antibolic 演示文稿
罗贝洛兰模板1 优质课件
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蓝色扁平化学术答辩ppt模板
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毕业设计第二次汇报，小清新，XXXXXXX大学XX学院 21
4.最后的最后是接下来的安排 计划一
小清新准备在3月前完成《毕业设计完全攻略》，也就是10套不 同专业的毕设免费模板，现在一直在做我自己的专业的，因为感觉大家 对毕设答辩什么的不太重视，想要别的专业的来看看，效果很差。
计划二
1.这页照片墙就别放文字了，靠嘴吹就可以了
毕业设计第二次汇报，小清新，XXXXXXX大学XX学院 06
1.在此输入一些酷炫的文字
不要问我为什么图 这么多，其实你也 可以这么多图，没 这么多图也没办法， 谁让你要用这套模 板呢，你有没有发 现我实际上在凑字 数，好了版面够了。
毕业设计第二次汇报，小清新，XXXXXXX大学XX学院 07
这里是放置方案一存在的
不足，跟右边对比显得方案一 比较挫。
这里是放置新方案的优点
的，显得比原来的方案要碉堡， 不然你怎么证明你这个月是干 活了呢。
毕业设计第二次汇报，小清新，XXXXXXX大学XX学院 12
03
PART THREE
在此输入一些酷炫的文字
Lorem ipsum dolor sit amet
酷炫碉堡方案
KuXuan DiaoBao Scheme
小清新 XXXXXXX大学 XX学院
Duan childe,School of Aeronautics, Northwestern Polytechnical university.
CONTENT
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在此输入一些酷炫的文字
Lorem ipsum dolor sit amet
1.在此输入一些酷炫的文字
我的毕设要用到轮子
Kn2-7 Antibacterial Peptide
Antibacterial Activity and Mechanism of a Scorpion Venom Peptide Derivative In Vitro and In VivoLuyang Cao.,Chao Dai.,Zhongjie Li,Zheng Fan,Yu Song,Yingliang Wu,Zhijian Cao*,Wenxin Li* State Key Laboratory of Virology,College of Life Sciences,Wuhan University,Wuhan,P.R.ChinaAbstractBmKn2is an antimicrobial peptide(AMP)characterized from the venom of scorpion Mesobuthus martensii Karsch by our group.In this study,Kn2-7was derived from BmKn2to improve the antibacterial activity and decrease hemolytic activity.Kn2-7showed increased inhibitory activity against both Gram-positive bacteria and Gram-negative bacteria.Moreover,Kn2-7exhibited higher antibacterial activity against clinical antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus(MRSA).In addition,the topical use of Kn2-7effectively protected the skin of mice from infection in an S.aureus mouse skin infection model.Kn2-7exerted its antibacterial activity via a bactericidal mechanism.Kn2-7killed S.aureus and E.coli rapidly by binding to the lipoteichoic acid(LTA)in the S.aureus cell wall and the lipopolysaccharides(LPS)in the E.coli cell wall,respectively.Finally,the hemolytic activity of Kn2-7was significantly decreased,compared to the wild-type peptide BmKn2.Taken together,the Kn2-7peptide can be developed as a topical therapeutic agent for treating bacterial infections.Citation:Cao L,Dai C,Li Z,Fan Z,Song Y,et al.(2012)Antibacterial Activity and Mechanism of a Scorpion Venom Peptide Derivative In Vitro and In Vivo.PLoS ONE7(7):e40135.doi:10.1371/journal.pone.0040135Editor:Paulo Lee Ho,Instituto Butantan,BrazilReceived April24,2012;Accepted June4,2012;Published July5,2012Copyright:ß2012Cao et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Funding:This work was supported by grants from Natural Science Foundation of China(NSFC)(No.31071942),the China Specific Project for Developing New Drugs(Nos.2009ZX09103-612and2011ZX09102-001-32),and the National Basic Research Program of China(Nos.2010CB529800and2010CB530100).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.Competing Interests:The authors have declared that no competing interests exist.*E-mail:zjcao@(ZC);liwxlab@(WL).These authors contributed equally to this work.IntroductionDrug resistance poses an increasing threat to global public health,and new antibiotic-resistant pathogens have continued to emerge[1,2].Methicillin-resistant Staphylococcus aureus(MRSA)is considered one of the most threatening pathogens due to the high mortality rate and increased medical costs associated with treating it.[3,4].New types of antimicrobial agents are urgently needed to respond to the threat of pathogens that evolve resistance against conventional antibiotics[5].AMPs are distributed among a wide range of species,including insects,plants,humans,and even single-celled organisms[6].The structures of AMPs generally present highly amphiphilic topolo-gies,in which hydrophilic and hydrophobic side chains are located on opposite faces of the molecule[7,8].These peptides are potent antimicrobial agents against bacteria,fungi,viruses,and parasites, and several AMPs have been reported to inhibit the growth of MRSA[9,10,11].Their broad spectrum activity and low potential to induce resistance make AMPs an attractive family of compounds with the potential to be developed as therapeutics agents.During the last decade,several antimicrobial peptides have been investigated as therapeutic agents[12,13].Scorpion venom contains a diversity of bioactive peptides that represent a tremendous resource for use in drug design and development[14,15].Moreover,several AMPs have been found in scorpion venom,including hadrurin[16],scorpine[17],opisto-porins,parabutoporin[18],ISCTs[19],mucroporin[9]and StCT1[20].These scorpion venom peptides commonly exhibit cytolysis or microbial inhibition functions.In a previous study by our group,we characterized an antimicrobial peptide,BmKn2,derived from the venom of the scorpion Mesobuthus martensii Karsch[21].In the present study,a derivative of BmKn2,was designed to increase the antibacterial activity and reduce the hemolytic activity.Kn2-7showed increased inhibitory activity against both Gram-positive bacteria and Gram-negative bacteria in contrast to BmKn2.Meanwhile, Kn2-7could inhibit HIV-1by direct interaction with viral particle [22].Moreover,the hemolytic activity of Kn2-7was significantly lower than that of BmKn2.In addition,Kn2-7exhibited higher antibacterial activity against clinical antibiotic-resistant strains. Topical application of Kn2-7effectively