A cryptic sensor for HIV-1 activates antiviral innate

Vol39 No3May , 2021第39卷第3期2021年5月影像科学与光化学Imaging Science and Photochemistryhttp : //www. yxkxyghx. org一种荧光探针检测水溶液中碘离子刘炉英】，范远东】，王 静2,王静珊】，谢浩源】，谢丽君1.广东第二师范学院化学系，广东广州510303；2.中山大学孙逸仙纪念医院，广东广州510120摘 要：本文选用无水柠檬酸和氨乙基-广氨丙基)甲基二甲氧基硅烷,利用水热法制得碳量子点，此碳量子点发蓝光，激发波长和发射波长分别为370 nm 、466 nm 。

由于碘的重原子效应和碘 离子易于给出电子的能力，当碳量子点在水溶液中遇到碘离子时会发生荧光猝灭，通过测量荧光寿命，发现碳量子点和碘离子之间存在动态猝灭效应。

利用这一特性,建立一种选择性检测水溶液中 碘离子的荧光探针。

优化分散溶剂对碳量子点荧光强度的影响，以及响应时间和溶液pH 值对猝灭效率的影响。

在最优实验条件下:荧光变化值(F °—F )与碘离子浓度的对数呈线性，线性方程为Y = 427. 60X +646. 88D 2=0 9945,碘离子浓度的线性范围为5X10-7〜5 X 10-4 mol/L,检出限为3. 2X10-8 mol/L 。

选用三个浓度加标检测自来水样时，回收率范围为101%〜105%。

关键词：碳量子点；碘离子；重原子效应；动态猝灭效应；荧光探针doi ： 10. 7517/issn 1674-0475. 201006A Fluorescent Probe for Detecting the Iodide Ion in Aqueous SolutionLIU Luying 1, FAN Yuandong 1, WANG Jing , WANG Jingshan 1, XIE Haoyuan 1, XIE Lijun 1*1. Department of Chemistry , Guangdong University of Education , Guangzhou 510303,Guangdong , P. R. China ； 2. Sun Yat-Sen Memorial Hospital , Sun Yat-Sen University ,Guangzhou 510120!Guangdong !P C D C ChinaAbstract : In this paper, anhydrous citric acid and N-(-aminoethyl--aminopropyl) methyldimethoxysilane were used to preparecarbon quantum dots by h ydrothermal method. The carbon quantum dot emits blue light, and the excitation wavelength and emissionwaveleng8hwere370nmand466nm !respecively.Due8o8heheavya8ome f ec8ofiodineand8heabiliyofiodineions8oeasilydona8eelec8rons !fluorescencequenchingoccurswhencarbonquan8umdo8sencoun8eriodideionsinanaqueoussoluion.By measuring8hefluorescencelifeime !i wasfound8ha88here was a dynamic quenching e f ec8be8ween carbon quan8um do8s anding8hisfea8ure !afluorescen8probeforselecivede8ecionofiodideionsinaqueoussoluion wases8ablished.Opimize8heinfluenceof8hedispersionsolven8on8hefluorescencein8ensiyofcarbonquan8umdo8s !and8heinfluenceofresponseimeandpHon8hequenchinge f iciency.Under8heopimalexperimen8alcondiions : 8hefluorescencechangevalue (F 0-F ) wihthe logarithm of iodide ion concentration was linear , the linear equation was Y =427. 60X +646. 88 , R 2 =0. 9945 , and the linear range was 5X10-7-5X10-4 mol/L , the detection limit was 3. 2X 10-8 mol/L. The recovery range was 101 %-105% when threeconcent:ationsspikedwe:eusedtotestthewate:samples.Keywords ： carbon quantum dots ； iodine ion ； heavy atom effect ； dynamic quenching effect ； fluorescent probe2020-10-13 收稿，2021-01-07 录用广东省青年创新人才项目(2018KQNCX178, 2017KQNCX143)、广东省大学生创新实验项目(S201914278031)、广东省基础与应用基础研究基金项目(2020A1515010788)资助"通讯作者354第3期刘炉英等:一种荧光探针检测水溶液中碘离子355碳量子点(carbon dots, CDs)是由碳材料衍生而来的一类高级纳米材料,2004年被发现,逐渐引起研究者们的极大兴趣与传统的有机染料和半 导体量子点相比,CDs 在化学 、可调节的光致发光特性、出色的生物相容性及低毒性方面均表现出 色，因而在生物医药、环境监测、食品安全等领域中被广泛应用，并表现出检测的高灵敏度23。

LAB 14ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)STUDENT GUIDEGOALThe goal of this laboratory lesson is to explain the concepts and technique of enzyme linked immunosorbent assay (ELISA).OBJECTIVESAfter completing this lab the student should be able to:1.Define epitope.2.Describe how an ELISA works, including an explanation of its sensitivity.3.Explain the importance of a positive control for ELISA.4.Explain why the onset of AIDs takes several years for those infected with HIV.5.Explain why anti-HIV-1 IgG is screened instead of HIV.6.Explain how HIV targets T H cells.7.Describe common causes of false positive and false negative results in ELISA.pare and contrast a Western immunoblot and an ELISA.9.Explain why a Western immunoblot is performed to confirm a positive ELISA testfor HIVTIMELINEPrep will take 1.5 hours; the lab 1 hour.BACKGROUNDAn epitope is the area of the antigen that stimulates a specific immune response. There is approximately one epitope, also called an antigenic determinant, for each 5 kDa of a protein, but glycoproteins, polysaccharides, lipopolysaccharides, lipids and nuceleoproteins all contain epitopes that can elicit immune responses. Epitopes are usually on the surface of antigens and are the site of antibody attachment.The Enzyme Linked Immunosorbent Assay (ELISA) test is performed by first incubating a specific antigen in the wells of a plastic microtiter plate. The antigen binds to the wells, excess antigen is removed, and the sample suspected to contain antibodies against this antigen is applied to the antigen in the wells. After a wash to remove any unbound sample antibodies, a secondary antibody (an anti-antibody) specific to the antibody being investigated, is applied. This secondary antibody has been conjugated with an enzyme, such as horseradish peroxidase, and after incubation and washing to remove any that is unbound, the enzyme‟s substrate is added. The slightest amount of linked or sandwiched antibodies in the well will result in detectable amounts of color when the colorless substrate is cleaved to a colored product by the conjugated enzyme. See Figure 1, below.Figure 1. How an ELISA worksThe key to this chromogenic reaction (produces a colored product) is the addition of hydrogen peroxide, the substrate for the conjugated enzyme, peroxidase. Hydrogen peroxide is a very reactive and toxic compound that would kill a cell if not for peroxidase. The conjugated enzyme in this experiment is horseradish peroxidase and aminosalicylate (RH2in the equation, below) is the co-substrate, a compound that donates hydrogen atoms in the reaction that converts the hydrogen peroxide to water. The enzyme and co-substrate used in colorometric assays like ELISA can vary, but all work to form a colored product (R in the equation) that can be observed and measured.RH2 + H2O2 R + 2 H2O(peroxidase)The ELISA is used as an initial clinical test of blood for HIV because only inexpensive generic anti-human antibodies need to be used. Many types of HIV antigens are purified and placed in the test well. Then antibodies in the serum sample of the donor are added. If the donor is newly infected, the HIV may have already elicited an immune response of anti-HIV antibodies, but there may not be many HIV particles in the blood – they will be inside infected cells, slowly producing progeny. The titer of HIV may not be high enough to detect, but the anti-HIV antibody titer may be detectable. The ELISA test is extremely sensitive because it is an enzyme-linked test – it only requires a minute amount of bound human antibody epitopes to bind many secondary generic conjugated antibodies, which in turn catalyze the reaction that hydrolyzes substrate molecules to easily detected colored product.Careful, meticulous lab technique is absolutely required or cross contamination will give false result. In clinical practice, the ELISA is performed in triplicate. If the results are positive, a more sensitive test is performed to verify the results because false positives occur very frequently. There are several reasons why false positives occur. If the technician has poor technique, cross contamination may occur, giving a false positive. Cross-reactivity can also occur in immuno assays. This means that an individual may possess antibodies directed against Human Leukocyte Antigens (HLA), which are presentin the host cells that were used when HIV was propagated in the laboratory by the ELISA test manufacturer. (See the background information on HIV and AIDS given in the appendix of this manual for a discussion of how HIV propagates and steals some of the lipid bilayer of the infected cell for each new viral particle produced.) Women who have had multiple pregnancies may carry HLA antibodies against the HLA of the children that they carried that match the HLA in the test. People that have had organ transplants, blood transfusions, gamma globulin immunization, anti-carbohydrate antibodies, and anti-collagen antibodies (found in gay men, hemophiliacs, those with leprosy, and Africans of both sexes) may all test positive if the ELISA test kit includes any of these antigens along with the HIV antigens. A recent cold or other viral infection can also produce antibodies that cross react with the HIV antigens in the test.False negatives can also occur. Poor laboratory technique can result in insufficient incubation of the HIV antigen in the test wells, which fails to allow enough antigen to coat the well. Even if the lab technique is adequate, 1/1000 tests give false negative results. One cause is a lack of sero-conversion, the increase in the titer of anti-HIV antibodies that takes some time to occur after the body is first infected. The ELISA does not always detect such low numbers of anti-HIV antibodies. A confirmatory test, such as the clinical immunoblot or Western blot should be performed after an ELISA gives positive results.A Western immunoblot, illustrated in Figure 2, uses a mixture of monoclonal antibodies, each against an epitope of HIV. The HIV epitopes must be present in the patient serum sample in order to get a positive result. In contrast to the ELISA, it is the HIV antigens themselves and not the patient‟s HIV antibodies that are being tested for. This makes the immunoblot a more definitive test for the presence of HIV. Since monoclonal antibodies are used, this test is more expensive and is used only if an ELISA, a less expensive test, shows a positive result.pale yellow, soluble substrate purple, insoluble product2 antibody1 monoclonal antibody mix against HIV epitopesFigure 2. Western (clinical) immunoblot to confirm HIVLABORATORY OVERVIEWThis lab will be performed in one lab period.▪Prep▪Addition of simulated HIV antigen to the wells of a micro-titer plate▪Washing of wells and addition of simulated HIV antibodies or negative simulated serum samples▪Washing and addition of secondary antibody conjugated to horseradish peroxidase (HP) enzyme▪Color detection of bound secondary antibodies with HP substrate solution SAFETY GUIDELINESGood Laboratory Practice requires wearing safety glasses, lab coat and gloves. MATERIALSPer class:Water bath set at 37CEDVOTEK Kit #271HIV antigens (simulated –component A)Positive control (1 antibodies –component B)Donor 1 serum (simulated –component C)Donor 2 serum (simulated –component D)Anti-IgG-peroxidase conjugate(2 antibody –component E) Hydrogen peroxide (component F)Amiosalicylic acid (peroxide co-substrate –component G) Phosphate buffered saline concentrate (component H) Microtiter plates Microcentrifuge tubes – 1.5 ml Two 50 ml plastic tubes for mixing 2 antibody and substrateScissorsSharpie markersColored pencilsTubes or bottles to hold 25 ml – one per student up to 10 per class (for PBS) Beakers for waste solutions – one per two students1 Corning bottle to hold 270 mlGraduated cylinder to measure 270 mlElectric serological pipette pumps – one per two students1 ml serological pipettes – one per student1 25 ml serological pipetteBottles of dH2O – one per two studentsPROCEDUREEach student can perform his or her own lab. All prep should be done so that everyone receives each of the reagents.Part I. PrepTeam 1Make sure the water bath for incubation is turned on and set at 37C. Cut the micro-titer plates, as shown below, so that each student receives a small micro-titer plate with 12 wells – 4 rows of three wells. Distribute to class members. (If there are only 4 Teams in the class, do the Team 5 prep, also.)Team 2Aliquot reagents A-D for each class member into 1.5 ml microcentrifuge tubes as given below and distribute one set to each person.Positive control (1 antibodies –component B) Team 3Prepare the phosphate buffered saline (PBS) by measuring 270 ml of dH2O and adding to a labeled Corning bottle. Add all of Component H, Phosphate Buffered Saline concentrate, to the water. Invert to mix. Label one small bottle for each person as “PBS” and dispense 25 ml into each and distribute one to each class member. Give the bottle of all remaining diluted PBS to Team 4.Team 4Prepare the 2antibody solution.1.Add 0.3 ml of diluted PBS (from Team 3) to the tube of Anti-IgG-peroxidaseconjugate –component E. Mix thoroughly by tapping and inverting the tube.2.Place 15 ml of diluted PBS (from Team 3) into the 50 ml plastic tube that came in thekit. Label the tube “2Antibody.”3.Transfer the entire contents of component E to the 15 ml of PBS in the labeled 50 mltube and invert to mix.bel a 1.5 ml microcentrifuge tube for each student as …2° Antibody‟ and d ispense1.4 ml to each.5.Give the remaining PBS (from Team 3) to Team 5 (or Team 1, if there are only 4Teams).Team 5 (or Team 1, if there are only 4 Teams)Peroxidase Substrate Solution (Prepare just before the final incubation step in the procedure.)1.Dispense 13.5 ml of the dilute PBS (from Team 3) to a plastic tube provided in thekit.2.Add all of the Aminosalicylic acid (peroxide co-substrate –component G). Cap andmix thoroughly by shaking and vortexing. There may be undissolved materialremaining.3.Add 1.5 ml of the Hydrogen peroxide (component F). Cap and mix.4.Dispense 1.4 ml into 1.5 ml microcentrifuge tubes labeled “Substrate” and dispenseone tube to each student during the 15 minute incubation with the 2 antibody.Each person will need the following:One tube of simulated HIV serum (the antigens)One tube of positive control serumOne tube of simulated donor serum #1 (the antibodies)One tube of simulated donor serum #2 (the antibodies)One tube of 2 antibody solution (the anti-antibodies)25 ml PBSSubstrate solution (to be mixed immediately prior to use)P-1000 automatic micropipetter and sterile tipsP-100 automatic micropipetter and sterile tipsBeaker for used tips (share with your partner)Beaker for waste solutions (share with your partner)Part II. Experimental Procedure1. Label a beaker for waste solutions.2. Label your micro-titer plate with your team number. Label the wells of your micro-titer plate as shown:3. Into all 12 wells, add 100 l of HIV serum (the antigens).4. Incubate for 5 minutes at room temperature.5. Using a micropipetter and sterile tips, remove all the HIV antigens and dispose into the waste beaker.6. Add 200 l PBS buffer to the wells in all rows to wash.7. Remove all the liquid from each well, changing tips each time. Dispose the liquid into the waste beaker.(In research labs, the next step would be to add a blocking solution of protein, such as BSA to saturate all sites not occupied in the well with the antigen. The kitmanufacturers have designed this experiment so that it works without this important step.)8. Use an automatic micropipetter to add the following reagents. Be sure you use a new tip for each solution.9. Incubate for 15 minutes by floating your micro-titer plate in a 37C water bath.(NOTE: be careful that your lab coat sleeve does not tip over another Team‟s plate!)Row 1 Row 2Row 3 Row 4 Row 1: 100 l PBS (negative control) Row 2: 100 l “+” (positive control)Row 3: 100 l DS #1 Row 4: 100 l DS #210.Remove all the solutions from each well. Be sure to change tips when you changerows or you will cross contaminate and get false results.11.Add 200 l PBS to each well without touching the wells with the tip. If you touch thetip to the wells, change tips.12.Remove the PBS from each well using a different tip for each row.13.Add 100 l of the 2 antibody solution to each well. If you touch the tip to the wells,change tips.14.Incubate for 15 minutes by floating your micro-titer plate in a 37C water bath.This is the time to mix the substrate solution.15.Remove all the liquid from each well using a different tip for each row.16.Add 200 l PBS to each well without touching the wells with the tip.17.Remove the PBS from each well using a different tip for each row.18.Add 100 l substrate solution to each well.19.Incubate by floating your micro-titer plate in a 37C water bath for 5 minutes, whichshould be enough time to see results.20.Draw your results in your notebook or use the scanner to record all ELISA resultsfrom the class.DATA ANALYSISLabel your results and describe what you observe in each well.QUESTIONS1.Define epitope.2.Explain how the ELISA test works and why is it so sensitive.3.Why can the onset of AIDS take several years?4.Why is anti-HIV-1 IgG screened instead of the virus itself?5.Why are there so many immunological variants of HIV?6.What can cause a false positive in an ELISA?7.What can cause a false negative in an ELISA?pare and contrast a Western immunoblot and an ELISA.9.Explain why a Western immunoblot is performed to confirm a positive ELISA testfor HIV.。

