaction at the interface. The former role is similar to other additives, such as nanofillers and ionic liquids, whereas the latter role is only performed by G4. Low and stable interface resistance between lithium metal and polymer electrolytes is essential for the electrochemical performance of WSLEs and to reduce lithium dendrite formation . Figure  28 days . The intd et al. , which cd et al. . These i18LiTFSI-2G4 motivated us to examine its role as the protective layer in a WSLE. The impedance of the Li/PEO18LiTFSI-2G4/LTAP/sat. LiCl aqueous solution/Pt, air cell was measured at 60 °C, using a platinized platinum reference electrode. 2 was obtained, which is comparable with those reported previously; the cell resistance for the Li/PEO18LiTFSI/LTAP/1 M LiCl/Pt, air cell was 539 Ω·cm2 [2 for the Li/PEO18LiTFSI-40 nm BaTiO3/LTAP/1 M LiCl aqueous solution/Pt, air cell [2 for the Li/PEO18LiTFSI-1.44 N-methyl-N-propylpiperdinium-bis(trifluromethanesulfonyl)imide/LTAP/1 M LiCl aqueous solution/Pt, air cell [Rb), the interfacial resistance between the polymer electrolyte and lithium metal electrode (Rf1), the interfacial resistance between the polymer electrolyte and the LTAP plate (Rf2), the charge-transfer resistance (Rc), and the Warburg impedance (W1) [Rb was quite stable at around 38 Ω·cm2 during the storage period. The fitting results suggest that Rf2 increased from 19.4 to 29.3 Ω·cm2, Rc decreased from 18.0 to 7.4 Ω·cm2, and Rf1 increased from 3.8 to 5.8 Ω·cm2 over the 28-day storage period.The high lithium ionic diffusion coefficient and low interface resistance with lithium metal for PEOair cell  and 130 nce (W1) . Rb was 18LiTFSI-2G4 and LTAP was investigated in an aqueous electrolyte using platinized platinum electrodes as the counter and reference electrodes. 18LiTFSI-2G4/LTAP/1 M LiCl-4 mM LiOH aqueous solution/Pt, air cell at current densities in the range of 0.5 to 4.0 mA·cm−2 at 60 °C. This WSLE exhibited quite low lithium plating and stripping overpotentials at high current densities up to 4.0 mA·cm−2. The lithium stripping and plating overpotentials at 1.5 mA·cm−2 were 0.10 and 0.15 V, respectively, which are lower than the best results obtained for the Li/PEO18LiTFSI-18 wt % PEGDME/LTAP/1 M LiCl-4 mM LiOH aqueous solution/Pt, air cell (0.29 V for plating and 0.21 V for stripping) in our previous studies [18LiTFSI-2G4.The electrochemical performance of a WSLE protected by PEO studies . The low18LiTFSI-2G4, a long period of lithium deposition at a constant current of 1 mA·cm−2 was performed using the Li/PEO18LiTFSI-2G4/LTAP/saturated LiCl aqueous solution/Pt, air cell, where the thickness of PEO18LiTFSI-2G4 was around 100 μm and the impedance of the WSLE was measured at every 4 h polarization. versus time curves as a function of the polarization period. The lithium electrode potential increased suddenly after a 25 h polarization. This potential increase may be due to lithium deposition on LTAP by lithium dendrite formation, which would result in the formation of a high resistance layer by the reaction of lithium and LTAP [2 by the short circuit of the lithium metal electrode with LTAP. The short circuit period of 25 h is approximately 2.6 times longer than that measured for a Li/PEO18LiTFSI/LTAP/10 M LiCl-4 mM LiOH aqueous solution/Pt, air cell [18LiTFSI-2G4/LTAP/saturated LiCl aqueous solution/Pt, air cell could be explained by the high lithium ion transport number, high salt diffusion coefficient, and low interface resistance [To study the lithium dendrite formation at the interface of Li and PEOair cell . The lonsistance ,23.18LiTFSI-2G4/LTAP/saturated LiCl aqueous solution/Pt, air cell for lithium deposition and stripping at a constant current density of 1.0 mA·cm−2 was measured at 60 °C, where the current was passed for 2 h. The cell voltage versus time profile is shown in 18LiTFSI-2G4. The cyclability of the Li/PEOv ≈ 600,000 g·mol−1) and LiTFSI  were dried overnight under vacuum at 50 and 160 °C, respectively, and then transferred to an Ar-filled glove box without exposure to air. G4  was immersed into activated 0.3 nm molecular sieves for 1 week and the same pretreatment by molecular sieves was performed 3 times before G4 was stored in a glove box. The composite polymer electrolytes of PEO18LiTFSI-xTEGDME in the range of x = 0–3 were prepared using a casting method [ca. 100 μm thick.PEO  in the frequency range from 0.1 Hz to 1 MHz at temperatures in the range of 25–80 °C. Z-plot software was employed for data analysis.ca. 250 μm thick) was supplied by Ohara Inc., Japan. The electrical conductivity of the LTAP plate at 60 °C was 1.4 × 10−3 S·cm−1. A Li/CPE/Li cell was assembled to investigate the interfacial resistance between lithium and the CPE. The active surface area was 1.00 cm2. The WSLE was prepared by sandwiching lithium metal foil (200 μm thick) with a Cu thin film lead, CPE, and the LTAP plate in a plastic envelope. The envelope was evacuated, heat-sealed apart from a 0.35 × 0.35 cm2 area, and then subjected to an isostatic pressure of 150 MPa for 15 min to ensure good contact. The WSLE was immersed into an aqueous solution of 1 M (or 10 M) LiCl with 4 mM LiOH. A platinized Pt electrode was used as both the counter and reference electrodes. Electrochemical measurements were conducted using a multichannel potentiostat-galvanostat (Biologic Science Instruments VMP3). The lithium ion transport number tLi+, was determined using the Li/CPE/Li cell with a combination of AC impedance spectroscopy and the DC polarization method, as originally proposed by Evans and Vincent, and later refined by Abraham and Jiang [et al. [The LTAP plate (nd Jiang ,25. The  [et al. . The Li/18LiTFSI-2G4 electrolyte exhibited a high lithium ion conductivity of 8.91 × 10−4 S·cm−1, a high diffusion coefficient of 3.37 × 10−7 cm2·S−1 at 60 °C, and a low interfacial resistance in contact with lithium metal. The Li/PEO18LiTFSI-2G4/LTAP/saturated LiCl aqueous solution/Pt, air cell exhibited excellent cyclability. The overpotentials for lithium deposition and stripping at 2.0 mAh·cm−2 for 2 h polarization at 60 °C were increased slightly from 0.10 to 0.15 V and from 0.08 to 0.13 V after 100 cycles, respectively. Therefore, the CPE is an attractive material for the interlayer between lithium metal and LTAP for the water stable lithium electrode in lithium-air batteries.The PEO