protected skin from infection in an S.aureus mouse skin infection model.Finally,the antibacterial mechanism of Kn2-7was clarified in this study. ResultsAntimicrobial Screening of BmKn2DerivativesSeven BmKn2-derived peptides were designed based on the amino acid sequence of BmKn2(Figure1A).The secondary structures of BmKn2and its mutants were predicted to be100% a-helical in nature.The helical wheels of BmKn2and its mutants were divided into two parts,and the hydrophobic face and the hydrophilic face were labeled(Figure1B).These peptides were used to determine the inhibitory activities against the representative Gram-positive bacteria S.aureus AB94004and the representative Gram-negative bacteria E.coli AB94012.The antimicrobial functional screening results showed局部的杀菌两亲性拓扑结构细胞溶解局部的that the Kn2-7mutant exhibited the strongest inhibitory activity (Figure 1C).The peptide Kn2-7had little hemolytic activity at high concentration (Figure 1D).According to the method of Ka ¨ber modified by Achmarine,the HC 50values of the mutant peptide Kn2-7and the wild-type peptide BmKn2were 90.27m g/mL and 17.13m g/mL,respectively (Figure 1D).Therefore,the hemolytic activity of Kn2-7was significantly decreased,compared to the wild-type peptide BmKn2.In Vitro Antibacterial Activity of Kn2-7In view of the in vitro antibacterial activity of Kn2-7,we selected it as the object of further study.Kn2-7not only exhibited enhanced inhibitory activity against both Gram-positive bacteria and Gram-negative bacteria in contrast to wild-type BmKn2(Table 1).The antibiotic-resistant pathogens used in this study were clinical isolates,all of which were tested with corresponding antibiotics to verify their resistance prior to conducting experi-ments.As shown in Table 2,Kn2-7was more effective against the antibiotic-resistant pathogens than the wild-type BmKn2.Kn2-7was not only able to inhibit the growth of standard strains,but it was also able to effectively inhibit the growth of clinically isolated antibiotic-resistant strains.As shown in Figure 2,at a concentration of MICs,Kn2-7and BmKn2both inhibited the growth of S.aureus completely.Additionally,Kn2-7andBmKn2Figure 1.Screening,antimicrobial and hemolytic activities of Kn2-7in vitro .(A)Multiple alignments of Kn2-7and its seven derivatives.(B)Predicted secondary structure of Kn2-7and its seven derivatives.(C)Antibacterial activity screening of Kn2-7and its seven derivative peptides.(D)Hemolytic activity of Kn2-7.The hemolytic activities of the Kn2-7and BmKn2peptides were estimated by monitoring the increase in the absorbance at 570nm after incubating human red blood cells with different peptide concentrations at 37u C for 1h.The positive control was 0.1%Triton X-100,and 0.85%saline was used as a blank.doi:10.1371/journal.pone.0040135.g001Table 1.MICs of Kn2-7against Gram-positive and Gram-negative strains.StrainsMIC (m g/mL)Kn2-7BmKn2Gram-positive bacteria S.aureus AB94004 3.136.25S.aureus ATCC 25923 3.13 6.25B.subtilis AB91021 6.2512.5B.thuringiensis AB92037 6.2512.5M.luteus AB93113 6.256.25Gram-negative bacteria E.coli AB94012 6.25.100E.coli ATCC 2592225.100P.aeruginosa AB930662550P.aeruginosa A09299450.100P.aeruginosa A093052100.100P.aeruginosa A09305650.100P.aeruginosa A09308550.100P.aeruginosa A093115100.100doi:10.1371/journal.pone.0040135.t001inhibited the growth of MRSA P1386completely.At a concen-tration of6.25m g/mL,the mutant peptide Kn2-7and kanamycin both inhibited the growth of E.coli AB94012,while the wild-type BmKn2peptide had no effect against E.coli AB94012.In Vivo Antibacterial Activity of Kn2-7To determine the in vivo antibacterial activity of Kn2-7,an S. aureus mouse skin infection model was established.After infection, the mice were observed daily.As shown in Figure3A,one day after infection,the skin of the mice in the group treated with Kn2-7had become scabbed over,while the wounded skin sections of the group treated with placebo were wet and presented with blistering.Additionally,blisters and light tissue fluid leakage could be observed in the wounded skin sections of the group that was not treated.Four days after infection,the skin sections of the group treated with Kn2-7were healed,and their scabs were desqua-mated.However,the blisters in the skin sections in the group treated with placebo became larger.Furthermore,the skin sections exhibited a great amount of tissue fluid leakage in the group without treatment.A group of mice was euthanized4hours after infection,and their skin viable counts were measured as a control.Other groups were euthanized4days after infection.As shown in Figure3B,the colony counts in mice treated with the peptide Kn2-7were clearly lower than those of the placebo-treated mice and the mice that were untreated.The histological morphology of the skin sections was observed. As shown in Figure3C,most of the epidermis of the scratched skin sections was defective,while other parts of the skin were normal compared to normal skin with intact structure.Four days after the skin of the mice was scratched,the epidermis recovered,and the biopsy specimens were observed to be normal.Four days after infection,the untreated group and the group treated with placebo lost nearly all of their epidermis,and the dermis was infected to a certain extent.However,the skin sections treated with Kn2-7 recovered almost fully and presented nearly intact structures.Antibacterial MechanismSecondary structure analysis.As