A magnetic switch for the control of cell deathsignalling iA magnetic switch for the control of cell death signalling in in vitro and in vivo systemsMi Hyeon Cho 1?,Eun Jung Lee 1,2?,Mina Son 2?,Jae-Hyun Lee 1,Dongwon Yoo 1,Ji-wook Kim 1,Seung Woo Park 3,Jeon-Soo Shin 2,4*and Jinwoo Cheon 1,2*The regulation of cellular activities in a controlled manner is one of the most challenging issues in ?elds ranging from cell biology to biomedicine 1,2.Nanoparticles have the potential of becoming useful tools for controlling cell signalling pathways in a space and time selective fashion 3,4.Here,we have developed magnetic nanoparticles that turn on apoptosis cell signalling by using a magnetic ?eld in a remote and non-invasive manner.The magnetic switch consists of zinc-doped iron oxide magnetic nanoparticles 5(Zn 0.4Fe 2.6O 4),conjugated with a targeting antibody for death receptor 4(DR4)of DLD-1colon cancer cells.The magnetic switch,in its On mode when a magnetic ?eld is applied to aggregate magnetic nanoparticle-bound DR4s,promotes apoptosis signalling pathways.We have also demonstrated that the magnetic switch is operable at the micrometre scale and that it canbe applied in an in vivo system where apoptotic morphological changes of zebra?sh are successfully induced.Cell signalling is an important process in biological systems for exchanging information through networks of various signal molecules to control cellular activities,such as differentiation,growth,metabolism and death 6.Owing to their newly developed high precision and accuracy,physical stimuli using optical,electrical and magnetic methods have been devised to regulate cell signalling 1–4.Among these,magnetic techniques are uniquely advantageous because magnetic fields can penetrate deeply with negligible attenuation into biological tissues 7,8.Consequently,it has distinctive benefits for in vivo applications.Moreover,when coupled with magnetic nanoparticles,magnetic fields can be transformed into other forms of energy,such as heat and mechanical force 9–16.The magnetic heat induction has been used for gating of the thermosensitive ion channel 9as well as for hyperthermia therapy 10.Although relatively large mechanical force (in the piconewton range)has been used in in vitro and in vivo systems for direct stretching of ion channels and cytoskeletal stimulation 11–13,two recent in vitro studies have revealed that the induction of calcium influx 15and tubulogenesis 16using receptor clustering is also possible by usingnanoparticles.Magnetic nanoparticles can exert a gentle force (in the femtonewton range)on membrane receptors to induce their clustering without disturbing the rheological and cytoskeletal properties.Furthermore,the nanoscale dimensions of nanoparticles conjugated with targeting molecules make them beneficial for probing cellular sensory structures and functions at the molecular level and for inducing specific cellular activation processes 7,8.Nonetheless,the nanoscale magnetic switching technique for receptor clustering is still at too1Departmentof Chemistry,Yonsei University,Seoul 120-749,Korea,2Graduate Program for Nanomedical Science,Yonsei University,Seoul 120-749,Korea,3Department of Internal Medicine,Institute of Gastroenterology,College of Medicine,Yonsei University,Seoul 120-752,Korea,4Department ofMicrobiology,Severance Biomedical Science Institute,Institute for Immunology and Immunological Diseases,College of Medicine,Yonsei University,Seoul 120-752,Korea.?These authors contributed equally to this work.*e-mail:******************;****************.kr.early a stage of development to guarantee that it will begenerally applicable to the control of cell signalling in other biologically important systems.In addition,it is not known whether it will be effective in in vivo systems.Apoptosis,programmed cell death,is known to be a major factor in maintaining homeostasis and removing undesired cells 17–19.Recently,an extrinsic apoptosis signalling pathway that is initiated by death receptors has emerged as one of the main targets for cancer therapy 20–22.Extrinsic apoptosis signalling is usually activated by clustering of death receptors through docking of biochemical ligands,such as the TNF-related apoptosis inducing ligand (TRAIL)that is a potent inducer of apoptosis 23,24.However,direct use of such ligands in clinical applications is limited by the short plasma half-life and the ease of degradation 25,26.In the study described below,we have developed a magnetic switch for apoptosis signalling,and demonstrate its in vivo feasi-bility through the receptor clustering process (Fig.1).The mag-netic switch for cell signalling consists of DR4monoclonal anti-body conjugated to magnetic nanoparticles (Ab–MNPs).DR4s are highly expressed on tumour cells 20–27and magnetic nanoparticles are designed to bind DR4s through a specific monoclonal anti-body interaction.Zinc-doped iron oxidemagnetic nanoparticles (Zn 0.4Fe 2.6O 4MNPs,15nm)are chosen (Fig.2a)owing to their high saturation magnetization value (161e .m .u .g ?1),which is essential for effective utilization of magnetic force 5(Supplementary Section S1).For preparation of the Ab–MNPs,protein A is conjugated to thiolated MNPs through a sulpho-SMCC (sulphosuccinimidyl-4-[N -maleimidomethyl]cyclohexane-1-carboxylate)crosslinker.TheDR4antibody is then conjugated to protein A on MNPs in a DR4antibody/MNP stoichiometric ratio of 1:1(Fig.2b and Supple-mentary Section S1).。

医护英语考试题及答案一、选择题（每题2分，共20分）1. What is the most common symptom of the common cold?A. FeverB. CoughC. Sore throatD. All of the above答案：D2. Which of the following is NOT a vital sign?A. Blood pressureB. PulseC. Respiratory rateD. Body temperature答案：D3. What does the acronym "ICU" stand for?A. Intensive Care UnitB. International Clinical UnitC. Immediate Care UnitD. Inpatient Clinical Unit答案：A4. The term "diabetes" refers to a condition characterized by:A. High blood sugar levelsB. Low blood sugar levelsC. High blood pressureD. High cholesterol levels答案：A5. A patient is said to be "anemic" if they have:A. Too much red blood cellsB. Too few red blood cellsC. Too much white blood cellsD. Too few platelets答案：B6. What is the medical term for a surgical incision?A. IncisionB. AmputationC. BiopsyD. Excision答案：A7. Which of the following is a method of sterilization?A. Washing with soap and waterB. BoilingC. Using alcohol swabsD. All of the above答案：D8. The abbreviation "MRI" stands for:A. Magnetic Resonance ImagingB. Medical Radioactive ImagingC. Multiple Radioactive IndicatorsD. Medical Radio Imaging答案：A9. What is the primary function of the liver?A. To filter bloodB. To produce bileC. To regulate blood sugar levelsD. To produce red blood cells答案：B10. A "thermometer" is used to measure:A. Blood pressureB. Body temperatureC. Respiratory rateD. Pulse答案：B二、填空题（每题1分，共10分）11. The medical term for a broken bone is ____________.答案：fracture12. A person with a severe allergy to penicillin would be given a warning to avoid contact with this medication, known as a(n) ____________.答案：allergy alert13. The abbreviation "HIV" stands for Human Immunodeficiency ____________.答案：Virus14. A healthcare professional who specializes in the diagnosis and treatment of diseases of the heart is called a ____________.答案：cardiologist15. The process of removing waste products from the body is known as ____________.答案：excretion16. A patient's medical history is recorded in their____________.答案：medical record17. The practice of washing hands with soap and water to prevent the spread of disease is called ____________.答案：hand hygiene18. A(n) ____________ is a healthcare professional trained to provide emergency medical services.答案：paramedic19. The abbreviation "OT" stands for Occupational____________.答案：Therapy20. A patient's condition is assessed and monitored throughregular ____________.答案：check-ups三、简答题（每题5分，共30分）21. What are the four stages of the nursing process?答案：The four stages of the nursing process are assessment, planning, implementation, and evaluation.22. Explain the difference between a virus and a bacterium.答案：A virus is a microscopic infectious agent that can only replicate inside the living cells of an organism, while a bacterium is a single-celled microorganism that can exist independently and can be beneficial, neutral, or harmful to humans.23. What is the purpose of a stethoscope in medical practice?答案：A stethoscope is used by healthcare professionals to listen to the sounds produced by the body, such as the heartbeat and breathing, to diagnose or monitor various conditions.24. Describe the role of a registered nurse in a hospital setting.答案：A registered nurse in a hospital setting providesdirect patient care, administers medications, monitors patients' conditions, collaborates with physicians and other healthcare professionals, and educates patients about theirhealth conditions and treatments.四、翻译题（每题5分，共20分）25. 请将以下句子翻译成英文：医生建议他每天服用阿司匹林以预防心脏病。

·综述·基金项目：“十三五”国家科技重大专项（2018ZX10302104）；重庆市医学科研重点项目（2019ZDXM012）通信作者：陈耀凯，E-mail: yaokaichen ＠引用格式：陈力，何小庆，陈耀凯.隐球菌荚膜抗原检测在隐球菌病中的诊断价值[J].新发传染病电子杂志，2021，6（1）：58-61. Chen Li, He Xiaoqing, Chen Yaokai.The value of detecting cryptococcal antigen in the diagnosis of cryptococcosis[J].Electronic Journal of Emerging Infectious Diseases,2021,6(1):58-61.陈力1，何小庆2，陈耀凯2（1.重庆市公共卫生医疗救治中心急诊科，重庆 400036；2.重庆市公共卫生医疗救治中心感染科，重庆 400036）隐球菌荚膜抗原检测在隐球菌病中的诊断价值 【摘要】隐球菌病是由隐球菌感染人体引起的一种侵袭性真菌病，是艾滋病患者及实体器官移植者中常见的感染和致死原因。

10年来，隐球菌病的诊断方法已从真菌培养、墨汁染色，到目前的隐球菌荚膜抗原（cryptococcal capsular antigen，CrAg）检测。

CrAg检测操作简便快速，花费少，敏感性及特异性高，因此在世界范围内得到广泛应用。

世界卫生组织推荐对高危人群进行CrAg筛查，对CrAg阳性患者进行抢先治疗，从而降低病死率。

本文就CrAg检测在隐球菌病中的诊断价值展开综述。

【关键词】隐球菌荚膜抗原；隐球菌病；诊断价值 DOI：10.19871/ki.xfcrbzz.2021.01.013The va l ue of detect i ng cryptococca l ant i gen i n the d i agnos i s of cryptococcos i sChen Li 1, He Xiaoqing 2, Chen Yaokai 2(1. Department of Emergency, Chongqing Public Health MedicalCenter, Chongqing 400036, China; 2. Department of Infectious Diseases, Chongqing Public Health Medical Center, Chongqing 400036, China) 【Abstract】 Cryptococcosis is an invasive fungal disease caused by cryptococcosis,it is a common cause of infection and death among AIDS patients and solid organ transplant recipients.In the past ten years, the diagnostic methods for cryptococcosis have ranged from fungal culture and ink staining to the current detection of cryptococcal capsular antigen (CrAg).CrAg detection is simple and fast, inexpensive, high sensitivity and specificity, so it is widely used worldwide.The World Health Organization recommends CrAg screening for high-risk groups and preemptive treatment of CrAg-positive patients to reduce the mortality rate.This article reviews the value of cryptococcal capsularantigendetecting in the diagnosis of cryptococcosis. 【Key words】 Cryptococcal capsular antigen; Cryptococcosis; Diagnosis 隐球菌是一种溶组织酵母菌，为条件致病菌，主要通过呼吸道传播，人因吸入鸽粪污染的空气而感染。

艾滋病未解之谜的英语作文In the 20years since the first cases of AIDS were detected, scientists say they have learned more about this viral disease than any other.Yet Peter Piot, who directs the United Nations AIDS program, and Stefano Vella of Rome, president of the International AIDS Society, and other experts say reviewing unanswered questions could prove useful as a measure of progress for AIDS and other diseases.Among the important broader scientific questions that remain：Why does AIDS predispose infected persons to certain types of cancer and infections？A long-standing belief is that cancer cells constantly develop and are held in check by a healthy immune system. But AIDS has challenged that belief. People with AIDS are much more prone to certain cancers like non-Hodgkins lymphomas and Kaposi’s sara , but not to breast, colon and lung, the most mon cancers in the United States. This pattern suggests that an impaired immune system, at least the type that ours in AIDS, does not allow mon cancers to develop.What route does HIV take after it enters the body to destroy the immune system？When HIV is transmitted sexually, the virus must cross a tissue barrier to enter the body. How that happens isstill unclear. The virus might invade directly or be carried by a series of different kinds of cells.Eventually HIV travels through lymph vessels to lymph nodes and the rest of the lymph system. But what is not known is how the virus proceeds to destroy the body’s CD-4cells that are needed to bat invading infectious agents.How does HIV subvert the immune sys-tem？Although HIV kills the immune cells sent to kill the virus, there is widespread variation in the rate at which HIV infected people bee ill with AIDS. So scientists ask：Can the elements of the immune system responsible for that variability be identified？If so, can they be used to stop progression to AIDS in infected individuals and possibly prevent infection in the first place？What is the most effective anti-HIV therapy？In theory, early treatment should offer the best chance of preserving immune function. But the new drugs do not pletely eliminate HIV from the body so the medicines, which can have dangerous side effects, will have to be taken for a lifetime and perhaps changed to bat resistance. The new policy is expected to remend that treatment be deferred until there are signs the immune system is weakening.Is a vaine possible？There is little question that an effective vaine is crucial to controlling the epidemic . Yet only one has reached the stage of full testing, and there is wide controversy over the degree of protection it will provide. HIV strains that are transmitted in various areas of the world differ geically. It is not known whether a vaine derived from one type of HIV will confer protection against other types.In the absence of a vaine, how can HIV be stopped？Without more incisive , focused behavioral research, prevention messages alone will not stop the global epidemic.。

临床和实验医学杂志2021年3月第2。

卷第5期-453-[17]张爱华，邹天骥，石继来，等.老年性白内障晶状体上皮细胞的人晶状体上皮细胞凋亡中的实验研究[D].郑州：郑州大学,TFAR19蛋白表达及临床意义[J].海南医学，2019,30(3)：257-2012.255.(收稿日期：2020-l1-19) [15]杨鑫.JNK/j-J uv通路及其靶基因Bimi与PUMA在氧化应激诱导DOI：10.3969/j.issn.1471-4695.2021.05.002文章编号：1071-4695(2021)05-0453-04亚低温辅助运动通过抑制脑神经凋亡改善缺氧缺血性脑损伤大鼠幼崽脑神经功能的机制探讨孙晓燕1李润东i陈艳丽2(青岛市海慈医疗集团1针推康复科；2重症医学科山东青岛266600)【摘要】目的探讨亚低温辅助运动通过抑制脑神经凋亡改善缺氧缺血性脑损伤大鼠幼崽脑神经功能的机制。

方法选择无特定病原体(SPF)级新生7日龄Wistar大鼠，采用手术结扎左颈总动脉法建立缺氧缺血性脑损伤幼鼠模型。

按照随机数字表法分为假手术组(假手术幼鼠，正常饲养不进行任何干预)、HIE模型组(HIE模型幼鼠，正常饲养不进行任何干预)和HIE模型+亚低温和运动干预组(HIE模型幼鼠，亚低温和运动干预、，每组大鼠幼崽各5只，共干预63d。

分别于治疗前以及治疗后第42天和第63天，通过旋转脚架性能测试检测3组幼鼠平衡能力变化、去除胶带测试检测3组幼鼠行为能力变化；治疗结束后，采用蛋白质印迹(Western blotting)法检测治疗前后模型幼鼠的脑部凋亡相关蛋白因子细胞色素c(Cyt C)、活化型半胱天冬酶-3(cleaveX-Caspase3)、半胱天冬酶-3前体(pro-Caspase3)、聚ADP核糖聚合酶1(PARP-1)、蛋白酶激活受体(PAR)的表达情况。

结果第42天和第63天，与假手术组相比，HIE模型组和HIE模型+亚低温和运动干预组幼鼠平衡维持时间均显著缩短，去除胶带所用时间、海马组织中凋亡相关蛋白Cy)C、cleaveX-Caspase3、PARP-1和PAR相对表达量均显著增加，差异均有统计学意义(P vOD)与HIE模型组相比，HIE模型+亚低温和运动干预组幼鼠平衡维持时间均显著延长，去除胶带所用时间、海马组织中凋亡相关蛋白Cyt C'CleaveX-Caspase3、PARP-1和PAR相对表达量均显著降低，差异均有统计学意义(P vOD)。

学习科研设计切入点（2012-1）没事翻译一下，共108项（仅作内部学习用）"Bulletin Board" For Broadcasting Vaccine Supply/DemandTopic: Design New Approaches to Optimize Immunization SystemsArun Ramanujapuram of Logistimo, Inc. in India proposes to develop a mobile-phone based "bulletin-board" for capturing and broadcasting availability and demand information for vaccines and medicines. By bringing real-time visibility to these essential goods, stock can be appropriately redistributed to areas of need, and waste can be reduced.A Biotic Stress Sensor Printed on Maize LeavesTopic: Protect Crop Plants from Biotic Stresses From Field to MarketHideaki Tsutsui of the University of California, Riverside in the U.S. will develop a low-cost stamp to directly print biosensors on maize leaves for colorimetric detection of biotic stresses. The strategy is to develop an immunochromatographic assay using microneedle probes while printing an easily-read color-change detector.A Device for Self-Sampling of Blood for Infectious DiseaseTopic: Explore New Solutions in Global Health Priority AreasIan Matthews of Cardiff University in the United Kingdom proposes to develop a self-sampling micro-needle patch device for the collection of small volumes of blood. Micro-needles will be fabricated using Deep Reactive Ion Etching. The device will permit non-refrigerated transport of collected blood for subsequent assays for diagnosis of infectious disease.A Diagostic Test for Poor Anti-TB Drug BioavailabilityTopic: Explore New Solutions in Global Health Priority AreasChristopher Vinnard of Drexel University in the U.S. proposes to develop a low-cost point-of-care urine test that can safely and accurately identify tuberculosis patients who poorly absorb anti-TB drugs. Testing patients for inadequate drug bioavailability could enable better drug dose optimization and decrease transmission rates.A Microbial Platform for the Biosynthesis of New DrugsTopic: Apply Synthetic Biology to Global Health ChallengesChristina Smolke of Stanford University in the U.S. will develop synthetic biology platforms to improve the scale and efficiency of microbial systems used to discover, develop, and produce drugs based on natural products. Such new biosynthesis approaches could lead to new and less expensive drugs for global health.A Mobile Cloud System to Achieve Universal VacccinationTopic: Design New Approaches to Optimize Immunization SystemsAlain Labrique of Johns Hopkins Bloomberg School of Public Health in the U.S. will develop and field test in rural Bangladesh a cloud-based mobile phone system that will allow for universal access to vaccination records, send vaccine reminders and messaging, and provide incentives toparents and health care workers via a phone application. This new strategy could increase the reach, coverage, and public acceptance of immunization.A Passive Solar Thermal Standard for Vaccine Storage RoomsTopic: Design New Approaches to Optimize Immunization SystemsLoriana Dembele of Eau et Vie Jiduma in Mali proposes to develop new architecture and construction guidelines for vaccine storage rooms in hot climates that incorporate passive solar thermal technologies to keep vaccines at recommended temperatures. The team will construct and test prototype storage facilities using a variety of building materials to determine new standards that prevent vaccine spoilage, reduce operating costs, and improve refrigeration capabilities.A Predictive Model for Vaccine Testing Based on AptamersTopic: Apply Synthetic Biology to Global Health ChallengesAlexander Douglas of the Jenner Institute, University of Oxford in the United Kingdom will use synthetic nucleic acid molecules known as aptamers to develop a model that can be used to predict the success or failure of new vaccines in clinical trials. This work could help to remove some of the uncertainty in the early-stage development of new vaccines.A Probiotic-based Approach To Improve Child NutritionTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenAlip Borthakur of the University of Illinois at Chicago in the U.S. will characterize the effects of probiotics on epithelial uptake of the fatty acid butyrate in vitro and in a mouse model to inform therapeutic strategies to cure and prevent acute diarrhea and malnutrition in children.A Single Strategy to Attack Insect Vector and Transmitted VirusTopic: Protect Crop Plants from Biotic Stresses From Field to MarketStéphane Blanc of the Institut National de la Recherche Agronomique (INRA) in France will target a newly described structure at the tip of aphid stylets, the acrostyle, to block transmission of plant viruses and disturb insect feeding. The proposed work will identify specific proteins in the acrostyle for targeting in future interventions.A Synthetic Biosensor to Find Drugs Targeting TB PersistenceTopic: Apply Synthetic Biology to Global Health ChallengesRobert Abramovitch of Michigan State University in the U.S. proposes to use a synthetic biosensor strain and high-throughput screening to discover compounds that inhibit tuberculosis persistence. Study of these compounds may lead to new drugs that limit the establishment of chronic tuberculosis infections.A Transformational Vaccine Platform for RotavirusTopic: Explore New Solutions in Global Health Priority AreasAnton Middelberg of the University of Queensland in Australia proposes to develop a new vaccine for rotavirus by the directed self-assembly of a safe virus-like particle in industrial reactors. The approach uses low-technology engineering methods suitable for the developing world, ensuring relevance to the communities most in need of vaccine.Accelerating Vaccine Development Against P.vivax MalariaTopic: Explore New Solutions in Global Health Priority AreasJames Beeson and Damien Drew of the Burnet Institute in Australia propose to generate chimeric Plasmodium falciparum that expresses the antigens of another malaria parasite, P. vivax, allowing them to be evaluated as vaccine candidates. Because laboratory culturing of P. vivax is costly and technically difficult, this new method could help accelerate the development of vaccines against malaria caused by P. vivax.Aerosol Delivery of Synthetic Lung SurfactantTopic: Explore New Solutions in Global Health Priority AreasFrans Walther of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center in the U.S. will develop an aerosol formulation of an advanced synthetic lung surfactant to treat lung surfactant deficiency and improve lung function in premature infants who are supported with non-invasive ventilation for prematurity-related breathing problems.An "Oral Contraceptive" For Male Anopheles MosquitoesTopic: Explore New Solutions in Global Health Priority AreasLuna Kamau of the Kenya Medical Research Institute in Kenya will investigate how feeding on selected compounds affects male Anopheles mosquito fertility and subsequently, mating competiveness. The compounds could be presented in sugar meals or introduced into larval breeding sites to control mosquito population densities, thereby reducing malaria transmission.Arginine Metabolism in Indian, Jamaican and American WomenTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenFarook Jahoor of Baylor College of Medicine in the U.S. will conduct studies to test whether healthy women in India produce less arginine, an amino acid critical for a successful pregnancy, than pregnant Jamaican and American women and will research the underlying mechanisms to inform possible interventions to reduce the number of low birth weight babies in India.Bi-directional Membrane Device for Child DeliveryTopic: Explore New Solutions in Global Health Priority AreasSergey Shevkoplyas, Lakhinder Kamboj, and Noshir Pesika of Tulane University in the U.S. will develop a microfabricated bidirectional membrane that can be placed in the mother‟s vagina just prior to delivery to facilitate the baby‟s passage through the birth canal. This simple-to-use device could significantly improve outcomes during vaginal deliveries in resource limited settings.BioClay - Managing Biotic Stresses From Field to MarketTopic: Protect Crop Plants from Biotic Stresses From Field to MarketNeena Mitter of The University of Queensland in Australia will develop a …BioClay‟ technology to deliver biological agents that kill crop pathogens and pests. This technology could provide broad spectrum protection to crops without the need for transgenic plants or the use of chemicals.Biomarkers of in utero Aflatoxin Exposure and Child GrowthTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenZdenko Herceg of the International Agency for Research on Cancer (IARC) in France will evaluate the impact of seasonal dietary aflatoxin exposure of pregnant women in rural Africa by identifying epigenetic biomarkers of exposure, growth impairment, and disease risk. This will facilitate the development of interventions to improve child health.Biomarkers of Nutrition Related Cognitive DevelopmentTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenClare Elwell of University College London in the United Kingdom proposes to use a non-invasive optical brain imaging technology (near-infrared spectroscopy) to study cognitive function in malnourished infants and children. This approach could be used to determine the impact of malnutrition on the developing brain and guide nutrition-related interventions.Breeding the Epigenome in CropsTopic: Protect Crop Plants from Biotic Stresses From Field to MarketSally Mackenzie of the University of Nebraska-Lincoln in the U.S. will establish a strategy to exploit crop phenotype variations that are controlled by epigenetic changes for breeding. The strategy is to disrupt the MSH1 gene in millet and maize, releasing a large amount of variation derived from heritable epigenetic changes that can be used to select for abiotic and biotic stress tolerance.Business Card-based Bacteriophage TherapyTopic: Protect Crop Plants from Biotic Stresses From Field to MarketMichael DuBow of UniversitéParis-Sud in France will develop a biodegradable, water-soluble business card impregnated with bacteriophages targeting bacterial pathogens. Cards would be enclosed in a plastic sleeve and be printed with pictographic instructions for diagnosis and use.Cereal Drying Unit To Combat Aflatoxin ContaminationTopic: Protect Crop Plants from Biotic Stresses From Field to MarketMeshack Obonyo of Egerton University Njoro in Kenya and Aman Bonaventure of Masinde Muliro University in Kenya will develop a simple, cost-effective, solar-powered grain drying unit for prevention of aflatoxin contamination in stored grain. The prototype will use locally available materials and be tested in the laboratory and the field.Checkmating PolioTopic: Explore New Solutions in Global Health Priority AreasPanduranga Rao and Nagendra Hegde of Ella Foundation in India will develop and test for use in a vaccine a live single-cycle poliovirus that has been modified to eliminate the gene essential for replication. This highly disabled virus will be tested for its immunogenicity and its inability to re-emerge as vaccine-derived poliovirus (VDPV).Choline and Optimal DevelopmentTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenSteven Zeisel of the University of North Carolina at Chapel Hill in the U.S. will test thehypothesis that adequate levels on choline in the diets of pregnant and nursing mothers is needed for optimal brain development of fetuses and infants. He will develop and validate a methodology to measure infant memory and cognition for use in a larger study on the effects of dietary choline on infant development in The Gambia.Deciphering the Role of Bioactive MicroRNAs in MilkTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenBogdan Mateescu of ETH University in Switzerland will explore the role of mammalian microRNAs maternally transmitted to infants through milk. This project could lead to the development of innovative nutritional supplements for correcting unhealthy growth or protecting infants from pathogens.Defeating Antibiotic Resistance Before It EmergesTopic: Explore New Solutions in Global Health Priority AreasPaul de Figueiredo and colleagues at Texas A&M University in the U.S. propose to develop a system whereby drugs for defeating new antibiotic-resistant strains of Mycobacterium tuberculosis can be developed in the laboratory before these strains emerge in human populations.Defeating Ug99 Wheat Stem RustTopic: Protect Crop Plants from Biotic Stresses From Field to MarketWalter Messier of Evolutionary Genomics, Inc. in the U.S. will discover genes in wheat that underlie resistance to Ug99 stem rust by looking for genes that have evolutionary signatures of adaptation. The strategy involves high quality sequencing of RNA from close wild relatives of wheat and doing a comparative analysis, looking for genes under positive selection.Development & First Field Testing Battery Free Solar FreezerTopic: Design New Approaches to Optimize Immunization SystemsSteve McCarney of the Solar Electric Light Fund (SELF) in the U.S. proposes to accelerate the development and field testing of two solar powered, battery-free icepack freezers to provide a missing link in the cold chain where outreach efforts require frozen packs to cool vaccines during transport and immunization sessions.Development of a Microorganism to Produce ArtemisininTopic: Apply Synthetic Biology to Global Health ChallengesJay Keasling of Zagaya in the U.S. will explore the production by an endophytic fungus of artemisinin, a key ingredient in malaria treatments. If the fungus produces artemisinin in the absence of light, an enzymatic mechanism is likely involved. This mechanism could be harnessed for a new production method to reduce treatment costs for malaria patients in developing countries.Development of Alternative Strategies of Chemotherapy for TBTopic: Explore New Solutions in Global Health Priority AreasKanury Rao of International Centre for Genetic Engineering and Biotechnology in India is testing small molecule inhibitors of host proteins required for M. tuberculosis to survive and replicatewithin host cells. Identifying and optimizing compounds that target host proteins could lead to new drug therapies for tuberculosis that are effective even against multidrug-resistant bacterial strains.Development of Extremophile Bacteriophage-based VaccinesTopic: Explore New Solutions in Global Health Priority AreasSamuel M. Duboise of the University of Southern Maine in the U.S., along with colleagues in Kenya, will develop a vaccine platform that uses bacteriophages that are structurally stable in one of Earth‟s most extreme environments –the hypersaline alkaline soda lakes of Kenya‟s Great Rif t Valley. Vaccines produced using these engineered bacteriophages are expected to be inexpensive, stable, and easy to produce in regionally dispersed locations using common microbiological and biochemical methods that are highly scalable and adaptable for "just-in-time" production.Development of Portable Technology for Rapid Aflatoxin DetectionTopic: Protect Crop Plants from Biotic Stresses From Field to MarketHaibo Yao of Mississippi State University in the U.S. proposes to develop portable and cost effective spectral-based technology for rapid and non-invasive detection of aflatoxin contamination in whole maize ears for use by farmers in rural areas of developing countries.Discovering New Anti-Microbial Peptides Against MycobacteriaTopic: Apply Synthetic Biology to Global Health ChallengesErdogan Gulari of the University of Michigan in the U.S proposes to design and produce a large library of antimicrobial peptides (AMPS) that will be tested against Mycobacterium tuberculosis strains to identify potential new drugs that can damage the bacterial membrane and be less susceptible to evasion by the development of resistance.DNA Nanodevice For Pathogen DetectionTopic: Apply Synthetic Biology to Global Health ChallengesEric Henderson of Iowa State University in the U.S. will build an inexpensive and robust nanodevice that uses DNA as a scaffold to interact with proteins and nucleic acid markers of target pathogens. When this interaction occurs, the movement will be detected by a reader embedded in the device to create a visual readout of pathogen detection.Effect of HDAC InhibitorsTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenGalia Gat-Yablonski of Tel Aviv University in Israel will test in a rat model whether histone deacetylase (HDAC) inhibitors can enhance catch-up growth after nutrient restriction, providing evidence for epigenetic pathways that might underlie stunting in malnourished children.Effects of Placental Transfusion on Early Brain DevelopmentTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenJudith Mercer of the University of Rhode Island in the U.S. will conduct a randomized study to test whether delaying umbilical cord cutting after birth by as little as five minutes allows the placenta to transfer iron-rich blood cells to the newborn, reducing iron deficiency and anemia inthe first year of life and promoting myelination of the brain to aid neurodevelopment.Efficient Analytical Methods for Breastmilk MicronutrientsTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenLindsay Allen of ARS Western Human Nutrition Research Center in the U.S. will use recently developed analytical methods to simultaneously analyze multiple micronutrients in human milk to evaluate how nutritional interventions during lactation affect micronutrients in milk and subsequent infant status.Eliminate the Cold Chain with Low-Cost Liquid Viral VaccinesTopic: Explore New Solutions in Global Health Priority AreasTarit Mukhopadhyay of University College London in the United Kingdom and Stephen Ward of Stabilitech are working to apply a new liquid stabilizing technology to create thermostable vaccines that can withstand extremes of temperature and eliminate the need for a cold chain.Epidermal Electronics for Continuous Pregnancy MonitoringTopic: Explore New Solutions in Global Health Priority AreasTodd Coleman of the University of California, San Diego in the U.S. along with John Rogers of the University of Illinois will develop wireless tattoo-like electronics to continuously monitor vital signs of the pregnant mother and fetus. The devices have the potential to be inexpensively mass produced, which could advance epidemiological studies of preterm birth.Establishing an Anti-Vaccine Surveillance and Alert SystemTopic: Design New Approaches to Optimize Immunization SystemsSeth Kalichman of the University of Connecticut in the U.S. will establish an internet-based global monitoring and rapid alert system for finding, analyzing, and counteracting misinformation communication campaigns regarding vaccines to support global immunization efforts.Feeding Tools to Improve Infant Nutrition and GrowthTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenAmy Webb Girard of Emory University in the U.S. will develop and pilot test the use of feeding bowls with illustrations of appropriate feeding practices during pregnancy, lactation, and early childhood, along with sieved spoons that can be used to test the appropriate thickness of infant meals, in an effort to improve nutrition and growth during the critical first 1,000 days of infant development.Fetal Inflammation, Cachexia, Iron Uptake, and LBWTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenJennifer Friedman of the Center for International Health Research at Rhode Island Hospital in the U.S. will examine the role that chronic placental and fetal exposure to infectious diseases during gestation plays in low birth weight. This work will form the basis of further studies to identify biomarkers for fetal inflammation in the maternal circulation for possible use in diagnostic tests to identify at-risk pregnancies.Field Selection of New Disease Resistance AllelesTopic: Protect Crop Plants from Biotic Stresses From Field to MarketJonathan Jones of the Sainsbury Laboratory in the United Kingdom will enhance mutation rates of R gene loci in a targeted fashion by creating transgenic plants with a mutagenic transgene. The mutagenic transgene has sequence-specific DNA binding domains fused to deaminases that mutate cytidine to thymidine (changing C:G base pairs to T:A base pairs), increasing mutation rates specifically at R gene loci. Resulting plants would then be screened for resistance with promising lines backcrossed to remove the mutagenic locus. Proof of concept will be completed in yeast, Arabidopsis, and then wheat.Field-Deployable Nutrient-Rich Matrix for Crop ProtectionTopic: Protect Crop Plants from Biotic Stresses From Field to MarketJulie A. Willoughby of North Carolina State University in the U.S. proposes to develop a biodegradable cellulose matrix platform comprising active ingredients for seed treatment. Widespread distribution of crop protection agents could ultimately increase crop yield without interfering with subsistence farming practices.Fluorescent Protein Sensor to Diagnose HIV at Low CostTopic: Explore New Solutions in Global Health Priority AreasShengxi Chen of Arizona State University in the U.S. will design and prepare a fluorescent sCD4 protein that changes color when it binds to the HIV pg120 protein. By directly detecting a virus protein instead of antibodies or RNA, which take days to months to accumulate sufficiently to detect, HIV infection can be diagnosed immediately to help prevent the spread of the epidemic.Generating Whitefly-Resistant PlantsTopic: Protect Crop Plants from Biotic Stresses From Field to MarketSaskia Hogenhout of The John Innes Centre in the United Kingdom will generate whitefly-resistant plants by engineering transgenic cassava plants with RNAi targeted against genes essential to whitefly Bemisia tabaci survival and reproduction.Geospatial Optimization ToolTopic: Design New Approaches to Optimize Immunization SystemsPayal Kamdar of VSolvit in the U.S. proposes to develop a customizable Geographical Information System web application platform that integrates existing data in a particular region (e.g., population, locations of vaccines stores, health care facilities, transportation options, even weather) to maximize delivery of vaccines to target populations.Good Bugs: Sustainable Food for Malnutrition in ChildrenTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenAaron Dossey of All Things Bugs, LLC in the U.S. proposes to develop a method for the efficient production of nutritionally dense food using insect species. Dossey and collaborators will identify candidate species and test the nutritional value, shelf-stability, and palatability of a dried powder made from these insects for use in food products to help eliminate malnutrition in children.Growing Nutrient-Rich Potatoes for an Impoverished WorldTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenRichard Veilleux and Mark Williams of Virginia Polytechnic Institute and State University in the U.S., along with Merideth Bonierbale of the International Potato Center in South America, will examine genetic and environmental influences that affect the accumulation of phytonutrients, specifically iron and zinc, in potatoes. Understanding the interaction between microorganisms in the soil and genes in the plant could lead to strategies to alleviate human micronutrient deficiencies in populations dependent upon the potato as a staple.Hollow Microcapsules with pH-Responsive MacroporesTopic: Explore New Solutions in Global Health Priority AreasCarlo Montemagno of the University of Cincinnati in the U.S. proposes to develop hollow microcapsules with pores that only form at neutral pH, allowing vaccines in the capsules to be released only after bypassing the acidic stomach environment and arriving at the mucosal tissues of the lower gastrointestinal tract. This new oral vaccine delivery system could allow highly immunogenic vaccines to be administered with increased efficacy and allow for vaccine dose-sparing.Improving Fetal Growth Rates in Developing CountriesTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenLaura Woollett of the University of Cincinnati in the U.S., in collaboration with the MRC International Nutrition Group in The Gambia, will test the theory that low levels of plasma cholesterol in pregnant mothers is a factor limiting fetal growth in poor populations. Determining the relationship between the cholesterol levels of the mothers and the size of their newborns could lead to better nutritional supplementation to reduce the number of low birth weight infants.Improving Health of Low Birth Weight Infants Via Nutritional EpigeneticsTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenLin Xi and Jack Odle of North Carolina State University in the U.S. propose to use metabolomic and epigenetic analysis in a pig model to explore the mechanisms by which dietary choline and DHA (docosahexanoate) supplementation in gestating mothers could alleviate the effects of malnutrition on infants. The data could inform the design of nutritional supplements for mothers to improve the developmental outcomes of low birth weight infants.Killing a Virus With Another Virus: Tales of a Suicidal VirusTopic: Protect Crop Plants from Biotic Stresses From Field to MarketCarl Spetz of Bioforsk in Norway will eliminate sweet potato feathery mottle virus (SPFMV) from infected sweet potato plants by co-infecting the plants with cucumber mosaic virus (CMV) engineered with RNAi against SPFMV. Because CMV can only survive on sweet potato in the presence of SPFMV, it is itself eliminated as it eliminates SPFMV.Leishmania Virus--A Diagnostic and Therapeutic ToolTopic: Explore New Solutions in Global Health Priority AreasCatherine Ronet and Mary-Anne Hartley of the University of Lausanne in Switzerland will targeta naturally occurring RNA virus in Leishmania parasites (LRV) that not only aids parasite survival, but also causes a destructive inflammatory response in humans. They will develop and refine a LRV diagnostic device and formulate the virus as a vaccine candidate for the prevention and treatment of the leishmaniasis.Low-Cost, Mobile Phone-Based Detection of Neonatal JaundiceTopic: Explore New Solutions in Global Health Priority AreasChetan Patil of Vanderbilt University in the U.S. will test the ability of a mobile phone platform to perform measurements of bilirubin with sufficient sensitivity to accurately identify jaundiced newborns. By using the phone‟s camera and simple applications to detect levels of bilirubin through the skin, a quantitative assessment can be made so that simple treatment can be initiated.Materials for Food Storage with Antiseptic PropertiesTopic: Protect Crop Plants from Biotic Stresses From Field to MarketEwa Kowalska of Ulm University in Germany will develop photoactive materials with antiseptic and antifungal properties for food storage safety. Titanium oxide (titania) will be evaluated for its antiseptic properties, the ultimate goal being a paint that could be applied to food storage containers.Modulation of AHR Activity for Intestinal Health in ChildrenTopic: Explore Nutrition for Healthy Growth of Infants and ChildrenAndrew Patterson and Gary Perdew of the Pennsylvania State University in the U.S. will investigate the ability of the aryl hydrocarbon receptor (AHR) to modulate intestinal inflammation and general intestine health, and they will test in a mouse model whether natural AHR activators given to nursing mothers provide protection to pups from experimental intestinal inflammation.More Vaccination, Less Debris: Developing Compostable Vaccine PackagingTopic: Design New Approaches to Optimize Immunization SystemsClaire Dillavou of the University of California, Los Angeles in the U.S. will develop compostable vaccine packaging to diminish the environmental impact of residual debris from mass vaccination campaigns in developing countries lacking adequate disposal infrastructure.Nature-Inspired Nanoswitches For HIV Antibodies DetectionTopic: Apply Synthetic Biology to Global Health ChallengesFrancesco Ricci of the University of Rome, Tor Vergata in Italy and collaborator Alexis Vallee-Belisle of the University of California, Santa Barbara propose to develop molecular nanoswitches that provide a visual cue when they bind to HIV antibodies for use in a rapid (one minute) diagnostic test to detect and quantify HIV antibodies in serum samples.Net Zero Energy Warehousing Systems for Drugs and VaccinesTopic: Design New Approaches to Optimize Immunization SystemsJonathan Colton of Georgia Institute of Technology in the U.S., with John Lloyd, Andrew Garnett and Steve McCarney, will solicit proposals from industry to create a full set of requirements and engineering specifications for the development of a new “net zero energy” warehouse and。