shown in Figure4A and Figure4B,Kn2-7and the wild-type peptide BmKn2displayed typical a-helical profiles in the structure-promoting solution TFE (30%and70%).However,both Kn2-7and BmKn2displayed random coil profiles in water.Table2.MICs of Kn2-7against clinical isolated antibiotic-resistant bacterial strains.Strains MIC(m g/mL)Kn2-7BmKn2Vancomycin Penicillin CefotaximePenicillin resistantP1383 6.2512.5 6.2510000 6.25P1389 6.2512.5 6.2510000 3.13Methicillin resistantP1374 3.1312.5 6.255000400P1369 3.1312.5 6.2520000400P1381 6.2512.5 3.135000400P1386 6.2512.5 3.1310000100Penicillin sensitiveP1111 3.13 6.25 3.1325 6.25All strains were clinical isolates obtained from the302nd Military Hospital,Beijing,China.All data are from experiments that were repeated at least threetimes with an MIC of no greater than2fold.doi:10.1371/journal.pone.0040135.t002Figure2.Gowth inhibitory activities of Kn2-7.(A)Growth curvesof S.aureus AB94004treated with Kn2-7,BmKn2or antibiotics.(B)Growth curves of MRSA P1386treated with Kn2-7,BmKn2or antibiotics.(C)Growth curves of E.coli AB94012treated with Kn2-7,BmKn2orantibiotics.doi:10.1371/journal.pone.0040135.g002Enzyme release assay of Kn2-7.The enzymatic activities ofthe supernatants were measured via an enzyme release assay immediately after treatment with Kn2-7or BmKn2.As shown in Figure 4C,the supernatant from S.aureus AB94004treated with ampicillin exhibited increasing catalase activity over time.However,the supernatant treated with Kn2-7or BmKn2showed almost the same catalase activity at each time point.As shown in Figure 4D,the supernatant from E.coli AB94012treated with kanamycin presented increasing catalase activity over time.However,the supernatant treated with Kn2-7showed nearly the same high enzymatic activity at each time point.In contrast,the supernatant treated with the wild-type peptide BmKn2showed low catalase activity at each time point and did not exhibit increasing catalase activity over time.Bactericidal effect of Kn2-7.Kn2-7exerted bactericidal effects on both S.aureus AB94004and E.coli AB94012.As shown in Figure 5A and Figure 5B,when the peptide concentration increased,the killing rate also increased.Additionally,Kn2-7wasFigure 3.In vivo antibacterial activity of Kn2-7.(A)Mice were observed after being treated with the peptide on days 1and 4.(B)Cutaneous viable counts in treated mice.Eight mice per group were euthanized,and the viable counts of the surviving S.aureus bacteria were then determined.(C)Histological morphologies of the skin in treated mice.(a)Normal dorsal skin of mice.(b)Immediately after the skin was scratched.(c)Four days after the skin was scratched.(d)Four days after S.aureus infection in skin treated with Kn2-7.(e)Four days after S.aureus infection in skin treated with BmKn2.(f)Four days after S.aureus infection in skin treated with a placebo.(g)Four days after S.aureus infection in untreated skin.Numbered arrows indicate the following:1,corneum;2,epidermis;3,dermis;and 4,muscular layer.doi:10.1371/journal.pone.0040135.g003Figure4.Secondary structure and enzyme release assay of Kn2-7.(A)and(B)Secondary structure analysis of the Kn2-7and BmKn2peptides.(A)Circular dichroism spectra of0.1mg/mL of Kn2-7in water,30%TFE/H2O or70%TFE/H2O.(B)Circular dichroism spectra of0.1mg/mL of BmKn2in water,30%TFE/H2O or70%TFE/H2O.(C)and(D)Enzyme release assay.(C)S.aureus AB94004treated with Kn2-7or BmKn2was harvested,and the catalase activities of the supernatants were measured;0.9%saline was used as the negative control,and ampicillin sodium was used as the antibiotic control.(D)E.coli AB94012treated with Kn2-7or BmKn2was harvested,and the catalase activities in the supernatants were measured;0.9%saline was used as the negative control,and kanamycin was used as the antibiotic control.doi:10.1371/journal.pone.0040135.g004able to kill E.coli AB94012rapidly,whereas the wild-type peptide BmKn2was not able to kill E.coli AB94012(Figure 5C).Transmission electron microscopy.Semi-thin sections of S.aureus AB94004and E.coli AB94012treated with Kn2-7or BmKn2were prepared to observe the structural changes in the cell walls and membranes of the bacteria (Figure 6).Cracks were observed in the cell walls of the bacteria.Additionally,membrane disruption was observed in the peptide-treated bacteria.Further-more,semi-thin sections of bacteria treated with Kn2-7or BmKn2showed that there was a release of cell content upon cell wall disruption.As shown in Figure 5B,BmKn2had no effect against E.coli AB94012.However,Kn2-7accumulated on the surface of E.coli AB94012,combined with the cell wall and membrane,and then formed microspheres that surrounded the bacteria.Biolayer interferometry.BLI was performed to identify the target of Kn2-7in S.aureus and E.coli .LTA is the main component of the peptidoglycan layer surrounding Gram-positive bacteria,while LPS is the main component of the cell wall surrounding Gram-negative bacteria.As shown in Figure 7A and Figure 7B,curve B3and curve E3indicated that the designed peptide Kn2-7bound strongly to LTA,as did the wild-type peptide BmKn2.Meanwhile,curve A3and curve D3indicated that only the Kn2-7mutant was able to bind to LPS,while wild-type BmKn2could not.LTA confer a portion of the negative charge associated with the peptidoglycans Gram-positive bacteria,and LPS confer a portion of the negative charge associated with the outer membrane surrounding Gram-negative bacteria,respectively.LTA