The human genome contains only about 21,000 distinct protein-coding genes1, which is substantially fewer than originally expected. However, owing to alternative splicing mechanisms and many post-translational modifications, these ~21,000 genes are estimated to produce more than one million different proteins2. Although the proteome (that is, the whole set of proteins that are expressed in a specified cell, tissue or organism) is highly complex, it is outnum-bered by the many smaller peptides that are generated from full-length proteins by pro-teolytic cleavage. The largest fraction of the peptidome (that is, all of the peptides that are present in a specific cell, tissue, organ-ism or system) comprises peptide fragments that are generated by the proteolysis of larger precursor molecules. Only a small fraction of the peptidome has been functionally char-acterized, owing to its enormous complexity and the lack of broadly applicable methods that enable its unbiased analysis.For many years, the peptidome was commonly regarded as ‘biological trash’. However, it has recently been recognized that the expression and activities of the more than 500 human proteases that generate these peptides are altered under pathologi-cal conditions3. Furthermore, accumulat-ing evidence suggests that some cleavage products of larger precursor proteins exert specific and sometimes highly unexpectedactivities against human pathogens4–14. Thus,the human peptidome is a rich source ofdisease-specific biomarkers. It has long beenknown that humans and other mammalianspecies have evolved various antimicrobialpeptides as a first line of defence againstviruses, bacteria and fungi15–18. Although therole of some antimicrobial peptides, such asdefensins and cathelicidins, in innate immu-nity, immune modulation and inflamma-tion is well-established15–18, it seems highlylikely that many important peptidic immunemodulators and effectors in the human bodyremain to be identified.Thousands of antimicrobial peptideshave been isolated from various natu-ral sources (for example, bacteria, fungi,plants and animals). Furthermore, severalinhibitors of bacterial infections have beenisolated from human immune cells (forexample, α-defensins)19, skin (for exam-ple, β-defensins)20, saliva (for example,histatins)21, nasal secretions (for exam p le,lysozyme)22, seminal plasma (for example,cathelicidins)23,24 and sweat (for exam p le,dermicidin)25. The various types of anti-microbial peptides15–25 and their possibleclinical applications26–31 have been the topicsof several recent reviews. In this Innovationarticle, we discuss the discovery of novelantimicrobial peptides by the systematicscreening of highly complex peptide librariesthat are derived from human bodily fluidsor tissues. In particular, we focus on theisolation of endogenous agents that affectHIV-1 infection by unexpected mechanismsand that may have implications beyondHIV/AID S. Furthermore, we present theadvantages and disadvantages of this strategy.Finally, we discuss how endogenous peptidesthat have been isolated from the human pep-tidome can be optimized for therapeutic orbasic research applications, and we highlightfuture directions and challenges.Human-derived peptide librariesPeptide libraries that have been generatedfrom human bodily fluids or tissues, suchas blood or lymphoid tissues, contain essen-tially all peptides and small proteins of therespective source in their final processedand physiologically relevant forms32–34. Thus,they are an excellent source for the identifi-cation of as-yet-unknown bioactive peptides.Bioactive peptides from human sources.Most clinically approved drugs are smallmolecules, and most of these compoundshave a molecular weight of much less than1 kDa. Such a small size is one of the require-ments for uptake through the gastrointesti-nal tract and thus oral application. By con-trast, large protein drugs, such as antibodies,have molecular weights of up to 150 kDaand need to be administered parenterally.The size of peptides, which are usually upto 50 amino acids in length, is betweenthese two drug classes, although there isno strict demarcation28–31. Antimicrobialmolecules are found in all three size classes,including small molecules such as penicil-lin (which is 0.334 kDa), the glycopeptidevancomycin (which is 1.449 kDa) and largerabies-specific immunoglobulins (which areabout 150–170 kDa)28. Other examples ofclinically approved antimicrobial peptidesare the antibiotics polymyxin B and poly-myxin E (also known as colistin) and theantiviral agent interferon-α. Owing to theincreasing resistance of microorganisms toconventional antibiotics, there is currently agrowing interest in the development of anti-microbial peptides for clinical applications,and several candidates are in clinical trials orI N N OVAT I O NDiscovery of modulators of HIV‑1infection from the human peptidomeJan Münch, Ludger Ständker, Wolf-Georg Forssmann and Frank KirchhoffAbstract | Almost all human proteins are subject to proteolytic degradation, whichproduces a broad range of peptides that have highly specific and sometimesunexpected functions. Peptide libraries that have been generated from human bodilyfluids or tissues are a rich but mostly unexplored source of bioactive compounds thatcould be used to develop antimicrobial and immunomodulatory therapeutic agents.In this Innovation article, we describe the discovery, optimization and application ofendogenous bioactive peptides from human-derived peptide libraries, with aparticular focus on the isolation of endogenous inhibitors and promoters of HIV-1infection.PERSPECTIVES NATURE REVIEWS |MICROBIOLOGY ADVANCE ONLINE PUBLICATION |1 Nature Reviews Microbiology| AOP, published online 11 August 2014; doi:10.1038/nrmicro3312antibacterial and antiviral peptides that have responses than small-molecule or protein drugs, particularly if they are derived from a human source16. However, the isolation of individual bioactive peptides from highly complex human bodily fluids or tissues is a challenging task.Generating peptide libraries from human haemofiltrate.Human tissues and bodily fluids are usually only available in very lim-ited quantities, and standardized methods to purify bioactive agents from the large number of highly diverse peptides and proteins that are contained in these tissues and fluids are mostly unavailable. Large quantities of starting materials are advanta-geous from a technical and experimental standpoint. One abundant source of human peptides is haemo f iltrate, which is a waste product of dialysis that contains essentially all blood components that have a molecu-lar weight of less than 20–30 kDa (BOX 1) and is available from patients with chronic renal failure at quantities of thousands of litres. A combination of ultrafiltration, fol-lowed by cation-exchange separation and reverse-phase chromatography, enables the standardized separation and concentration of all of the peptides and small proteins that are present in haemofiltrate into about 300–500 fractions32,33(BOX 1). The analysis of these fractions does not require the high-throughput methodologies and facilities that are required for the testing of other libraries, which often comprise hundreds of thou-sands of compounds. For example, specific assays that test the inhibition of a particular pathogen, the modulation of specific cel-lular functions or the induction of selected immune factors can be used to identify and purify the most active agents that are present in these fractions. Such peptide libraries are a useful source for the discovery of novel bioactive agents, as they represent the enor-mous structural and functional diversity of the human peptidome and the peptides are present in their final processed, and thus bioactive, forms32–35. Notably, haemofiltrate contains not only endocrine peptides but also peptides that function in a paracrine or autocrine manner, as a small fraction of these peptides are released into the extracel-lular space and are found in the blood34,35. Thus, haemofiltrate-derived peptide librar-ies contain essentially the entire circulating blood peptidome in a lyophilized, bioactive and highly concentrated form. However, in addition to peptides that naturally exist in the human body, these libraries may also contain proteolytic cleavage products that specifically arise and accumulate during the collection or storage of body fluids orP E R S P E C T I V E S2 | ADVANCE ONLINE PUBLICATION /reviews/microtissues. Nonetheless, these peptide libraries are an excellent source for the identification of endogenous bioactive peptides.Alternative human rge quantities of starting material are advantageous but not always obligatory for the identification of novel endogenous bioactive peptides. In fact, many peptides or proteins only become active and exert their respective functions in specific compartments or at sites of infec-tion and/or inflammation; for example, saliva, genital fluid, milk and sweat contain particularly large numbers of antimicrobial peptides16. To isolate agents that are not cir-culating in the bloodstream, it is important to generate peptide libraries from sources other than haemofiltrate, and in principle, they can be produced from any tissue or bodily fluid. Using ultrafiltration and cation exchange followed by reverse-phase chroma-tography, peptide libraries have been gener-ated from human plasma, milk, placenta, sweat, seminal plasma and saliva (J.M., L.S., W.-G.F. and F.K., unpublished observations), and some antimicrobial peptides that have been isolated from these libraries are listed in TABLE 1.Human peptides that affect HIV-1 infection Most clinically used antiretroviral drugs are small molecules that have been selected to specifically target and inhibit viral proteins that are essential for HIV-1 replication.Enfuvirtide (also known as T-20; Trimeris)is the only currently licensed peptidicantiretroviral drug36. Enfuvirtide is derivedfrom an α-helical region in the viral trans-membrane glycoprotein known as heptadrepeat 2 (HR2). By mimicking HR2 andcompetitively binding to another α-helicalregion of gp41, known as HR1, enfuvirtideprevents the interaction between HR1 andHR2 and thus prevents the conformationalchange that is required for virion fusionwith the host cell37. Although this proteinis not from a human source, enfuvirtideprovides an example of the feasibility ofdeveloping peptide antiretroviral agents.In addition, chemokines, such as CXC-chemokine ligand 12 (CXCL12; also knownas SDF-1) and CC-chemokine ligand 3(CCL3; also known as MIP-1α), CCL4 (alsoknown as MIP-1β) or CCL5 (also known asRANTES), bind to the G protein-coupledreceptors (GPCRs) CXC-chemokine recep-tor 4 (CXCR4) and CC-chemokine recep-tor 5 (CCR5), respectively, and they preventinfection by HIV-1 strains that use the samereceptors for viral entry. In fact, these inhibi-tory activities were crucial for the discoveryof CCR5 as the main co-receptor for HIV-1entry38,39. Modified derivatives of CCL5 areextremely potent inhibitors of R5-tropicHIV-1 strains in vitro40, but they have a lowefficiency in vivo. Several intracellular hostproteins that inhibit HIV-1 replication at dif-ferent steps of the viral life cycle41 — whichare known as restriction factors — have morerecently been discovered, and it seems highlylikely that additional antiretro v iral peptidesand/or proteins remain to be discovered.Identification of novel endogenous HIV‑1inhibitors.An initial screen of a haemo-filtrate-derived peptide library for CCR5ligands identified a naturally occurringtruncated form of the abundant humanchemokine CCL14 as a potent agonist ofthe receptor, which inhibits infection byR5-tropic HIV-1 strains6,42. Notably, otherchemokines, such as CXCL4 and specificfragments of CXCL4, also inhibit HIV-1(REF. 43)and other important pathogens,such as Plasmodium falciparum44. Screen-ing of further haemofiltrate-derived peptidefractions identified another HIV-1 inhibitor:a 20-residue C-proximal subfragment of theserine protease α1-antitrypsin, which hasbeen designated virus-inhibitory peptide(VIRIP). This peptide was purified by mul-tiple rounds of peptide separation and anti-viral screening8(FIG. 1). VIRIP specificallyinteracts with the viral fusion peptide thatis located at the amino terminus of the gp41transmembrane domain, which is a regionthat is distinct from the HR1 region that istargeted by enfuvirtide. VIRIP blocks anentry step before membrane fusion — thatTable 1 |Modulators of microbial infections identified from human peptide libraries*peptide 2; E.­coli, Escherichia­coli; hBD1, human β-defensin 1; hBNP, human brain-type natriuretic peptide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCMV, human cytomegalovirus; hHEMβ , human β-chain of haemoglobin; LEAP1, liver-expressed antimicrobial peptide 1; S.­carnosus, Staphylococcus­carnosus; PAP, prostatic acid phosphatase; VIRIP, virus-inhibitory peptide. *This table only lists peptides that have been identified by the systematic screening of human peptide libraries, which is outlined in FIG. 1. Many other important antimicrobial peptides have been discovered by different approaches and are described in recent reviews15–18. Peptides that are in clinical development or have recently been approved by the FDA are described in REFS 28–31,91.P E R S P E C T I V E S NATURE REVIEWS |MICROBIOLOGY ADVANCE ONLINE PUBLICATION |3is, penetration and insertion of the viral fusion peptide into the host cell membrane (FIG. 2). This is a novel and unexpected mech-anism, as the fusion peptide only becomes accessible during the viral entry process after conformational changes of gp41 (REF. 45). Thus, it was assumed that this anchoring step cannot be inhibited45. The finding that viral fusion peptides can be targeted by an antiviral agent may offer perspectives beyond HIV/AIDS, as many enveloped viruses, such as influenza virus, hepatitis C virus, filovirus, coronaviru s, arenavirus and hepadnaviruses use fusion peptides to infect their target cells46–50. Owing to this distinct inhibitory mechanism, VIRIP is active against HIV-1 strains that are resistant to other antiretro-viral drugs, including other entry inhibitors8.A proteomics approach identified VIRIP in plasma from patients with acute HIV-1 infec-tion, which suggests that it may contribute to the earliest systemic antiviral responsein HIV-1 infection51. Thus, screening ofh a emofiltrate-derive d peptide libraries enabled the isolation of two novel HIV-1 inhibitors: a CCR5 agonist and a peptide that targets the HIV-1 gp41 fusion peptide (FIG. 2). It is remarkable that this approach did not identify the well-known CCR5 ligand CCL5 but instead identified a novel agonist, and this suggests that other relevant peptide ligands of GPCRs that are targets of many modern drugs remain to be discovered. Endogenous peptides that promote HIV‑1infection.The success of screening haemo-filtrate-derived peptide libraries encouragedthe screening of smaller-scale libraries fromother sources for novel antiviral agents. Theefficiency of virus transmission by sexualcontact is surprisingly low, which suggeststhat inhibitors of HIV-1 infection are presentin semen. Furthermore, it has been reportedthat semen contains various antimicrobialagents23,24,52–55 and cationic peptides thathave anti-HIV-1 activity56. However, unex-pectedly, screening of a semen-derivedpeptide library only identified fractions thatpromoted HIV-1 infection57, which mightreflect the specific adaptation of the virus forefficient transmission in this environment.Further analyses showed that C-proximalfragments of prostatic acidic phosphatase(PAP), which is an abundant enzyme insemen, self-assemble into cationic amyloidfibrils57. These fibrils, which are known assemen-derived enhancers of virus infection(SEVIs), increase the infectious virus titre incell culture by several orders of magnitude57and can facilitate virus infection in non-human primate models after exposure tolow viral doses that approximate the in vivosituation58. Virus attachment is usually inef-fective as the densities of the viral envelopeglycoproteins on the virion and the CD4receptor on the target cell are low and thesurfaces of viral and cellular membranesare negatively charged and usually repeleach other. Both SEVI fibril formation andits positive surface charge are crucial forincreased HIV-1 infection59, which suggeststhat SEVI promotes the attachment of thevirus to target cells by neutralizing the repul-sion between negatively charged viral andcellular membranes (FIG. 2). Interestingly, fol-low-up studies showed that an N-proximalfragment of PAP, as well as peptides that arederived from semenogelin 1 and semeno-gelin 2, which are the most abundant pro-teins in semen, also form amyloid fibrils thatincrease HIV-1 infection60,61. Endogenousamyloid fibrils have recently been detectedand structurally characterized in freshnon-modified ejaculates62. Notably, theirlevels correlate with the efficiency of semen-mediated promotion of HIV-1 infectionin vitro61,63. Thus, semen, which is the mainvector for HIV-1 transmission, containsamyloid fibrils that boost viral infectivityand are therefore novel targets to reduce therisk of sexual transmission. In fact, severalagents that block the formation or infection-promoting activity of semen-derived fibrilshave been reported64–68. Such amyloids mayalso be exploited by other pathogens, asrecent data show that semen amyloids alsoincrease cytomegalovirus (CMV) infec-tion69. Semen is the first bodily fluid that hasbeen identified to naturally contain amyloidfibrils in a non-diseased state62. The physi-ological role of seminal amyloids in healthyindividuals remains to be defined; however,it is tempting to speculate that they mightbe involved in both innate immunity70 andfertility.Optimization and development.VIRIPand CCL14(9–74) are both active againstHIV-1 in the lower micromolar range. Thisconcentration may well be physiologicallyrelevant, as it can readily be achieved byproteolytic cleavage of their highly abun-dant α1-antitrypsin and CCL14 precursors.However, this concentration is suboptimalfor therapeutic applications, which usuallyrequire activities in the nanomolar range71.Specific modifications of these naturalHIV-1 inhibitors increased their antiviralactivity by about two orders of magnitude.Chemical modifications of the N termi-nus of CCL14(9–74) rendered the peptideresistant to degradation and thereby stronglyincreased its antiretroviral potency72. In thecase of VIRIP, the introduction of aminoacid alterations that stabilize the active con-formation of the peptide and increase itshydrophobic interactions with the HIV-1fusion peptide greatly increased its antiviralgraphy. Each cycle of separation and screening reduces the complexity of the peptide samples untilthe purified bioactive agent can be identified by mass spectrometry analysis and peptide sequencing.VIRIP was shown to be a 20-residue (LEAIPMSIPPEVKFNKPFVF) fragment of the serine proteaseα1-antitrypsin8. The identified peptides can be functionally characterized and optimized for clinicaldevelopment.P E R S P E C T I V E S4 | ADVANCE ONLINE PUBLICATION /reviews/micropotency 8. Notably, short-term monotherapy with one of these optimized derivatives (known as VIR-576) was well tolerated and reduced plasma viral loads in treatment-naive patients with HIV-1 (REF . 73). This provides proof-of-concept that naturally occurring antiviral peptides can be opti-mized for therapeutic applications. Owing to their mode of application (that is, injection) and the high costs that are involved in pep-tide synthesis, VIRIP derivatives are not yet competitive with the many highly effec-tive and orally available small-molecule drugs that are available for the treatment of HIV/AIDS. Several strategies are underway to further increase the efficacy and applica-tion of VIRIP derivatives, including packag-ing them into nanoparticles to improve their delivery and stability, as well as structure-based optimization.Semen-derived amyloid fibrils have also been developed for further applications; as they boost HIV-1 infection, they could be used to improve retroviral gene delivery. Indeed, SEVI increases the efficiency of retroviral gene transfer, although only with moderate and variable efficiency 74. On the basis of these findings, broader screens were carried out for amyloidogenic peptides that function as promoters of retroviral transduc-tion. These studies led to the development of a synthetic 12-residue peptide known as EF-C (Protransduzin; Pharis Biotec GmbH)that instantaneously self-assembles into nanofibrils and promotes retroviral gene transfer more efficiently than semen-derived fibrils 75. Furthermore, EF-C captures virions and thus enables retroviral particles to be concentrated by conventional low-speed centrifugation instead of the more labori-ous ultracentrifugation, which has so far been used for virus concentration. These developments pave the way for a new class of promoters of retroviral gene transfer in basic research and clinical applications.Proteolytic cleavage as a common defence mechanism? It is remarkable that a single assay system (that is, the inhibition of HIV-1 infection) led to the discovery of three novel endogenous agents that affect HIV-1 entry by distinct mechanisms (FIG. 2). As discussed above, these peptides are structurally and functionally distinct from other positively charged amphipathic antimicrobial peptides and might have implications in addition to HIV/AIDS in innate and adaptive immunity as well as in sperm function and fertility. It is also striking that these newly identified modulators of HIV-1 infection are generated from highly abundant precursor proteins: α1-antitrypsin, PAP and semenogelins. The proteases that generate the bioactive pep-tides remain to be fully defined. However, at least in some cases, these proteases are released from immune cells and are inducedor activated by inflammation; for example, VIRIP is generated by matrix metallopro-teinase 9 (MMP9)8 and CCL14(9–74) is generated by serine proteases 6. Furthermore, these endogenous peptides interact with their respective targets in a highly specific manner, and subtle structural changes at the N terminus (for example, removing or add-ing single amino acid residues) disrupt their functional activities 6,8. Thus, it is tempting to speculate that the cleavage of abundant precursors may be a more common, fast and effective means to generate immunomodula-tors and antimicrobial peptides at the loca-tions and times that they are actually needed, and it is likely that such peptides have roles in various biological processes. Moreover, the rapid generation of specific effectors by the proteolysis of abundant precursor proteins instead of de novo protein synthe-sis may be particularly advantageous for innate defence mechanisms against invading pathogens for which immediate responses are crucial for effective counteraction.Notably, the local extracellular acidification that activates various proteases is currently emerging as a key regulatory concept of innate immunity 76,77.Other antimicrobial peptides. The use of peptide libraries from human sources for the discovery of novel factors that modu-late HIV-1 infection is a prime example of| MicrobiologyEnhanced attachment:PAP fibrilsInsertion inhibitor: VIRIPFusion inhibitor:EnfuvirtideCo-receptor binding inhibitor: CCL14(9-74)Figure 2 | Endogenous peptides that affect HIV-1 infection. Overview of the HIV-1 entry steps that are targeted by peptides. Amyloidogenic peptides that are derived from human semen, such as fragments of pros-tatic acidic phosphatase (PAP), can self-assemble into amyloid fibrils that promote the attachment of the virus to target cells by neutralizing the repulsion between negatively charged viral and cellular membranes 8,61. Unlike these semen-derived enhancing agents, human peptides that are isolated from haemofiltrate have antiviral activity. The truncated form of the abundant human chemokine CC-chemokine ligand 14 (CCL14(9–74)) inhibits entry of R5-tropic HIV-1 strains by binding to the CC-chemokine receptor 5 (CCR5) co-receptor 6,42. Human-derived virus-inhibitory peptide (VIRIP; which is a subfragment of the serine protease inhibitor α1-antitrypsin) also targets the fusion peptide by specifically interacting with the amino terminus of its transmembrane domain to block penetra-tion and insertion of the viral fusion peptide 8. Enfuvirtide is a synthetic peptide that is derived from an α-helical region in the HIV-1 transmem-brane glycoprotein 36. This inhibitor binds to helical repeat region 1 (HR1) of gp41, which is a region that is distinct from the region that is targeted by VIRIP , and prevents the formation of a hairpin structure that is required for membrane fusion, thereby blocking HIV-1 infection. The target sites for VIRIP and enfuvirtide in gp41 are indicated. From Forssman, W.-G. et al . Short-term monotherapy in HIV-infected patients with a virus entry inhibitor against the gp41 fusion peptide. Sci.­Transl.­Med . 2, 63re3 (2010). Reprinted with permission from AAAS.NATURE REVIEWS | MICROBIOLOGYADVANCE ONLINE PUBLICATION | 5peptide drug discovery; however, in prin-ciple, such libraries can be used to identify endogenous peptides that have any activ-ity of interest. The main prerequisite is a robust bioassay that enables the reliable screening of several hundreds of peptide fractions. A list of antiviral and antibacterial peptides that have been identified by screen-ing human peptide libraries is provided in TABLE 1. These agents were isolated from var-ious sources, such as haemofiltrate, placental tissue, milk and semen, and target various bacterial and viral pathogens. This list also includes human β-defensin 1 (REF. 78) — the first member of the family of β-defensins to be discovered in humans — which medi-ates the resistance of epithelial surfaces to microbial colonization. Several agents that have been isolated from human-derived peptide libraries show the typical features of most antimicrobial peptides — that is, they are cationic and amphipathic, and destabilize the membranes of pathogens (TABLE 1). The microbicidal action of other endogenous peptides that were identified in this screen was less predictable; for example, a recently discovered derivative of the neutrophil-activating peptide 2 (known as CYVIP) inhibits human CMV (HCMV) infection by binding to heparan sulphate proteoglycans, which function as attachment receptors for CMV79. Moreover, an endogenous human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) peptide has been isolated from human placental tissue. This fragment is internalized by Candida albicans and kills the pathogen by inducing rapid apoptotic death13. As these discoveries are based on only a limited number of antimicrobial screens, this suggests that the human pepti-dome might yield more surprising findings.Obstacles to purifying bioactive peptides. Currently, a key challenge is the purification and identification of the active compounds from the complex peptide mixture in active fractions of the generated peptide libraries. However, rapid progress has been made in the development of improved methodolo-gies for peptide separation and analysis80; for example, state-of-the-art mass spectrometry methods enable the convenient deduction of peptide sequences from complex fragmenta-tion spectra. Furthermore, highly sensitive time-of-flight (TOF) mass spectrometers for matrix-assisted laser desorption–ionizatio n (MALDI) and electrospray ionization (ESI) already enable accurate mass determina-tion of peptides at femtomolar concen-trations. The combination of automated high-throughput sample handling, together with miniaturized liquid chromatographyapproaches and powerful mass spectrometrictechnologies, will greatly facilitate the iden-tification and characterization of bioactiveendogenous peptides in future studies.Endogenous peptides in the clinicThere are several disadvantages that limit theuse of peptides as therapeutic agents, includ-ing the fact they are usually not orally availa-ble and are often unstable. Nonetheless, theyare already used in the treatment of cancer,diabetes and cardiovascular diseases28,29.There is a growing interest in using peptidesin other therapeutic areas, particularly asantimicrobial agents, owing to the increas-ing number of multidrug-resistant bacteriaand viruses. Many antimicrobial peptides arebroadly active, as they often target conservedcomponents of bacterial membranes15–30.Furthermore, such peptides neutralize endo-toxins and it is difficult for most pathogensto develop resistance81; for example, HIV-1variants only showed reduced sensitivity toVIRIP after more than 1 year of cell culturepassage in the presence of the inhibitor82.Accumulating evidence also suggests thatantimicrobial peptides have immunomodu-latory and antineoplastic properties andthus offer prospects for the treatment ofinflammation and cancer83–85. Other poten-tial advantages of peptides are that they areusually easy to synthesize and can be readilymodified to optimize their activity, specific-ity and stability. Notably, the costs of peptidesyntheses are rapidly decreasing and noveldelivery methods may increase their efficacyand eventually even enable oral application.As a consequence, the number of peptidiccompounds that are entering clinical trialsis steadily increasing and many candidatesare in clinical and preclinical development29.Thus, although low stability under physi-ological conditions and lack of oral bioavail-ability are still important challenges26–31, thestrategy to optimize bioactive endogenouspeptides for clinical applications seems tobe highly promising. Alternatively, it maybe possible to induce or apply the proteasesthat generate the active peptides from theirabundant precursors to treat infectious orinflammatory diseases.Conclusions and perspectivesThe human peptidome probably containsmany unknown factors that may havekey roles in antimicrobial immunity andinflammatory processes. These bioactivepeptides cannot be identified by genomicsand transcriptomics approaches, as they donot detect post-translational modifications.Peptide libraries that are derived from bodilyfluids or tissues will facilitate the systematicand unbiased identification of these ‘hid-den treasures’. Some activities may certainlybe missed because they are relatively weakor because some antimicrobial agents maybe degraded or inactivated during sampleprocessing or storage. Nonetheless, thismethodology seems to be suitable to identifythe most potent and stable endogenous anti-microbial peptides. In the longer term, weenvision the highly reproducible generationof peptide libraries from multiple sources incombination with robust assays for the iden-tification of factors that affect key pathogensand biological activities. We also speculatethat novel powerful techniques for thepurification, optimization, production anddelivery of endogenous peptides will greatlyfacilitate their future clinical development.Jan Münch, Ludger Ständker, Wolf-Georg Forssmannand Frank Kirchhoff are at the Institute of MolecularVirology, Ulm University Medical Center,89081 Ulm, Germany.Jan Münch, Ludger Ständker and Frank Kirchhoffare also at Ulm Peptide Pharmaceuticals,Ulm University, 89081 Ulm, Germany.Wolf-Georg Forssmann is also at Pharis Biotec GmbH,30625 Hannover, Germany.Correspondence to F.K.e-mail: frank.kirchhoff@uni-ulm.dedoi:10.1038/nrmicro3312Published online 4 August 20141. Lander, E. S.et al. Initial sequencing and analysis ofthe human genome. Nature 409, 860–921 (2001).2.Jensen, O. N. Modification‑specific proteomics:characterization of post‑translational modifications bymass spectrometry. Curr. Opin. Chem. Biol. 8,33–41(2004).3.Petricoin, E. F., Belluco, C., Araujo, R. P. & Liotta, L. A.The blood peptidome: a higher dimension ofinformation content for cancer biomarker discovery.Nature Rev. Cancer 6, 961–967 (2006).4.Zucht, H. D., Raida, M., Adermann, K., Mägert, H. J. &Forssmann, W. G. Casocidin‑I: a casein‑αs2derivedpeptide exhibits antibacterial activity. FEBS Lett. 372,185–188 (1995).5. Mägert, H. J.et al. LEKTI, a novel 15‑domain type ofhuman serine proteinase inhibitor. J. Biol. Chem. 274,21499–21502 (1999).6. Detheux, M.et al. Natural proteolytic processing ofhemofiltrate CC chemokine 1 generates a potent CCchemokine receptor (CCR)1 and CCR5 agonist with anti‑HIV properties. J. Exp. Med. 192, 1501–1508 (2000).7. Liepke, C.et al. Human hemoglobin‑derived peptidesexhibit antimicrobial activity: a class of host defensepeptides. J. Chromatogr. B Analyt. T echnol. Biomed.Life Sci. 791, 345–356 (2003).8. Münch, J.et al. Discovery and optimization of anatural HIV‑1 entry inhibitor targeting the gp41 fusionpeptide. Cell 129, 263–275 (2007).9. Påhlman, L. I.et al. Antimicrobial activity of fibrinogenand fibrinogen‑derived peptides — a novel linkbetween coagulation and innate immunity. Thromb.Haemost. 109, 930–939 (2013).10.Papareddy, P., Mörgelin, M., Walse, B.,Schmidtchen, A. & Malmsten, M. Antimicrobial activityof peptides derived from human ß‑amyloid precursorprotein. J. Pept. Sci. 18, 183–191 (2012).11. Papareddy, P. et al. C‑terminal peptides of tissue factorpathway inhibitor are novel host defense molecules.J. Biol. Chem. 285, 28387–28398 (2010).12. Kalle, M. et al. Proteolytic activation transformsheparin cofactor II into a host defense molecule.J. Immunol. 190, 6303–6310 (2013).P E R S P E C T I V E S6 | ADVANCE ONLINE PUBLICATION /reviews/micro。

科学家发现近视基因或研制早期预防药物Genetic code linked to short sight foundDrugs to be given to children at genetic risk of myopia may now be a real possibility.()Get Flash PlayerScientists have discovered strands of genetic code linked to short sight, the most common eye disorder in the world.The findings shed light on what goes awry to make distant objects look blurred, and raises the prospect of developing drugs to prevent the condition.Understanding the biological glitches behind short-sightedness could help researchers develop eye drops or tablets that could be given to children to stop their vision from failing as they get older.Short-sightedness, or myopia, usually starts to manifest early on in life. The extent to which genes are to blame varies, but for those with the worst vision, around 80% of the condition is caused by genetic factors. Two separate studies, published in Nature Genetics journal, found variations in DNA that were more common in people with short sight. Chris Hammond, at King's College, London, found one section of DNA on chromosome 15 was more common in people with myopia. Caroline Klaver, at Erasmus Medical Centre in Rotterdam, found another strand, also on chromosome 15, linked to short sight.The variations in DNA amount to misspellings in the genetic code. These alter the activity of three genes that control the growth of the eyeball and ensure light entering the eye is converted into electrical pulses 脉冲 in the retina. 视网膜The discovery helps scientists piece together how a healthy eye becomes short-sighted and points the way to medicines to prevent it in children. "My hope is that we can identify a pathway that we can block with eyedrops or tablets that will stop the eye growing too much and without interfering with normal brain development or other processes in the body," Hammond told the Guardian.(Read by Renee Haines. Renee Haines is a journalist at the China Daily Web site.)科学家最近发现了一组与近视有关的遗传密码。

有心传宗接待猜谜防艾滋病用语英文回答：As an ambassador for HIV/AIDS prevention, it is crucial to communicate effectively and engage with the audience. One way to do this is through the use of riddles and puzzles. These can serve as a fun and interactive way to educate people about the importance of safe practices and prevention methods. Let me provide some examples of riddles and their corresponding explanations.Riddle 1: "I am a tiny creature that can be transmitted through unprotected sex. What am I?"Explanation: The answer to this riddle is "HIV." By using the term "tiny creature," it helps to create a visual image of the virus that is small and invisible to the naked eye. This riddle can be used as a conversation starter to discuss the risks associated with unprotected sex and the importance of using condoms.Riddle 2: "I am a word that starts with 'C' and ends with 'S.' I can prevent the spread of HIV. What am I?"Explanation: The answer to this riddle is "Condoms." By using a word that starts with 'C' and ends with 'S,' it creates a sense of anticipation and curiosity. This riddle can be used to emphasize the effectiveness of condoms in preventing the transmission of HIV and other sexually transmitted infections.Riddle 3: "I am a behavior that involves sharing needles and can increase the risk of contracting HIV. What am I?"Explanation: The answer to this riddle is "Needle sharing." By using the term "behavior," it highlights the importance of individual choices and actions. This riddle can be used to discuss the dangers of needle sharing among drug users and the need for harm reduction strategies such as needle exchange programs.中文回答：作为艾滋病防治的传宗接待者，有效沟通和与观众互动至关重要。