and LPS competition binding assay.To confirm the results of the BLI titrations,250m g/mL of Kn2-7or BmKn2was mixed with an equal volume of 1mg/mL LTA or LPS.Then,the MICs of Kn2-7or BmKn2mixed with LTA against S.aureus and E.coli were measured.As shown in Figure 7C,the MICs of Kn2-7and BmKn2against S.aureus AB94004were 3.13and 6.25m g/mL,respectively.The MIC of the Kn2-7against E.coli AB94012was 6.25m g/mL.LTA and LPS exhibit no inhibitory activity against S.aureus or E.coli .The MICs of Kn2-7and BmKn2treatedFigure 5.Bactericidal assays of the Kn2-7and BmKn2peptides against S.aureus and E.coli AB94012.Set point indicates untreated bacteria,and 0minutes was defined as the time of the first sample collection,which was immediately after mixing bacteria and Kn2-7or BmKn2.The other samples were collected at 5min,15min,30min or 60min.All of the counts were the average of three dishes,and the experiment was repeated at least three times.(A)Time-killing curve of Kn2-7against S.aureus AB94004.(B)Time-killing curve of BmKn2against S.aureus AB94004.(C)Time-killing curve of Kn2-7against E.coli AB94012.doi:10.1371/journal.pone.0040135.g005Figure6.Transmission electron microscopy of S.aureus and E.coli treated with Kn2-7.(A)Transmission electron microscopy was performed with Kn2-7-treated S.aureus.(A1),(A2)and(A3)were negative controls.(A4),(A5)and(A6)show S.aureus treated with Kn2-7for2min. (A7),(A8)and(A9)show S.aureus treated with BmKn2for2min.(B)Transmission electron microscopy was performed with Kn2-7-treated E.coli.(B1), (B2)and(B3)were the negative controls.(B4),(B5)and(B6)show E.coli treated with Kn2-7for2min.(B7),(B8)and(B9)show E.coli treated with BmKn2for2min.doi:10.1371/journal.pone.0040135.g006Figure7.Binding assay of Kn2-7and LTA or LPS.(A)Interaction of10m g/ml of Kn2-7with500m g/ml of LPS or500m g/mL of LTA.Curve A3is LPS,curve B3is LTA and curve C3is buffer.(B)Interaction of10m g/mL of BmKn2with500m g/mL of LPS or500m g/mL of LTA.Curve D3is LPS,curve E3is LTA and curve F3is the buffer.(C)MICs of Kn2-7and BmKn2treated with LTA or LPS against S.aureus and E.coli.doi:10.1371/journal.pone.0040135.g007with LTA against S.aureus were25and12.5m g/mL,respectively. The MIC of Kn2-7treated with LTA against E.coli AB94012was at least25m g/mL.Meanwhile,the MICs of the Kn2-7and BmKn2treated with LPS against S.aureus were12.5and6.25m g/ mL,respectively,and the MIC of the Kn2-7mutant treated with LPS against E.coli was at least25m g/mL.All of these results further confirmed that both the Kn2-7and BmKn2peptides bind to LTA,but only the mutant Kn2-7peptide was able to bind to LPS.DiscussionThe structure-function relationship of some antimicrobial peptides has been analyzed to unravel the regular pattern of antibacterial activity,hemolytic activity and toxicity[23].How-ever,the regular patterns of antibacterial activity,hemolytic activity and toxicity have still need to be clarified.More recently, molecular design of antimicrobial peptides has become an important and attractive strategy for developing new antimicrobial drugs.In this study,six BmKn2-derived peptides were designed with the aim of increasing the proportion of polar residues at first. But the activity of the six deletion mutants did not improve.So Kn2-7peptide was designed to increase the net positive charge of BmKn2to improve its affinity for bacteria.The antibacterial activities of Kn2-7were better than those of BmKn2.Moreover, Kn2-7showed higher antibacterial activity than the other antimicrobial peptides identified from scorpion venoms to date [9,11,17,18,19,20].Furthermore,the hemolytic activity of Kn2-7 reduced observably compared to the wild-type peptide BmKn2. High hemolytic activity is always an obstacle to the application of AMPs.Increasing the net positive charge of AMPs may be a good way to improve the high hemolytic activity.Topical application of Kn2-7cured the skin of mice infected with pared with conventional antibiotics,Kn2-7has many advantages,such as broad-spectrum activities,rapid killing ability,low levels of induced resistance,and broad anti-inflammatory activities.Kn2-7 may have the therapeutic potential for topical use.New AMPs have been continuously isolated and identified. Several bilayer interaction and disruption models have been proposed for those AMPs that depend on membrane interference for their antibacterial activity,such as‘‘barrel-stave pore’’,‘‘carpet mechanism’’,‘‘toroidal pore’’and‘‘disordered toroidal pore’’[24]. New molecular models has been proposed to describe the mechanism more accurately,such as‘‘form ion channels’’[25] and‘‘internalization into the cytoplasm’’[26].In the present study,the results of secondary structure analysis,bactericidal assays,enzyme release assays provided solid evidence that the inhibitory effect of Kn2-7on the bacteria was mediated through rapid killing.However,the binding mode of Kn2-7to the surface of E.coli seems different from that of S.aureus.The results of biolayer interferometry and competition binding assay proved to be so.The findings of TEM indicated that the bacterial cell wall of S.aureus was disrupted immediately upon treatment with Kn2-7. Kn2-7accumulated on the surface of E.coli,combined with the outer membrane,and then formed microspheres surrounded the bacteria.This mode is consistent with the model of carpet mechanism in general.To the best of our knowledge,the structures of the microspheres were first observed in the