10.1128/AAC.45.5.1539-1546.2001.2001, 45(5):1539. DOI:Antimicrob. Agents Chemother. Matsuoka, Hiroshi Ohrui and Hiroaki MitsuyaMachida, Hiroyuki Gatanaga, Shiro Shigeta, Masao Ei-Ichi Kodama, Satoru Kohgo, Kenji Kitano, HaruhikoImmunodeficiency Virus Variants In VitroInhibitors of Multidrug-Resistant Human -Ethynyl Nucleoside Analogs: Potent ′4/content/45/5/1539Updated information and services can be found at: These include:REFERENCES/content/45/5/1539#ref-list-1at: This article cites 36 articles, 13 of which can be accessed free CONTENT ALERTSmore»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new /site/misc/reprints.xhtml Information about commercial reprint orders: /site/subscriptions/To subscribe to to another ASM Journal go to: on May 14, 2014 by YONSEI UNIV LIBRARY/Downloaded fromA NTIMICROBIAL A GENTS AND C HEMOTHERAPY ,0066-4804/01/$04.00ϩ0DOI:10.1128/AAC.45.5.1539–1546.2001May 2001,p.1539–1546Vol.45,No.5Copyright ©2001,American Society for Microbiology.All Rights Reserved.4Ј-Ethynyl Nucleoside Analogs:Potent Inhibitors of Multidrug-Resistant Human Immunodeficiency Virus Variants In VitroEI-ICHI KODAMA,1SATORU KOHGO,2KENJI KITANO,3HARUHIKO MACHIDA,3HIROYUKI GATANAGA,4SHIRO SHIGETA,5MASAO MATSUOKA,1,6HIROSHI OHRUI,2AND HIROAKI MITSUYA 4,6*Laboratory of Virus Immunology,Institute for Virus Research,Kyoto University,Kyoto 606-8507,1Department of Applied Biological Chemistry,Faculty of Agriculture,Tohoku University,Sendai 981-8555,2Biochemicals Division,Yamasa Corporation,Chiba 288-0056,3Department of Microbiology,Fukushima Medical University School of Medicine,Fukushima 960-1295,5and Department of Immunopathophysiology and Internal Medicine II,Kumamoto University School of Medicine,Kumamoto 860-0811,6Japan,and Experimental Retrovirology Section,Department ofDevelopmental Therapeutics,Medicine Branch,National Cancer Institute,Bethesda,Maryland 208924Received 6October 2000/Returned for modification 20December 2000/Accepted 15February 2001A series of 4؅-ethynyl (4؅-E )nucleoside analogs were designed,synthesized,and identified as being active against a wide spectrum of human immunodeficiency viruses (HIV),including a variety of laboratory strains of HIV-1,HIV-2,and primary clinical HIV-1isolates.Among such analogs examined,4؅-E -2؅-deoxycytidine (4؅-E -dC),4؅-E -2؅-deoxyadenosine (4؅-E -dA),4؅-E -2؅-deoxyribofuranosyl-2,6-diaminopurine,and 4؅-E -2؅-deoxy-guanosine were the most potent and blocked HIV-1replication with 50%effective concentrations ranging from 0.0003to 0.01␮M in vitro with favorable cellular toxicity profiles (selectivity indices ranging 458to 2,600).These 4؅-E analogs also suppressed replication of various drug-resistant HIV-1clones,including HIV-1M41L/T215Y ,HIV-1K65R ,HIV-1L74V ,HIV-1M41L/T69S-S-G/T215Y ,and HIV-1A62V/V75I/F77L/F116Y/Q151M .Moreover,these analogs inhibited the replication of multidrug-resistant clinical HIV-1strains carrying a variety of drug resistance-related amino acid substitutions isolated from HIV-1-infected individuals for whom 10or 11different anti-HIV-1agents had failed.The 4؅-E analogs also blocked the replication of a non-nucleoside reverse transcrip-tase inhibitor-resistant clone,HIV-1Y181C ,and showed an HIV-1inhibition profile similar to that of zidovudine in time-of-drug-addition assays.The antiviral activity of 4؅-E -thymidine and 4؅-E -dC was blocked by the addition of thymidine and 2؅-deoxycytidine,respectively,while that of 4؅-E -dA was not affected by 2؅-deoxya-denosine,similar to the antiviral activity reversion feature of 2؅,3؅-dideoxynucleosides,strongly suggesting that 4؅-E analogs belong to the family of nucleoside reverse transcriptase inhibitors.Further development of 4؅-E analogs as potential therapeutics for infection with multidrug-resistant HIV-1is warranted.Combination chemotherapy or highly active antiretroviral therapy (HAART)using two or more reverse transcriptase (RT)inhibitors (RTIs)and protease inhibitors (PIs)has dra-matically improved the quality of life and survival of patients infected with human immunodeficiency virus type 1(HIV-1)(10,23).However,the ability to provide effective long-term antiretroviral therapy for HIV-1infection has become a com-plex issue,since a number of patients who initially achieved very favorable viral suppression later experience treatment failure (40).Moreover,30to 40%of HIV-1-infected individ-uals who had received no prior antiviral therapy fail to achieve viral suppression to undetectable levels (9,17).In addition,10to 40%of antiviral therapy-naive individuals infected with HIV-1have persistent viral replication (plasma HIV RNA Ͼ500copies/ml)under HAART (11,12,37),possibly due to transmission of drug-resistant HIV-1variants (40).Thus,the development of novel compounds that are active against drug-resistant HIV-1variants and that prevent or delay the emer-gence of resistant HIV-1variants is urgently needed.Certain 4Ј-substituted nucleosides have been described inthe literature.Maag et al.(19)reported that 4Ј-azido-2Ј-de-oxythymidine (4Ј-AZT),the hydrogen atom at the 4Јposition of which was substituted for with an azido group,exerted potent activity against HIV-1in vitro.Subsequently,Chen and colleagues (4)reported that 4Ј-AZT was active against HIV-1through its DNA chain-terminating activity.More recently,Sugimoto et al.have reported that 4Ј-substituted nucleosides including 4Ј-ethynylthymidine exhibited potent activity against not only HIV-1,but also herpes simplex virus type 1(38).We recently designed and synthesized a series of 4Ј-ethynyl (4Ј-E )-2Ј-deoxynucleosides and their analogs and identified several highly potent anti-HIV-1compounds,including 4Ј-E -2Ј-deoxy-cytidine (4Ј-E -dC),4Ј-E -2Ј-deoxyadenosine (4Ј-E -dA),4Ј-E -2Ј-deoxyribofuranosyl-2,6-diaminopurine (4Ј-E -dDAP),and 4Ј-E -2Ј-deoxyguanosine (4Ј-E -dG).These 4Ј-E compounds,unlike all of the currently available nucleoside RTIs (NRTIs),lack the 2Ј,3Ј-dideoxyribose configuration but have a 2Ј-deoxyribose configura-tion (Fig.1and Table 1).All of these 4Ј-E compounds blocked the replication of a wide spectrum of laboratory and clinical HIV-1strains in vitro with low cellular toxicities.These compounds also suppressed the replication of various drug-resistant HIV-1clones,including HIV-1M41L/T215Y ,HIV-1L74V ,HIV-1K65R ,HIV-1M41L/T69S-S-G/T215Y ,HIV-1Y181C ,and HIV-1A62V/V75I/F77L/F116Y/Q151M .These 4Ј-E compounds also*Corresponding author.Mailing address:Medicine Branch,Build-ing 10,Room 5A11,National Cancer Institute,9000Rockville Pike,Bethesda,MD 20892.Phone:(301)402-3631.Fax:(301)402-0709.E-mail:hmitsuya@.1539on May 14, 2014 by YONSEI UNIV LIBRARY/Downloaded fromsuppressed various multidrug-resistant clinical HIV-1variants carrying a variety of drug resistance-related amino acid substi-tutions,which were isolated from patients for whom virtually all of the currently available antiviral regimens had failed.Furthermore,we demonstrate in this study that these 4Ј-E analogs most likely block HIV-1by functioning as NRTIs.MATERIALS AND METHODSAntiviral agents.3Ј-Azido-3Ј-deoxythymidine (AZT,or zidovudine),2Ј,3Ј-dideoxyinosine (ddI,or didanosine),and 2Ј,3Ј-dideoxycytidine (ddC,or zalcita-bine)were purchased from Sigma (St.Louis,Mo.).(Ϫ)-2Ј,3Ј-Dideoxy-3Ј-thiacy-tidine (3TC,or lamivudine)was a kind gift from R.F.Schinazi (Atlanta,Ga.).A series of 4Ј-position-substituted nucleosides were designed and synthesized as described elsewhere (16,27,28).Their basic formula is shown in Fig.1.Anon-NRTI (NNRTI),MKC-442,was a gift from Mitsubishi Kasei Corporation (Yokohama,Japan)(2).Cells.MT-4and H9cells were grown in an RPMI 1640-based culture medium,and Cos-7cells were grown in Dulbecco’s modified Eagle medium (DMEM);each of these media was supplemented with 10%fetal calf serum (FCS;HyClone Laboratories,Logan,Utah),2mM L -glutamine,50U of penicillin per ml,and 50␮g of streptomycin per ml.HeLa-CD4-LTR/␤-gal cells (14)were kindly provided by M.Emerman through the AIDS Research and Reference Reagent Program,Division of AIDS,National Institute of Allergy and Infectious Diseases (Be-thesda,Md.).Prior to use,HeLa-CD4-LTR/␤-gal cells were propagated in DMEM supplemented with 10%FCS,0.1ng of hygromycin B per ml,and 200␮g of Geneticin per ml.In the anti-HIV assay,cells were cultured in the DMEM-based culture medium with addition of 50U of penicillin per ml and 50␮g of streptomycin per ml instead of hygromycin B and Geneticin.Peripheral blood mononuclear cells (PBMCs)were obtained from healthy HIV-1-seronegative donors by Ficoll-Hypaque gradient centrifugation and were stimulated for 3days with phytohemagglutinin M (PHA;10␮g/ml;Sigma)prior to use.Viruses and construction of recombinant HIV-1clones.Three laboratory strains,HIV-1LAI ,HIV-2ROD ,and HIV-2EHO ,were employed.Multidrug-resis-tant clinical HIV-1strains were isolated from patients with AIDS who had been treated with 9to 11anti-HIV-1drugs for 39to 64months,as previously de-scribed (42).These clinical HIV-1isolates were passaged once or twice in PHA-stimulated PBMCs (PHA-PBMCs),and titers of the culture supernatants obtained were determined for infectivity and stored at Ϫ70°C until use.Recombinant infectious HIV-1clones carrying various mutations in the pol gene were generated as previously described (35,39,42).Briefly,the desired mutations were introduced into the Xma I-Nhe I region (759bp)of pTZNX1,which encoded Gly-15to Ala-267of HIV-1RT (strain BH 10),by the oligonu-cleotide-based mutagenesis method (42).The Xma I-Nhe I fragment was inserted into a pHXB2RIP7(a kind gift from M.Reitz,Jr.,National Institutes of Health,Bethesda,Md.)-based plasmid,pSUM9,generating various molecular clones with the desired mutations.Each molecular clone (10␮g/ml as DNA)was transfected into Cos-7cells (4ϫ105cells/100-mm-diameter dish)by the calcium phosphate method (Promega,Madison,Wis.).After 24h,MT-2cells (106cells/dish)were added and cocultured with Cos-7cells for an additional 24h.When an extensive cytopathic effect was observed,cell supernatants wereharvested,FIG.1.Structures of 4Ј-substituted nucleosides.All nucleoside an-alogs discussed here have substitutions at the 4Јposition of the sugar moiety shown here except for the two compounds 4Ј-E -araT and 4Ј-E -araC,which have an arabinofuranosyl sugar moiety.See Table 1.TABLE 1.Anti-HIV-1activity of 4Ј-substituted nucleosides in MT-4cells aCompound (abbreviation)BaseSugar modification at X(2Ј-)Y(3Ј-)Z(4Ј-)EC 50(␮M)bCC 50(␮M)bSelectivity index4Ј-Ethynylribosylthymine (4Ј-E -rT)Thymine -OH -OH -CCH Ͼ400Ͼ400ND c 4Ј-Ethynylthymidine (4Ј-E -T)Thymine -H -OH -CCH 0.83Ϯ0.46Ͼ400Ͼ4824Ј-Ethynylarabinofuranosylthymine (4Ј-E -araT)Thymine ␤-OH -OH -CCH 119Ϯ22Ͼ400Ͼ44Ј-Ethylthymidine (4Ј-Et-T)Thymine -H -OH -CH 2CH 3Ͼ400Ͼ400ND 4Ј-Vinylthymidine (4Ј-V-T)Thymine -H -OH -CHCH 23.9Ϯ1.0Ͼ400Ͼ1034Ј-Hydroxyethylthymidine (4Ј-HE-T)Thymine -H -OH -CH 2CH 2OH 7.0Ϯ4.3Ͼ400Ͼ574Ј-Ethynyl-5-iodo-2Ј-deoxyuridine (4Ј-E -IdU)5-Iodo-uracil -H -OH -CCH 0.29Ϯ0.063Ͼ400Ͼ1,3804Ј-Ethynyl-5-ethyl-2Ј-deoxyuridine (4Ј-E -EtdU)5-Ethyl-uracil-H -OH -CCH Ͼ400Ͼ400ND 4Ј-Ethynyl-5-bromovinyl-2Ј-deoxyuridine (4Ј-E -BVDU)5-Bromovinyl-uracil -H -OH -CCH Ͼ15 1.5ND 3Ј-Azido-3Ј-deoxythymidine (AZT)Thymine -H -N 3-H 0.032Ϯ0.001129.4Ϯ8.79,1904Ј-Ethynyl-2Ј-deoxycytidine (4Ј-E -dC)Cytosine -H -OH -CCH 0.0048Ϯ0.001 2.2Ϯ1.04584Ј-Ethynylarabinofuranosylcytosine (4Ј-E -araC)Cytosine ␤-OH -OH -CCH 0.043Ϯ0.011 2.0Ϯ0.446.54Ј-Methyl-2Ј-deoxycytidine (4Ј-Me-dC)Cytosine -H -OH -CH 30.015Ϯ0.063 1.0Ϯ0.4166.74Ј-Fluoromethyl-2Ј-deoxycytidine (4Ј-FMe-dC)Cytosine -H -OH -CH 2F 0.0068Ϯ0.00360.12Ϯ0.02182Ј,3Ј-Dideoxy-3Ј-thiacytidine (3TC)Cytosine-H none -H 0.10Ϯ0.035Ͼ100Ͼ1,0004Ј-Ethyl-N 6-methyladenosine (4Ј-E -NM-A)6-Methyladenine -OH -OH -CCH Ͼ400Ͼ400ND 4Ј-Ethynyladenosine (4Ј-E -A)Adenine -OH -OH -CCH Ͼ400Ͼ400ND 4Ј-Ethynyl-2Ј-deoxyadenosine (4Ј-E -dA)Adenine -H -OH -CCH 0.0098Ϯ0.004316Ϯ7.91,6304Ј-Ethynyl-2Ј-deoxyribofuranosylpurine (4Ј-E -dP)Purine-H -OH -CCH 135Ϯ25Ͼ400Ͼ34Ј-Ethynyl-2Ј-deoxyribofuranosyl-2,6-diaminopurine (4Ј-E -dDAP)2,6-Diaminopurine -H -OH -CCH 0.00034Ϯ0.000030.9Ϯ0.172,6004Ј-Ethynyl-2Ј-deoxyinosine (4Ј-E -dI)Hypoxanthine -H -OH -CCH 0.13Ϯ0.035137Ϯ391,0534Ј-Ethynyl-2Ј-deoxyguanosine (4Ј-E -dG)Guanine-H -OH -CCH 0.0015Ϯ0.00031.4Ϯ0.169334Ј-Ethynyl-6-chloro-2Ј-deoxyguanosine (4Ј-E -CldG)6-Chloroguanine-OH -OH -CCHϾ400Ͼ400NDa Anti-HIV-1activity was determined by the MTT method.AZT and 3TC served as controls.bThe data shown are mean values with standard deviations derived from the results of three independent experiments.cND,not determined.1540KODAMA ET AL.A NTIMICROB .A GENTS C HEMOTHER .on May 14, 2014 by YONSEI UNIV LIBRARY/Downloaded fromand the virus was further propagated in H9cells.The culture supernatant was harvested,the titer was determined for infectivity,and the sample was stored at Ϫ70°C until use.The presence of intended mutations and the absence of unin-tended mutations in infectious clones were confirmed by determination of the nucleotide sequence of proviral DNA isolated from the virus-producing H9cells. HIV-1HXB2D was generated by using pSUM9(35)and served as a wild-type infectious clone(HIV-1wt).Determination of drug susceptibility of HIV-1.The inhibitory effects of test compounds on HIV-1replication were monitored by the inhibition of virally induced cytopathicity in MT-4cells.Briefly,MT-4cells were suspended at105 cells/ml and exposed to HIV-1LAI at10050%tissue culture infectious doses (TCID50s).Immediately after viral exposure,the cell suspension(104cells in100␮l)was brought into each well of a96-wellflat microtiter culture plate(Costar, Cambridge,Mass.)containing various concentrations of test compounds.After incubation for5days,the number of viable cells was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)method as previ-ously described(15,30).The sensitivity of infectious clones to various RTIs was determined in the multinuclear activation of the galactosidase indicator(MAGI)assay(14),with some modifications using the viral preparations titrated as previously described (20).Briefly,target cells(HeLa CD4-LTR/␤-gal;104/well)were plated in96-well flat microtiter culture plates.On the following day,the medium was aspirated, and the cells were inoculated with HIV-1clones(70MAGI units/well,which gave70blue cells after48h of incubation)and cultured in the presence of various concentrations of drug in fresh medium.Forty-eight hours after viral exposure,all blue cells in each well were counted.The cytotoxicity of the com-pound was determined by the MTT method as previously described(15).All experiments were performed in triplicate.The drug susceptibility of HIV-1clinical isolates was determined as previously described(42).Briefly,PHA-PBMCs(106cells/ml)were exposed to each viral preparation at a TCID50of50and cultivated in200␮l of culture medium containing various concentrations of the drug in10-fold serial dilutions in96-well culture plates.All assays were performed in triplicate,and the amounts of p24 antigen produced by the cells into the culture medium were determined on day 7in culture with a commercially available radioimmunoassay kit(Du Pont, Boston,Mass.).The activity of test compounds is shown as the concentration that blocks HIV-1replication by50%(EC50),and cytotoxicity is shown as the concentration that suppresses the viability of HIV-1-unexposed cells by50%(CC50).The window between CC50and EC50is shown by the CC50/EC50ratios(selectivity indices).RESULTSActivity of4؅-substituted nucleosides against HIV-1in vitro. We designed and synthesized more than20novel4Ј-substi-tuted nucleoside analogs(16,27,28,38)and tested them for in vitro activity against HIV-1by using the MTT colorimetric assay employing MT-4cells.A thymidine analog,4Ј-ethynyl (E)-T,and a uracil analog,4Ј-E-IdU,were found to be mod-erately active against HIV-1LAI,with EC50s of0.83and0.29␮M,respectively(Table1).Conversion of the2Ј-deoxyribose of4Ј-E-T to ribose,generating4Ј-E-rT,nullified the antiviral activity of4Ј-E-T.Other4Ј-substituted thymidine and uridine analogs had marginal or no activity.All four4Ј-substituted cytidine analogs examined were active,among which4Ј-E-dC and4Ј-fluoromethyl(FMe)-dC were most potent against HIV-1,although the latter was substantially cytotoxic(Table 1).It was noted that4Ј-E-araC was moderately active. Among4Ј-E purine analogs,4Ј-E-dA,4Ј-E-dDAP,and4Ј-E-dG were highly potent against HIV-1,with subnanomolar to nanomolar EC50s(Table1).4Ј-E-dI was only moderately ac-tive against the virus.It is noteworthy that both4Ј-E-dDAP and4Ј-E-dA had a favorable toxicity profile,with selectivity indices of2,600and1,630,respectively(Table1).All three 4Ј-E purine analogs with a ribose configuration(4Ј-E-NM-A, 4Ј-E-A,and4Ј-E-CldG)were inactive.Activity of4؅-substituted nucleosides against HIV-1variants resistant to various RTIs.We also evaluated whether4Ј-sub-stituted nucleosides were active against HIV-1variants resis-tant to various nucleoside RT inhibitors(NRTIs)by using theMAGI assay.Four pyrimidine analogs(4Ј-E-dC,4Ј-E-araC,4Ј-Me-dC,and4Ј-FMe-dC)that were potent against HIV-1LAIwere selected and tested further against various infectiousHIV-1clones carrying resistance-conferring amino acid substitu-tions(Table2).It was noteworthy that all four of these cytidineanalogs examined suppressed the replication of ddI-and ddC-resistant HIV-1K65R and HIV-1L74V,AZT-resistant HIV-1M41L/ T215Y,and multi-dideoxynucleoside-resistant(resistant to AZT, ddI,ddC and d4T)variant HIV-1A62V/V75I/F77L/F116Y/Q151M(35,36).It was noted that among4Ј-substituted pyrimidine analogs,only4Ј-E-dC remained active against3TC-resistant variants HIV-1M184I and HIV-1M184V,but was less active against the multi-NRTI-resistant(resistant to AZT,ddI,ddC,d4T and3TC)vari-ant HIV-1M41L/T69S-S-G/T215Y(41),while4Ј-FMe-dC remainedpotent against HIV-1M41L/T69S-S-G/T215Y.However,all three4Ј-Epurine compounds(4Ј-E-dA,4Ј-E-dDAP,and4Ј-E-dG)were ac-tive against the infectious clones tested,including HIV-1M184Vand HIV-1M41L/T69S-S-G/T215Y.These purine analogs were alsoactive against an NNRTI-resistant infectious clone,HIV-1Y181C.When tested in HeLa-CD4-LTR/␤-gal cells,the CC50s of theanalogs examined were allϾ100orϾ200␮M,except for that of4Ј-E-dG.4؅-Ethynyl group is required for activity against HIV-1M184V and HIV-1M184I.To investigate whether and to what extent the 4Ј-E configuration was crucial for activity against HIV-1,three thymidine analogs and two2Ј-deoxycytidine analogs substi-tuted at the4Ј-position with different groups(vinyl,ethyl,hy-droxyethyl,methyl,orfluoromethyl)were synthesized.4Ј-eth-ylthymidine(4Ј-Et-T)was inert,but4Ј-vinylthymidine(4Ј-V-T) and4Ј-hydroxyethylthymidine(4Ј-HE-T)were active against HIV-1LAI,although they were less so than to4Ј-E-T(Table1). These two thymidine analogs were active against the control wild-type infectious clone HIV-1HXB2and a multidideoxynucleo-side-resistant infectious clone(HIV-1A62V/V75I/F77L/F116Y/Q151M), but were totally inert(Ͼ100␮M)against the3TC-resistant clone HIV-1M184V(Table2).Two2Ј-deoxycytidine analogs,4Ј-Me-dC and4Ј-FMe-dC,were as potent against HIV-1LAI as4Ј-E-dC. These analogs were also active against various drug-resistant in-fectious clones,but were less potent against HIV-1M184V and HIV-1M184I(Table2).Taken together,although the4Ј-E substi-tution is not essential for antiviral activity against HIV-1,the4Ј-E configuration appears to be required for activity against3TC-resistant HIV-1M184V and HIV-1M184I.Activity of selected4؅-substituted nucleosides against HIV-2 strains.We also examined selected4Ј-E analogs against two HIV-2strains,HIV-2ROD and HIV-2EHO,in the MAGI assay (Table3).Although the thymidine analog4Ј-E-T showed only moderate activity,three cytidine analogs,4Ј-E-dC,4Ј-E-araC, and4Ј-Me-dC,were highly active against HIV-2,and4Ј-E-dC was the most potent analog,with50%inhibitory concentra-tions(IC50s)ofϳ0.001␮M(Table3).Four4Ј-E purine ana-logs examined also suppressed the replication of both HIV-2 strains.AZT,tested as a control compound,was active against both HIV-2strains,as previously described(21).Considering that all of the currently known NNRTIs fail to suppress theV OL.45,20014Ј-ETHYNYL NUCLEOSIDES ACTIVE AGAINST HIV1541on May 14, 2014 by YONSEI UNIV LIBRARY /Downloaded fromreplication of HIV-2(6),it appears that 4Ј-substituted nucleo-sides do not belong to NNRTIs.Activity of 4؅-ethynyl-2؅-deoxynucleosides against HIV-1iso-lated from heavily drug-experienced patients.4Ј-E analogs were then evaluated for their activity against multidrug resis-tant clinical HIV-1isolates in vitro.In this study,we chose three most potent 4Ј-E compounds,4Ј-E -dC,4Ј-E -dA,and 4Ј-E -dDAP.Four clinical HIV-1strains were isolated from patients who received a variety of anti-HIV-1agents for 39to 64months and had failed to respond to any existing regimens of antiviral therapy (42).As shown in Table 4,all four clinical HIV-1strains contained a variety of drug resistance-conferring amino acid substitutions in the RT-and protease-encoding regions of HIV-1gene.All three 4Ј-E compounds suppressed the replication of these highly drug-resistant clinical strains as effectively as that of the wild-type clinical strain HIV-1ERS104pre (36)and two HIV-1clinical strains (HIV-1IVR205and HIV-1IVR207)isolated from drug-naive AIDS patients.It was noted,however,that 4Ј-E -dC was moderately active against HIV-1Pt6and HIV-1Pt9.There was no apparent asso-ciation of the observed reduced antiviral activity with amino acid substitutions identified in these clinical isolates (Table 4).Testing 4؅-ethynyl nucleosides in the time-of-drug-addition assay.The triphosphate form of 4Ј-E nucleosides is not pres-ently available,and how 4Ј-E nucleosides block the replication of HIV-1and HIV-2remains to be clarified.The time-of-drug-addition assays have been used to approximately determine what stage of the replication cycle of HIV-1the compound in question blocks (29).We therefore tested two potent 4Ј-E compounds,4Ј-E -dC and 4Ј-E -araC,in the assay together with three controls:a surface reactant,dextran sulfate 5000(DS5000);NRTI AZT;and NNRTI MKC-442.The concentrations of compounds tested were all fixed at their EC 90s.At indicated time points,compounds were added to the HIV-1LAI -exposed MAGI cells.As shown in Fig.2,DS5000,which like DS8000(24)inhibits the absorption of HIV and formation of syncytia,showed anti-HIV-1activity only when it was added early after viral exposure.AZT,which requires triphosphorylation before it becomes active (23),ex-erted its antiviral activity when its addition was delayed for up to 4h.MKC-442,which does not require activation,was ef-fective when its addition was delayed for up to 6h.The profiles of anti-HIV-1activity of 4Ј-E -dC and 4Ј-E -araC were quite similar to that of AZT (Fig.2).These results suggest that 4Ј-E nucleosides suppress the replication of HIV at or around the step of reverse transcription.Reversal of antiviral activity of 4؅-ethynyl nucleosides by natural 2؅-deoxynucleosides.It has been shown that biological effects such as antitumor or antiviral activity of nucleoside analogs are reversed by the addition of natural nucleosides (5,8,26).We first tested whether thymidine and 2Ј-deoxycytidineTABLE 2.Antiviral activity of 4Ј-substituted nucleosides against drug-resistant infectious clones aCompoundEC 50(␮M)bCC 50(␮M)bHXB2K65RL74VM41L/T215YM184VM184IM41L/T69S-S-G/T215YMDR cY181CPyrimidine analogs 4Ј-E -T 0.360.530.680.430.180.140.440.120.13Ͼ2004Ј-E -IdU 0.37ND d ND ND 0.21ND ND 0.32ND Ͼ2004Ј-V-T 8.1ND ND ND Ͼ100ND ND 9.7ND Ͼ1004Ј-HE-T 15.8ND ND ND Ͼ100NDND 20.7ND Ͼ1004Ј-Et-T Ͼ100ND ND ND ND ND ND Ͼ100ND Ͼ1004Ј-E -dC 0.00120.00080.00130.0060.00240.00260.0150.00120.0021Ͼ2004Ј-E -araC 0.00710.0150.0260.0260.710.480.170.00790.016Ͼ2004Ј-Me-dC 0.00580.00710.0062ND 0.20.74ND 0.0033ND Ͼ2004Ј-FMe-dC 0.00460.0650.00190.0035 2.0ND 0.0660.0039ND Ͼ200AZT 0.0220.020.020.30.010.017 1.615.30.014Ͼ1003TC 0.71ND ND ND Ͼ100Ͼ1009.9 1.1ND Ͼ100ddC 0.2 3.0 1.5ND 2.2ND 1.3 5.5ND Ͼ100Purine analogs 4Ј-E -dA 0.0080.0330.0040.0120.0470.0220.0650.00620.011Ͼ2004Ј-E -dDAP 0.00140.000350.00070.00170.00590.00270.00410.0010.0008Ͼ2004Ј-E -dI 0.810.250.61 1.316.6 1.5 2.20.51ND Ͼ2004Ј-E -dG 0.0070.0010.00120.0190.0080.00410.00680.00480.0152ddI3.912.719.5 3.610.1ND12.225NDϾ100a Anti-HIV activity was determined with the MAGI assay.The abbreviations are defined in Table 1.bThe data shown are mean values derived from the results of three independent experiments.cMultidideoxynucleoside-resistant HIV-1that contains mutations in the pol region:an Ala-62to Val substitution (A62V),V751,F77L,F116Y,and Q151M.dND,not determined.TABLE 3.Anti-HIV-2activity of 4Ј-substituted nucleosides inMAGI cells aCompoundEC 50(␮M)HIV-2RODHIV-2EHO4Ј-E -T 0.340.294Ј-E -dC 0.0010.00094Ј-E -araC 0.0120.0034Ј-Me-dC 0.0020.00134Ј-E -dA 0.00190.0014Ј-E -dI 0.290.374Ј-E -dDAP 0.0010.00084Ј-E -dG 0.0260.0042AZT0.00610.013aAnti-HIV-2activity was determined with the MAGI assay.The abbreviations are defined in Table 1.The data shown are mean values derived from the results of three independent experiments.1542KODAMA ET AL.A NTIMICROB .A GENTS C HEMOTHER .on May 14, 2014 by YONSEI UNIV LIBRARY/Downloaded fromreversed the antiviral activity of 4Ј-E -T and 4Ј-E -dC,respec-tively (Fig.3).The concentrations of compounds tested were all fixed at their EC 90s.Antiviral activity of 4Ј-E -T (2␮M)was suppressed by the addition of thymidine in a dose-dependent manner,and the antiviral activity of AZT (100nM)was also reversed by the addition of thymidine,in agreement with our previous published data (26),although 100␮M thymidine caused cytotoxicity (Fig.3A).Activity of 4Ј-E -dC (8nM)and 4Ј-E -dG (10nM)was similarly reversed by the addition of 2Ј-deoxycytidine and 2Ј-deoxyguanosine,respectively (Fig.3B and D).Likewise,the activity of ddC (2␮M)and ddG (10␮M)was reversed by the addition of dC and dG,respectively,in agreement with our previous data (25).In contrast,the antivi-ral activity of 4Ј-E -dA was not reversed by the addition of dA,a similar profile of the activity of ddI versus dA (25)(Fig.3C).Acid stability of selected 4؅-ethynyl nucleosides.Certain nu-cleoside analogs such as ddI are acid labile and require antacid upon oral administration,generating adverse reactions,such as abdominal discomfort and diarrhea (33).We therefore exam-ined the acid sensitivity of three selected 4Ј-E analogs (4Ј-E -dC,4Ј-E -dA,and 4Ј-E -dDAP).These compounds were ex-posed to 1N HCl for up to 20min and then neutralized with 1N NaOH and added to target cells exposed to HIV-1.As shown in Fig.4,ddI lost all of its antiviral activity following acid exposure for 20min;however,4Ј-E -dC,4Ј-E -dA,4Ј-E -dDAP,and the control acid-resistant AZT did not lose their antiviral activity.DISCUSSIONAll of the currently available NRTIs have a 2Ј,3Ј-dideoxyri-bofuranose configuration,and only after anabolic intracellular phosphorylation do they exert their antiviral activity against HIV by functioning as viral DNA chain terminators (22).In this study,we identified 4Ј-E nucleoside analogs potent against a variety of HIV strains,including laboratory strains ofHIV-1FIG.2.Antiviral activity of 4Ј-ethynyl nucleosides in the time-of-drug-addition assays.At indicated time points,4Ј-E -dC (open circles)and 4Ј-E -araC (closed circles)were added to the HIV-1LAI -exposed MAGI (HeLa CD4/LTR-␤-gal)cells,and the blue cells produced were counted at the completion of the 48-h period of incubation.DS5000(open diamonds),AZT (open squares),and MKC-442(open triangles)served as controls.TABLE 4.Antiviral activity of 4Ј-substituted nucleosides against clinical isolatesStrainAmino acid substitution(s)EC 50(␮M)aRT regionProtease region 4Ј-E -dC 4Ј-E -dA4Ј-E -dDAP AZTERS 104pre None None 0.00120.0130.000830.0056IVR205None K20M,M36I,D60E 0.00230.00340.00010.0015IVR207G190Q V77I 0.00460.0022Ͻ0.00010.0036Pt 1T69G,K70R,L74V,A98G,K103N,V179D,M184V,T215F,K219F L10I,L33I,M36I,M46I,L63P,A71,G73S,V82A,L90M 0.00064(0.5fold)0.00054(0.4fold)0.0011(1.3fold)0.029(52fold)Pt 6M41L,D67N,M184V,L210W,T215YL10I,K20R,L24I,M36I,M46L,I54V,L63P,V82A,L89M 0.013(11fold)0.040(3fold)0.0001(0.1fold)0.28(50fold)Pt7M41L,D67N,T69D,M184V,T215F L10I,K45R,I54V,L63P,A71V,V82T,L90M 0.0016(1.3fold)0.009(0.7fold)0.0005(0.6fold)1.9(340fold)Pt 9M41L,M184V,T215Y M46I,L63P,A71V,V77I,I84V,N88D,L90M0.023(19fold)0.029(2.2fold)0.0031(3.7fold)0.97(170fold)a EC 50were determined with PHA-PBMC.All assays were conducted in triplicate.Numbers in parentheses represent fold changes of EC 50s against each HIV-1isolate compared to those against the wild-type clinical HIV-1strain,ERS 104pre .The abbreviations are defined in Table 1.V OL .45,20014Ј-ETHYNYL NUCLEOSIDES ACTIVE AGAINST HIV 1543on May 14, 2014 by YONSEI UNIV LIBRARY/Downloaded from。