antibacterial mechanism study and our results provide conclusive evidence for the model of carpet mechanism.AMPs have both a cationic and amphiphilic nature.Their positive net charge ensures that AMPs can accumulate at the surface of bacteria that contain anionic polymers,such as LTA and LPS.Then,these peptides interact with the membranes of the microbes,resulting in membrane disruption[27].The results of BLI titrations and competition binding assays showed that Kn2-7 disrupted the cell walls of S.aureus and E.coli through binding to LTA and LPS,respectively.The question of why the mutant peptide Kn2-7can bind to both LTA and LPS while the wild-type peptide BmKn2can bind to only LTA is of particular interest. Kn2-7has five net positive charges while BmKn2only has two. Therefore we speculate that increasing the numbers of positively charged residues turned out to have improved the affinity to the bacterial surface.In conclusion,we described both in vitro and in vivo antimicrobial activities of Kn2-7,a scorpion venom peptide derivative,against Gram-positive bacteria and Gram-negative bacteria including clinical isolates,MRSA,MRCNS,PRSA and PRSE.The antimicrobial and hemolytic activities of Kn2-7peptide were notably improved compared to the wild-type peptide BmKn2.The binding mode of Kn2-7to the surface of E.coli was different from that of S.aureus.The peptide Kn2-7disrupted the cell surface structure of S.aureus and E.coli via binding to LTA and LPS, respectively.Taken together,these data strongly suggest that Kn2-7peptide can be developed as a topical therapeutic agent for treating bacterial infections.Materials and MethodsStrainsS.aureus AB94004,S.aureus ATCC25923,E.coli AB94012,E. coli ATCC25922,Pseudomonas aeruginosa AB93066,Bacillus thuringiensis AB92037,Bacillus subtilis AB91021,and Micrococcus luteus AB93113were purchased from the China Center of Type Culture Collection(CCTCC).Clinical isolated Pseudomonas aeruginosa strains A092994, A093052,A093056,A093085and A093115were obtained from Hubei Maternal and Child Health Hospital.Clinical isolates were obtained from the302nd military hospital of Beijing,China,including penicillin-resistant S.aureus(PRSA) P1383,penicillin-resistant S.epidermidis(PRSE)P1389,methicillin-resistant S.aureus(MRSA)P1381,P1386and P1374,methicillin-resistant coagulase-negative Staphylococcus(MRCNS)P1369,and penicillin-sensitive S.epidermidis(PSSE)P1111.BmKn2Derivatives and Chemical SynthesisSix deleted mutants and one point mutant were designed with the aim of enhancing the net positive charge of the hydrophilic side of the peptide while maintaining the amphipathic character of the peptides.BmKn2and the seven derivatives of this peptide noted above were synthesized with amidated C-termini by More Biotech(Wuhan)Ltd.,China.Hemolytic ActivityThe hemolytic activities of Kn2-7and BmKn2peptides were tested against human red blood cells[9].HC50values were obtained according to the method of Ka¨ber,modified by Achmarine[28].Saline solution at a concentration of0.85% was used as the negative control,and0.1%Triton X-100was used as the positive control.Antimicrobial Assays in VitroMinimum inhibitory concentration(MIC)analysis.The MIC was determined via a microdilution assay in96-well microtiter plates according to the broth microdilution guideline of Clinical and Laboratory Standards Institute(CLSI)[29]. Growth inhibitory assay.The time growth curves of S. aureus and MRSA treated with BmKn2or its derivatives were微稀释法determined based on measurements taken at630nm at each time point.Different concentrations of peptide were added to the tested strains,which were cultured in96-well plates using the same method as was used for determining the MIC.Antimicrobial assays in vivoExperimental animals.Female BALB/c mice weighing18–20g were purchased from the Hubei Research Center of Laboratory Animals.All animal studies were approved by the Institutional Animal Care and Use Committee of Wuhan University.Gel preparation.Kn2-7or BmKn2was prepared in gel form in0.5%hydroxypropylcellulose to a final concentration of 5mg/mL.Mouse skin abrasion and infection model.Mice were injected with150mg/kg of cyclophosphamide4days prior to infection.Subsequently,the mice were injected with100mg/kg of cyclophosphamide1day prior to infection.After being anesthe-tized,the dorsal skin of the mice was shaved,and abrasions were produced in a161cm2area using a blade.These abrasion wounds damaged only the stratum corneum and upper layer of the epidermis,but not the dermis.Five minutes later,the wounds were inoculated with20m l of S.aureus(107–108CFUs)using a micropipettor.A group of mice was killed4h after infection to control for the infectious dose.Four hours after infection,the wounds of the other mice were smeared with20m l of5mg/mL gel or a placebo gel,which was used as a negative control.The treatment lasted for4days,with two peptide gel applications being performed daily(morning and evening at8h intervals).After4 days of treatment,the mice were killed,and the wound area of the skin was immediately excised and homogenized.Suitable dilutions of the homogenates were plated on LB plates to determine the number of living bacteria(CFUs).Histological examination.After the animals were eutha-nized,biopsy specimens of the excised wound area of the skin were collected and immediately fixed in phosphate-buffered formalin (10%,pH=7.4).Then,the biopsy specimens were embedded in paraffin and stained with hematoxylin and eosin.Statistical analysis.In each experiment,the mean CFU was calculated using log10-transformed data.Based on the averages of three experiments,the mean,range,and coefficient of variation were calculated.Antibacterial MechanismSecondary structure analysis.The secondary structures of Kn2-7and BmKn2were analyzed using Heliquest(http:// rs.fr/cgi-bin/ComputParams.py)and circular dichroism(CD)assays.The CD assay was performed at room temperature in nitrogen-flushed cells using a2-mm path with a Jasco J-810spectropolarimeter.Kn2-7and BmKn2were measured at concentrations of0.1mg/mL in water,30%TFE/ H2O or70%TFE/H2O.The peptide configuration was obtained by analyzing the data with CD deconvolution software.Enzyme release assay.Bacterial suspensions and serial-diluted peptides were mixed at a ratio of4:1in different tubes with a final volume of2ml.The mixtures were then incubated at37u C with continuous shaking.At each time point,200m l of the treated bacterial suspension was transferred to sterilized tubes.After centrifugation,the supernatants were transferred to a sterilized 1.5-ml tube,and catalase activity was measured with a Catalase Assay Kit(Beyotime,Jiangsu,China)using the method for measuring peroxidase activity.Bactericidal assay.Bactericidal curves for Kn2-7and BmKn2were determined against S.aureus AB94004and E.coli AB94012.A400m l aliquot of the peptide solution was added to 1,600m l of a bacterial suspension.At each time point,200m l of the treated bacterial suspension was transferred to a sterilized1.5-ml tube.After centrifugation at1,0006g for5min,the superna-tant was removed,and the pellet was resuspended in200m l of LB medium.The bacterial suspensions were placed on agar plates and incubated at37u C until viable colonies could be counted. Transmission electron microscopy.Overnight-cultured S. aureus AB94004and E.coli AB94012were transferred to LB medium and cultured to the exponential phase.A200m l aliquot of the peptide solution was added to800m l of the bacterial suspensions at the final concentration of16MIC.The samples were cut into semi-thin sections.Microscopy was performed using a HITACHI H-8100Transmission Electron Microscope. Biolayer Interferometry.Biolayer Interferometry(BLI) experiments were performed on OCT RED(Forte´Bio,Inc.).A total of500m g/mL of lipoteichoic acid from S.aureus(LTA, Sigma,L2515),lipopolysaccharides from E.coli(LPS,Sigma, L2880)or buffer(10mM HEPES,pH7.4and0.14M NaCl)was dispensed into96-well microtiter plates at a volume of200m l per well.The operating temperature was30u C.Biosensor tips (Forte´Bio,Inc.,Menlo Park,CA)were pre-wetted with the buffer for60seconds at baseline.Then,10m g/mL Biotin-Kn2-7or Biotin-BmKn2was loaded onto the biosensor tips for900seconds. Baseline2was subsequently established for300seconds. Dissociation of Kn2-7or BmKn2occurred for900seconds in the buffer.Data were generated automatically by the Octet H User Software(version3.1)and were subsequently analyzed from the resultant text files using Excel2000.LTA and LPS competition binding assay.A total of 250m g/mL of each peptide solution was mixed with an equal volume of1mg/mL of the LTA or LPS solution.Then,the MICs of BmKn2and Kn2-7treated with LTA or LPS against S.aureus AB94004and E.coli AB94012were measured.Author ContributionsConceived and designed the experiments:LC ZC CD.Performed the experiments:LC CD ZL ZF YS.Analyzed the data:LC ZC YW WL. Contributed reagents/materials/analysis tools:LC ZC YW WL.Wrote the paper:LC ZC.References1.Allen HK,Donato J,Wang HH,Cloud-Hansen KA,Davies J,et al.(2010)Callof the wild:antibiotic resistance genes in natural environments.Nat Rev Microbiol8:251–259.2.Andersson DI,Hughes D(2010)Antibiotic resistance and its cost:is it possible toreverse resistance?Nat Rev Microbiol8:260–271.3.Hebert C,Weber SG(2011)Common approaches to the control of multidrug-resistant organisms other than methicillin-resistant Staphylococcus aureus (MRSA).Infect Dis Clin North Am25:181–200.4.Ippolito G,Leone S,Lauria FN,Nicastri E,Wenzel RP(2010)Methicillin-resistant Staphylococcus aureus:the superbug.Int J Infect Dis14Suppl4:S7–11.5.Brogden NK,Brogden KA(2011)Will new generations of modifiedantimicrobial peptides improve their potential as pharmaceuticals?Int J Antimicrob Agents38:217–225.6.Maroti G,Kereszt A,Kondorosi E,Mergaert P(2011)Natural roles ofantimicrobial peptides in microbes,plants and animals.Res Microbiol162:363–374.7.Godballe T,Nilsson LL,Petersen PD,Jenssen H(2011)Antimicrobial beta-peptides and alpha-peptoids.Chem Biol Drug Des77:107–116.8.Zasloff M(2002)Antimicrobial peptides of multicellular organisms.Nature415:389–395.环磷酰胺角质层接种过的成为均匀分布的微粒的组织学实验分光偏振器酶释放实验过氧化氢酶重悬生物膜干扰。
保护知识产权宣传教育(1)
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1-简洁美观幻灯片样式 2-恰当使用图表和数据展示 3-注重版式和排版规范 4-保持一致性和连贯性
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PART 04
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PART 03
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Antibiotics(7)
History
• Although for centuries preparations derived from living matter were applied to wounds to destroy infection, the fact that a microorganism is capable of destroying one of another species was not established until the latter half of the 19th cent. when Pasteur noted the antagonistic effect of other bacteria on the anthrax organism and pointed out that this action might be put to therapeutic use.