SeriesLipids and cardiovascular disease 1LDL cholesterol: controversies and future therapeutic directionsPaul M RidkerLifelong exposure to raised concentrations of LDL cholesterol increases cardiovascular event rates, and the use ofstati n therapy as an adjunct to di et, exerci se, and smoki ng cessati on has proven hi ghly eff ecti ve i n reduci ng thepopulation burden associated with hyperlipidaemia. Yet, despite consistent biological, genetic, and epidemiologicaldata, and evidence from randomised trials, there is controversy among national guidelines and clinical practice withregard to LDL cholesterol, i ts measurement, the usefulness of populati on-based screeni ng, the net benefi t-to-riskratio for diff erent LDL-lowering drugs, the benefi t of treatment targets, and whether aggressive lowering of LDL issafe. Several novel therapies have been introduced for the treatment of people with genetic defects that result in lossof function within the LDL receptor, a major determinant of inherited hyperlipidaemias. Moreover, the usefulness ofmonoclonal anti bodi es that extend the LDL-receptor li fecycle (and thus result i n substanti al loweri ng of LDLcholesterol below the levels achieved with statins alone) is being assessed in phase 3 trials that will enrol more than60 000 at-risk patients worldwide. These trials represent an exceptionally rapid translation of genetic observations intocli ni cal practi ce and wi ll address core questi ons of how low LDL cholesterol can be safely reduced, whether themechanism of LDL-cholesterol lowering matters, and whether ever more aggressive lipid-lowering provides a safe,long-term mechanism to prevent atherothrombotic complications.IntroductionIn mammals, the lipoprotein transport system serves many functions that are crucial for survival including the initial transport of dietary fats from the intestine to the liver, the secondary transport of processed cholesterol particles to peripheral tissues for steroid hormone production and membrane synthesis, and the processing of free fatty acids which ultimately serve as a source of fuel for immediate and future needs.1 The movement of cholesterol through plasma is mediated by lipoprotein particles that carry hydrophobic cholesteryl esters and triglycerides in a central core, enveloped within an external layer of hydrophilic phospholipids and free cholesterol. Each lipoprotein particle typically includes one of a set of highly conserved apolipoproteins that provide structural integrity for the complex, allow for its assembly and secretion, and provide a mechanism for receptor binding. Lipoproteins are traditionally classifi ed according to their size and density, with chylomicrons, chylomicron remnants, and VLDL being relatively large and light, whereas LDL and HDL are sequentially smaller and heavier. In human beings, LDL particles are the main carrier of cholesterol to peripheral tissues where they are internalised through the LDL receptor, a crucial mediator of plasma LDL concentrations.2 Genetic defects that result in loss of function within the LDL receptor are a major determinant of inherited hyperlipidaemias, and novelmonoclonal antibodies that can extend the lifecycle of theLDL receptor represent a major new direction in thetreatment of these disorders.3 The size of LDL particlesvaries such that particles with more triglycerides andfewer cholesteryl esters result in smaller, denser LDL. ForLDL cholesterol, the associated apolipoprotein moleculeis apolipoprotein B. Figure 1 schematically shows thestructural elements of lipoproteins and their relation tosize (diameter) and density.4For clinicians, few circulating molecular structures haveas much importance for daily practice as LDL cholesterol.Epidemiological evidence consistently shows thatincreased concentrations of LDL cholesterol are associatedwith an increased risk of myocardial infarction andvascular death.5 Findings from classic genetic studiessuggest that early exposure to excessive LDL cholesterol,which is often the result of mutations of the LDL receptor,results in markedly early atherothrombosis. Comparedwith healthy individuals in whom atherosclerosis istypically expressed in their 5th and 6th decades, individualswho inherit one copy of a defective LDL receptor-relatedgene (heterozygous familial hypercholesterolaemia) oftenhave clinical onset of symptoms in their 30s and 40s. Bycontrast, those who inherit two copies of a defective LDLreceptor-related gene or who inherit combined geneticdefects (homozygous familial hypercholesterolaemia)could have myocardial infarction and stroke in their teensand early 20s (fi gure 2).6 These data have led to the conceptof so-called cholesterol years of exposure and suggest thatreduction of LDL early in life might result in long-termgains. Results from mendelian randomisation studiesinfer that LDL cholesterol is likely to be a causal agent forplaque initiation and progression,7 data that are consistentwith the known cellular processes promoted by LDL thatLancet 2014; 384: 607–17This the fi rst in a Series of threepapers about lipids andcardiovascular diseaseHarvard Medical School, Centerfor Cardiovascular DiseasePrevention, Brigham andWomen’s Hospital Boston, MA,USA (Prof P M Ridker MD)Correspondence to:Dr Paul M Ridker, Center forCardiovascular DiseasePrevention, Brigham andWomen’s Hospital, Boston,MA 02215, USApridker@Search strategy and selection criteriaData for this Review were identifi ed through searches of PubMed, Google Scholar, and references from relevant articles published between 2000, and 2014, using the terms “low-density lipoprotein cholesterol”, “hyperlipidaemia”, “lipid lowering”, “cholesterol measurement”, and “familial hyperlipidaemia”.Seriesresult in cholesterol-laden activated macrophages, a hallmark of atherosclerotic plaques. Lastly, reduction of LDL cholesterol is strongly associated with reduced vascular event rates, particularly when that reduction is achieved with statin agents that block the rate-limiting step of cholesterol synthesis.8,9 If public health policies that are currently under debate are fully implemented, more than a third of all middle-aged or older adults in the USA and the UK will be recommended for statin therapy.10 Yet, despite LDL cholesterol being the most important and extensively studied risk factor for cardiovascular disease, substantial controversy remains in clinical practice with regard to its measurement and the net benefi t-to-risk ratio for diff erent LDL-lowering drugs. Furthermore, many new targets for LDL reduction which are being assessed have the potential to provide benefi ts beyond statin therapy.11 In this Review we outline several controversies that are relevant for the daily practice of medicine and highlight areas in which ongoing research is likely to be informative. When and how should lipid fractions be measured?For general screening purposes for which the goal is to identify individuals with high concentrations of LDL, indirect calculation of LDL-cholesterol with the Friedewald equation is an adequate technique. However, because the Friedewald equation uses a fi xed factor to estimate VLDL from triglyceride concentrations, calculated LDL concentrations will underestimate true con c entrations when triglycerides are high. This under-estimation is increased when untreated LDL con c en-trations are very low or when LDL is aggressively lowered by potent statins and other lipid-lowering interventions.12 Similar under e stimation of true concentrations of LDL can also occur when HDL concentrations are very high, an issue that has been reported in some studies of inhibitors of cholesteryl ester transfer protein (CETP).13 Thus, in settings where increased accuracy of measure-ment at low LDL concentrations is desired, alternative methods for LDL assessment should be considered.One alternative is to use an adjustable rather than a fi xed factor for the triglyceride-to-VLDL ratio.14 A second option is to measure LDL-cholesterol directly. In screening populations, direct measurements of LDL-cholesterol and Friedewald-estimated LDL-cholesterol concentrations are often highly correlated with each other (r>0·9 in a study ofFigure 1: The structural components of lipoproteins (A) and their relation to diameter and density (B)Adopted from Genest J, Libby P. Lipoprotein Disorders and Cardiovascular Disease. Braunwald’s Heart Disease: a textbook of cardiovascular medicine, ninth edition. Elsevier 2012, pp: 975–95. IDL=intermediate-density lipoprotein.Figure 2: Approximate age of onset of atherosclerotic symptoms for thosewith homozygous familial hypercholesterolaemia or heterozygous familialhypercholesterolaemia, and those without inherited defects of theLDL-receptorAdopted from Horton and colleagues.6 HoFH=homozygous familialhypercholesterolaemia. HeFH=heterozygous familial hypercholesterolaemia.Series27 000 healthy women).15 In other settings, such asdiabetes, marked obesity, or in individuals who have recently consumed a fat-rich meal, direct LDL-cholesterol measures are clinically slightly better.16 Compared withcalculated LDL, direct LDL measures could also be better for assessment of very low concentrations of LDL afteraggressive reduction with therapy.12 Direct LDL measure-ments have limitations and might not be as accurate inpatients with hepatic or renal dysfunction, paraproteins, and some heritable hyperlipidaemias.17–19non-fasting lipid concentrations generally provide a similar predictive measurement for incident cardio-vascular events (non-fasting triglycerides are better than fasting triglycerides for this purpose).20 For these reasons, and to reduce patient burden and increase clinicaleffi ciency, many centres now allow assessment of lipids in the non-fasting state.In addition to issues of fasting and time of day, there is vigorous debate about advanced lipid testing methods that provide clinicians with more detailed information than is routinely available from measures of total, LDL and apolipoprotein B and specifi c measures of LDL-particle size and number could improve risk prediction because of increased specifi city. In a meta-analysis of 165 000 participants in 37 prospective cohorts, theapolipoprotein B was slight when compared with thatalready available from assessment of total and H DLcholesterol.21 Similarly, screening studies of lipoprotein profi les measured by NMR have shown comparable butnot superior predictive use when compared with standard assessment of 26 lipid measures, the ratio of total to HDL cholesterol ratio was the single strongest predictor of vascular risk (fi gure 3).22Similar data have emerged from studies of patients treated with statin therapy for whom residual risk wasLDL-cholesterol, non-HDL-cholesterol, apolipoprotein B, and lipoprotein(a).23 By contrast, residual risk after statinon-treatment H DL-cholesterol than with the number ofon-treatment H DL particles.24 In a meta-analysis of 62 154 participants in eight statin trials, the strength of the association with recurrent vascular events was slightlygreater for on-treatment non-H DL-cholesterol than for on-treatment LDL-cholesterol or apolipoprotein B.25In some situations, there is discordance between riskbased on LDL cholesterol and risk based onapolipoprotein B, non-H DLC, or LDL particle number(LDL-P).26 In an assessment of lipid fractions in the prospective Women’s Health Study, one in four women were discordant between LDL-cholesterol and thenumber of LDL particles (discordance was defi ned as having one measure more than, and one measure less Figure 3: Direct comparison of 26 lipid fractions as predictors of fi rst-ever cardiovascular events in apparently healthy women Data are shown for the top versus bottom quintile of each lipid fraction, unless otherwise indicated. *Additionallyadjusted for total LDLnmr particle concentration. †Additionally adjusted for the other NMR proteins. Adoptedfrom Mora and colleagues.22Figure 4: Diff erential predictive usefulness of the atherogenic lipoproteins LDLC and apolipoprotein BAssessed in individuals where there was concordance and discordance between LDLC and apolipoprotein B . Adopted from Mora and colleagues.27LDLC=LDL cholesterol. ApoB=apolipoprotein B.Seriesthan, the study median for these two lipid markers). One in five were discordant between LDL-cholesterol and apolipoprotein B, and one in ten were discordant between LDL-cholesterol and non-H DL-cholesterol.27 For these discordant individuals, vascular risk diff ered substantially when calculated on the basis of either non-H DL-cholesterol, apolipoprotein B, or the number of LDL particles instead of LDL–cholesterol concentrations (fi gure 4).27Many of the limitations associated with measurement of LDL can be avoided with use of non-HDL-cholesterol concentrations for screening purposes and for on-treatment assessment.25 Non-H DL-cholesterol does not depend on VLDL estimation, consists of a simple measure of all cholesterol carried by the atherogenic apolipoprotein B-containing lipoproteins, and reduces the discordance diffi culty discussed earlier. Moreover, because non-HDL cholesterol can be directly calculated from measures of total and H DL-cholesterol, this approach is cost effective and avoids the need for advanced lipid testing. Movement towards non-HDL-cholesterol and away from LDL cholesterol will take substantial educational efforts but is likely to improve overall patient care.14How effective is LDL-cholesterol for identifi cation of those who will benefi t from statin therapy? Statin therapy is highly effective at reducing vascular event rates. In results from a comprehensive meta-analysis from the Cholesterol Treatment Trialists’ Collaboration of 27 randomised trials,8,9 statins reduced the risk of major coronary events by 24% for each 1 mmol/L reduction in LDL cholesterol (95% CI 0·73–0·79), stroke by 15% (0·80–0·89), and coronary revascularisation by 24% (0·73–0·79).8,9 The magnitude of these benefits is similar between women and men, smokers and non-smokers, in elderly and young people, and across all levels of obesity, blood pressure, and glucose. Moreover, the benefi ts of statin therapy accrue with no evidence of an increased risk of incident cancer (hazard ratio 1·00, 95% CI 0·96–1·04) or cancer mortality (0·99, 0·93–1·06). Although individual randomised trials28 and meta-analyses have shown a small increase in the risk of developing diabetes with statin therapy,29 the benefi ts of treatment in terms of vascular event reduction outweigh this adverse eff ect in patients both at low and high risk for diabetes. Indeed, statin therapy is the treatment of choice to prevent macrovascular events in those with diabetes.30 Potent statins, now widely recommended in US and European guidelines, result in regression of coronary atherosclerosis in some patients.31 Although not often discussed, the relative risk reductions observed in statin meta-analyses might be greater in patients with lower absolute risk than in those with higher absolute risk (fi gure 5).8,9 This is consistent with the biological view that inhibition of LDL cholesterol synthesis earlier in the disease process is likely to confer more protection than delayed treatment. Indeed, the only statin trials that have not shown clear event reduction were those initiated in the settings of heart failure and end-stage renal failure for which absolute risk is very high. Moreover, despite the overwhelming evidence of effi cacy associated with LDL cholesterol reduction after statin therapy, baseline LDL cholesterol concentrations are not a particularly eff ective marker to determine which patients might benefi t from treatment. In all major trials, the relative risk reductions from statin therapy are unrelated to baseline LDL cholesterol concentrations (fi gure 5).8,9 This is an important issue in understanding the discrepancy between LDL cholesterol as a biologically essential mediator of atherosclerosis on the one hand, and LDL cholesterol as a relatively modest biomarker of absolute risk on the other.Although a trial-based approach to statin allocation would ensure prescription for patients in whom evidence is certain,32 global risk-prediction models that are based on calculations of absolute risk remain the mainstay for both US and European guidelines for the use of statin therapy. This approach is based on the concept that higher absolute risk usually (but not always) results in higher absolute risk reductions. Thus, at least for patients without heart failureFigure 5: Relative risk reductions with statin therapyReductions are greater for patients at low levels of absolute vascular risk than for those at higher levels. p=0·04 for heterogeneity (A). By contrast, relative risk reductions with statin therapy are not related to baseline levels of LDL cholesterol; p=0·3 for heterogeneity (B). RR=relative risk. Adapted from Baigent and colleagues8 and Mihaylova and colleagues.9Seriesor renal failure, the number of vascular events avoided that were associated with reductions in LDL cholesterol with statin therapy will be greater in those at sequentially greater levels of absolute risk (fi gure 6).9 Unfortunately, no global risk score has been used as an entry criterion in any statin trial. Furthermore, allocation approaches that are based on epidemiological modelling invariably lead to recommendations to treat many when trial data do not exist or when absolute risk is driven almost entirely by older age, even in the absence of other vascular risk factors.10,33 Investigators of the largest, contemporary, primary prevention trial addressing statin therapy allocated treatment to patients with raised concentrations of high-sensitivity C-reactive protein (hsCRP), not elevated LDL cholesterol concentrations.34 For these reasons, several alternative approaches to statin allocation are being investigated, including the use of individualised prediction of treatment eff ects. Among such emerging methods is the individualised number needed-to-treat (iNNT) that seeks to balance the known benefits of therapy with potential weighted risks for individuals rather than populations.35 At the other end of the range is the approach of treating all individuals who are older than a fi xed age threshold, such as those older than 55 years.36 Thoughtful outcomes research is needed to address these approaches because they lead to markedly different numbers of individuals recommended for therapy.Are the LDL-cholesterol targets achieved after statin therapy relevant for clinical practice?Until quite recently, physicians in the USA were strongly recommended to treat-to-targets with regard to LDL cholesterol reduction, an approach that remains the mainstay for most other nations. This recommendation was made on the basis of post-hoc analyses suggesting greater event reductions in those with LDL cholesterol concentrations below specific targets such as less than 1·8 mmol/L. However, the main focus on LDL cholesterol from a biological perspective does not necessarily provide a rationale for use of LDL cholesterol targets in clinical practice because head-to-head statin trials compared different agents at different doses, not comparisons of diff erent concentrations of on-treatment LDL cholesterol.37 LDL cholesterol concentrations contribute only modestly to overall risk prediction, and no trial has shown that baseline or on-treatment LDL cholesterol concentrations alter the benefi cial eff ects of statin therapy.For these reasons, the most recent US guidelines no longer advocate treatment to specific LDL cholesterol targets, and instead advocate the use of higher-intensity statin agents in those with higher absolute risk. This change in policy away from LDL cholesterol targets should reduce the promotion and prescription of non-statin LDL-lowering drugs that have not shown evidence of reductions in clinical events. Despite this recommendation, many physicians will probably continue to measure on-treatment LDL cholesterol, if only as a measure of drug compliance. Current European and Canadian guidelines have chosento maintain LDL targets. A discussion of diff erencesbetween regional guidelines has recently been presented.38Are the anti-infl ammatory eff ects oflipid-lowering relevant for clinical practice?Infl ammation has a fundamental role in all stages of theatherothrombotic process. Statins, in addition to reducingLDL cholesterol, also have anti-infl ammatory properties.39The ability of statins to reduce hsCRP concentrationsrepresents the clinical expression of these anti-infl ammatory eff ects. In most40 but not all41 statin trials inwhich hsCRP concentrations were systematically assessedbefore and after therapy, participants with the largestreductions had the lowest residual risks for recurrentvascular events. In two statin trials that measuredatheroma progression by serial intravascularultrasound,42,43 progression was reduced in those with bothlowered hsCRP and LDL cholesterol. The primaryprevention JUPITER trial showed that statin therapyreduced myocardial infarction, stroke, and all-causemortality in patients with elevated hsCRP concentrationswho would not otherwise qualify for treatment because oftheir already-low concentrations of LDL cholesterol.34 Asin the secondary prevention trials, primary preventionparticipants in JUPITER who achieved lower on-treatment hsCRP concentrations had better clinicaloutcomes than those who did not reduce hsCRP.Imagingstudies further show that intensive statin therapy reducesatherosclerotic infl ammation.44 By contrast, not all statintrials have shown that on-treatment hsCRP stronglyFigure 6: Estimated numbers of major vascular events avoided according to estimated 5-year level of risk and the magnitude of LDL reduction achieved with statin therapyFrom Mihaylova and colleagues.9Seriespredicts residual risk 41 and CRP itself is a biomarker of systemic infl ammation, not a causal agent. Furthermore, although raised remnant cholesterol concentrations seem to be related to both low-grade infl ammation and vascular events, marked increases in LDL cholesterol seem to be associated with vascular events without the need for overt infl ammation.45,46Although current US and Canadian guidelines endorse the use of hsCRP screening in situations in which risk assessment is uncertain, the clinical usefulness of infl ammation assessment after initiation of statin therapy remains controversial. So far, the only evidence-based response to persistently elevated hsCRP despite statin therapy would be to increase the dose of the current statin, or to select a more potent one.Should non-statin approaches continue to beused for LDL reduction?In view of the robust trial evidence showing safety and event reduction, statin therapy is appropriately emphasised in current US and European guidelines as the main treatment to reduce LDL cholesterol. Statins, however, should be an adjunct rather than a substitute forstrong dietary and lifestyle interventions, which on their own can substantially reduce cholesterol in compliant individuals. Nonetheless, not all patients can tolerate statins, and statins are contraindicated in pregnancy. Physicians might also wish to add a second, non-statin, lipid-lowering agent to further reduce LDL when treating patients with familial hyperlipidaemias.In the pre-statin era, surgical approaches to hyper-lipidaemia such as partial ileal bypass were shown to lower LDL cholesterol. More recently, gastric bypass surgery has proven eff ective at reducing vascular event rates in obese patients, although this benefi t is more closely linked to improved glucose control than lipid changes.47,48Approved non-statin agents for LDL reduction include bile acid-binding resins (colestipol,cholestyramine, and colesevelam), which have the advantage of not being absorbed systemically and thus can be used in pregnancy; fi brates (fenofi brate, bezafi brate, and gemfi brozil) which mainly decrease triglycerides and increase H DL cholesterol; niacin which slightly decreases LDL cholesterol and triglycerides while increasing H DL cholesterol; the cholesterol-absorption inhibitor ezetimibe; and omega-3 fatty acid supplements. Although these agents can clearly improve lipid profi les in many patients, contemporary event reduction trials have shown little evidence to support their use either as monotherapy or as an adjunct to statins in the general population. As prominent examples, neither the AIM-HIGH 49 nor H PS-2-TH RIVE 50 trials showed effi cacy for niacin in reduction of vascular event rates, yet both trials showed hazards for gastrointestinal events and infection. Similarly, in the FIELD 51 and ACCORD 52 trials fenofi brate did not show effi cacy, although subgroup analyses suggest effi cacy in those with elevated triglycerides and reduced H DLC—a hypothesis that requires direct testing. The VA-HIT 53 study of gemfi brozil did show a reduction in vascular events but this eff ect was not clearly related to LDL reduction. Furthermore, gemfi brozil increases the risk of side-eff ects with statin therapy, and is generally not recommended in combination with statins. With regards to omega-3 fatty acid supplements, contemporary trial data are highly confl icting.54–57 Finally, the ENHANCE trial of ezetimibe did not show a reduction in the surrogate endpoint of carotid intimal medial thickness despite a reduction in LDL cholesterol,58 and there is little evidence so far to suggest that this agent improves outcomes compared with statins alone. The ongoing IMPROVE-IT 59 trial is nearing completion and will provide important data in this regard. In the SH ARP 60 trial of individuals with chronic renal failure, the combination of statin and ezetimibe reduced event rates, but ezetimibe alone was not investigated. Thus, use of these non-statin agents in most general practice settings should be restricted.What pharmacological strategies for LDL reduction beyond statins are emerging?Although statins are highly eff ective for reducing vascular events, not all LDL-lowering agents benefi cially reduce rates of myocardial infarction, stroke, and vascular death. Examples of agents that reduce LDL cholesterol but in clinical trials did not reduce vascular event rates include post-menopausal hormone-replace-ment therapy (HRT) and the CETP inhibitors torcetrapib and dalcetrapib.61,62 Whether the success of statins and the failure of HRT and the two CETP inhibitors in reduction of vascular events is because statins have additional anti-infl ammatory properties, but the other agents do not, ishypothetical. Different agents within the same class might have diff erential clinical benefi ts; with regard to CETP inhibition, two other agents, anacetrapib and evacetrapib (which both lower LDL cholesterol and raise H DL-cholesterol) are in phase 3 assessment and other CETP inhibitors are in earlier stages of development.63 The mechanism by which LDL cholesterol is lowered could matter for event reduction, therefore each new LDL-cholesterol lowering agent should be assessed in outcome trials.Several new approaches to LDL reduction are under aggressive clinical investigation, and two new agents were approved in 2013 as orphan drugs by the US Food and Drug Administration to treat patients with homozygous familial hypercholesterolaemia, a rare autosomal dominant condition that typically results from an inheritance from both parents of mutations in the LDL receptor. These new drugs are important clinical advances because individuals with homozygous familial hypercholesterolaemia (estimated prevalence rate onecase per 500000 people to one case per 1 million) will。