History
• Meanwhile the German chemist Paul Ehrlich developed the idea of selective toxicity: that certain chemicals that would be toxic to some organisms, e.g., infectious bacteria, could be killed but not other organisms, e.g., humans.
Antibiotics today
The antibiotics we take today are still produded by bacteria
Many are now chemically synthesized
第六章_商品质量监督与认证
2009年中国消费者协会消费维权十大举 措
§ 举措一：确定并开展“消费与发展”年主题 宣传活动，着力提升消费信心，促进经济平 稳较快发展
§ 举措二：加强消费教育，提升消费者自身的
素质，帮助消费者更加科学、合理的消费， 促进消费升级(《科学消费指引》， 《消费教 育大纲》)
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种104个高档样品酒进行检验，结果只有一种4个样
品为真酒，其他全是假酒。
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第六章_商品质量监督与认证
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§ 假冒伪劣产品严重诋毁了我国在国际上的商业 信誉。在山东烟台、青岛港外，外国货轮入港 时竟打出“当心山东水泥”、“小心中国水泥” 的大幅标语。在一些经济发达国家，“中国货” 一度成了质量低劣的代名词。俄罗斯的不少大 商场一度也以高悬“本商场无中国货”为荣， 证明自己的商品货真价实，东欧国家的一些商 团老板甚至因此拒绝与我国发生贸易往来。
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§ 加入WTO给质量技术监督带来更大的发展机遇：
§ (1)我国的农业、电信业、金融保险业、汽车制造业、商业和 服务性行业面临新的竞争环境和严峻挑战，为质量技术监督注入 新的活力，使之进入快速发展时期；
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抗生素的生产
概述 抗生素的分类 抗生素的应用 半合成抗生素 青霉素的生产和提取工艺
抗生素的生产
概述
定义：是生物在其生命过程中产生的，并在低浓度下 有选择性地抑制或杀灭其它微生物或肿瘤细胞的有机 物质。Microbial Products,effective at low 物质。 concentrations,which have the ability to kill or inhibit the growth of certain micro-organisms. History: 19世纪70年代，法国Pasteur,发现有些微生物可以抑制 炭疽杆菌，提出了利用微生物抑制另一种微生物的现 象来治疗一些感染性疾病。1928年，
主要抗生素的使用量（ 主要抗生素的使用量（Harvey, 1998)
抗生素种类 用量 (kg/year)
动物1996 动物1996
人类 1997/1998 314,498 47,500 5,409 47,696
头孢类 四环类 氨基糖类 大环内酯类
121,603 323,151 37,058 71,222
氨基糖苷类抗生素：包括链霉素、 氨基糖苷类抗生素：包括链霉素、庆大霉素含有氨基糖苷类或 氨基环醇 四环类抗生素：四环素类、土霉素以四个苯为母核。 四环类抗生素：四环素类、土霉素以四个苯为母核。 多肽类抗生素：多粘菌素、杆菌肽由产孢子杆菌产生。 多肽类抗生素：多粘菌素、杆菌肽由产孢子杆菌产生。 蒽环类抗生素：阿霉素、柔红霉素属于抗癌类抗生素。 蒽环类抗生素：阿霉素、柔红霉素属于抗癌类抗生素。 喹诺酮类抗生素，如环丙沙星、诺氟沙星。 喹诺酮类抗生素，如环丙沙星、诺氟沙星。
produced to treat 7 million per year.
Alexander Fleming
Discoverer of penicillin Born into a farming family in Scotland, moved to London at 13 Awarded Scholarship to study medicine
一个链霉菌效价单位为能在1ml肉汤培养基中完全抑制大肠杆菌 标准菌株所需的最小链霉菌素计量。 除了用效价单位之外，当物质被制成纯净的化学物质时，就可 以用质量表示，而且与效价单位之间有一定的换算关系 如1mg青霉素钠盐相当于1667个单位 1 mg链霉菌素碱相当于1000个单位
Industrial evolution of penicillin production
Zone of Inhibition
This picture illustrates the effect of penicillin on a fungus. Note the clear ring of inhibition. It was this observation which initiated Fleming’s further studies.
Common antibiotics and their sources
Bacitracin Cephalosporin(s)（头孢 Cephalosporin(s)（头孢 菌素） Chloramphenicol（氯霉 Chloramphenicol（氯霉 素） Cycloheximide（放线菌 Cycloheximide（放线菌 酮） Hygromycin Penicillin Streptomycin Tetracycline(s) Vancomycin Bacillus subtilis Cephalosporium sp. sp. S. venezuelae S. griseus S. hygromyces P. chrysogenum S. griseus S. aurofaciens S. orientalis
抗生素的分类
根据抗生素的来源分
放线菌产生的，占目前发现的抗生素的2/3。真菌产生的，主要包括青霉 放线菌产生的，占目前发现的抗生素的2/3。真菌产生的， 菌和头孢菌素。细菌产生的，多粘杆菌，枯草杆菌， 菌和头孢菌素。细菌产生的，多粘杆菌，枯草杆菌，芽孢杆菌产生的 粘多菌素。动植物产生的，蒜中的蒜素， 粘多菌素。动植物产生的，蒜中的蒜素，动物脏器中的鱼素 （ekmolin).
按照作用机制分类
抑制细胞壁合成的抗生素 影响细胞膜功能的抗生素 抑制病原菌蛋白质合成的抗生素， 抑制病原菌蛋白质合成的抗生素，如四环素 抑制核酸合成的抗生素如丝裂霉素C 抑制核酸合成的抗生素如丝裂霉素 抑制生物功能作用的抗生素， 抑制生物功能作用的抗生素，
根据抗生素的生物合成途径分类
氨基酸、肽类衍生物，如青霉素、 氨基酸、肽类衍生物，如青霉素、头孢霉素等寡肽抗生素 糖类衍生物， 糖类衍生物，如链霉素等糖苷类抗生素 以乙酸、丙酸为单位的衍生物， 如红霉素等丙酸衍。 以乙酸、丙酸为单位的衍生物， 如红霉素等丙酸衍。生物
根据作用谱分: 根据作用谱分:
广谱抗生素，如氨苄青霉素，可抑制G-&G+细菌 广谱抗生素，如氨苄青霉素，可抑制G &G+细菌 G+菌的抗生素 如青霉素。 菌的抗生素： 抗G+菌的抗生素：如青霉素。 菌的抗生素： 抗G-菌的抗生素：如链霉素 抗真菌的抗生素： 抗真菌的抗生素：如制霉菌素 抗病毒的抗生素：如四环类抗生素对立克次体积较大病毒有一定的作用。 抗病毒的抗生素：如四环类抗生素对立克次体积较大病毒有一定的作用。 抗癌抗生素：如阿霉素（ 抗癌抗生素：如阿霉素（Adriamycin) 目前在抗病毒、抗癌、等方面还没有理想的抗生素。 目前在抗病毒、抗癌、等方面还没有理想的抗生素。
Fleming noticed that the substance secreted was an inhibitor of many bacterial species He published a paper on the active ingredient which he called ‘penicillin’. This was the first demonstration that a substance produced by micromicro-org’s could inhibit or kill other microbes Fleming could not isolate penicillin. 10 yrs later scientists Florey and Chain successfully isolated penicillin. 1943-1945，scale1943-1945，scale-up production of pencillium. By 1945,enough penicillin was
到目前为止， 8000多种抗生素 2/3是 多种抗生素， 到目前为止，有8000多种抗生素，2/3是 由放线菌（ 产生的， 由放线菌（strepomyces）产生的，并 通过化学结构的改造，总共有3 通过化学结构的改造，总共有3万多种半 合成抗生素，它们的分子量为100-1200， 合成抗生素，它们的分子量为100-1200， 普遍应用的包括半合成和盐类的抗生素 350多种 其中以青霉素、头孢霉素、 多种， 有350多种，其中以青霉素、头孢霉素、 四环素类、 四环素类、氨基糖苷类和大环内脂类
Howard Florey
AustralianAustralian-born Studied Science & Medicine in Adelaide Awarded Scholarship at Oxford University Returned to England to complete a PhD
Some Clinically Important Antibiotics
Antibiotics Penicillin Producer Organism Activity GramGram-positive bacteria GramGram-positive bacteria Broad spectrum GramGram-negative bacteria Broad spectrum Site or mode of action Wall synthesis Wall synthesis Protein synthesis Protein synthesis Protein synthesis
抗生素的应用
对抗生素的评价： 对抗生素的评价：有较大的差异毒力 在体内发挥其抗生效能，而不被血液、脑肾液等破坏， 在体内发挥其抗生效能，而不被血液、脑肾液等破坏，不 大量与血清蛋白质产生不可逆结合。 大量与血清蛋白质产生不可逆结合。 给药后吸收快，并在被感染器官或组织中分布 给药后吸收快， 致病菌对抗生素不易产生耐药性 不引起过敏反应 理化性质稳定，便于提取、制剂和贮藏。 理化性质稳定，便于提取、制剂和贮藏。 抗生素的计量单位： 抗生素的计量单位： 效价单位：青霉素效价单位为能在 效价单位：青霉素效价单位为能在50ml肉汤培养基中完 肉汤培养基中完 全抑制金黄色葡萄球菌菌株发育的最小计量。 全抑制金黄色葡萄球菌菌株发育的最小计量。
Penicillin chrysogenum Bacitracin Bacillus subtilis Neomycin( Neomycin(新霉 Streptomyces 素） fradiae Streptomycin Streptomyces Griseus Tetracycline Streptomyces mediterranei
Ernst Chain
Of Russian- German Russiandescent Graduated in Chemistry and Physiology Left Berlin when Nazi’s came to power Hired by Florey to work on penicillin project