HS 2012 to SITC Rev.4 Correlation and conversion tables

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当表面活性剂遇到大环分子

114Univ. Chem. 2023, 38 (12), 114–119收稿：2023-06-27；录用：2023-08-01；网络发表：2023-08-11*通讯作者，Email:*****************.cn基金资助：2021年基础学科拔尖学生培养计划2.0研究课题(20211014)；天津市首批虚拟教研室试点建设项目(化学类交叉人才培养课程建设虚拟教研室)•专题• doi: 10.3866/PKU.DXHX202306051 当表面活性剂遇到大环分子阮文娟，李悦，耿文超，郭东升*南开大学化学学院，天津 300071摘要：近年来，表面和胶体化学与大环化学的结合引起了科学家的普遍关注。

将多样的大环结构引入表面活性剂分子，不仅极大地丰富了表面活性剂分子的种类，还可以赋予其大环的主客体识别功能。

由此所开发出的大环两亲和超两亲分子已在生物成像和药物递送中表现出很高的应用潜力。

从传统表面活性剂到大环两亲和超两亲分子的发展、应用表明，不同领域的交叉融合对科学研究的发展是非常重要的。

关键词：表面活性剂；胶束；大环结构；大环两亲分子；超两亲分子中图分类号：G64；O6Encountering of Surfactants with Macrocyclic MoleculesWen-Juan Ruan, Yue Li, Wen-Chao Geng, Dong-Sheng Guo *College of Chemistry, Nankai University, Tianjin 300071, China.Abstract: In recent years, the combination of surface and colloid chemistry with macrocyclic chemistry has garnered widespread attention among scientists. The integration of diverse macrocyclic structures into surfactant molecules not only greatly enriches the diversity of surfactants, but also imparts them with the host-guest recognition functionality of macrocycles. Macrocyclic amphiphiles and supra-amphiphiles, developed from this approach, have demonstrated high potential in applications such as bioimaging and drug delivery. The evolution from traditional surfactants to macrocyclic amphiphiles and supra-amphiphiles underscores the importance of interdisciplinary integration in advancing scientific research.Key Words: Surfactants; Micelles; Macrocycles; Macrocyclic amphiphiles; Supra-amphiphiles表面活性剂及其所构筑的胶束是表面和胶体化学中所涉及的一类非常重要的体系。

LetPub-JCR2012-期刊

0239-7528 1851-2372 1224-2780 2146-3123 1860-5397 1876-2883 1757-6180 1369-703X 1934-8630 0791-7945 1996-3599 0324-1130 0525-1931 0144-8617 0008-6223 1897-5593 1664-3828 1634-0744 1842-4090 2044-4753 0920-5861 1933-6918 2045-3701 1478-811X 2228-5806 2211-1247 0008-8846 1895-1066 1733-7178 2081-9900 1868-4904 0008-8994 0272-8842 0862-5468 0173-9913 1584-8663 0352-9568 1612-1872 1752-153X 1359-7345 0275-7540 0098-6445 1385-8947 0009-2347 0255-2701 0360-7275 0263-8762 0009-2509 0930-7516 0009-2460 0009-3122 1451-9372 0009-3068 0251-0790
B POL ACAD SCI-TECH B SOC ARGENT BOT BALK J GEOM APPL BALK MED J BEILSTEIN J ORG CHEM BENEF MICROBES BIOANALYSIS BIOCHEM ENG J BIOINTERPHASES BIOL ENVIRON BUILD SIMUL-CHINA BULG CHEM COMMUN BUNSEKI KAGAKU CARBOHYD POLYM CARBON CARDIOL J CARDIORENAL MED CARNETS GEOL CARPATH J EARTH ENV CATAL SCI TECHNOL CATAL TODAY CELL ADHES MIGR CELL BIOSCI CELL COMMUN SIGNAL CELL J CELL REP CEMENT CONCRETE RES CENT EUR J CHEM CENT EUR J ENERG MAT CENT EUR J GEOSCI CENT EUR NEUROSURG CENTAURUS CERAM INT CERAM-SILIKATY CFI-CERAM FORUM INT CHALCOGENIDE LETT CHEM BIOCHEM ENG Q CHEM BIODIVERS CHEM CENT J CHEM COMMUN CHEM ECOL CHEM ENG COMMUN CHEM ENG J CHEM ENG NEWS CHEM ENG PROCESS CHEM ENG PROG CHEM ENG RES DES CHEM ENG SCI CHEM ENG TECHNOL CHEM ENG-NEW YORK CHEM HETEROCYCL COM+ CHEM IND CHEM ENG Q CHEM IND-LONDON CHEM J CHINESE U

Toxicity of the Insecticide Fipronil and Its Degradates to Benthic

Toxicity of the Insecticide Fipronil and Its Degradates to Benthic Macroinvertebrates of Urban StreamsDonald P.Weston*,†and Michael J.Lydy‡†Department of Integrative Biology,University of California,Berkeley,California94720,United States‡Center for Fisheries,Aquaculture and Aquatic Sciences,Southern Illinois University,Carbondale,Illinois62901,United States *Supporting Informationtoxicity ofﬁpronil degradates,and demonstratesconcentrations posing a risk to a wide variety of streamThe dominant pesticides in agricultural and urban environ-ments change as new compounds are developed or use of existing compounds is restricted by regulatory action,typically because of unforeseen risks to human health or environmental quality.Insecticide use in urban environments provides an example.After most organochlorine insecticides were banned in the1970s and1980s,organophosphates,most notably diazinon and chlorpyrifos,became the dominant urban insecticides. However,because of human health concerns,urban-use diazinon and chlorpyrifos products were withdrawn from the U.S.market in the early2000s.As organophosphates were restricted,pyrethroids took their place.Nonagricultural pyrethroid use doubled in California from2000to2006.1 Pyrethroid use in the state declined from2006to2009, possibly because of factors related to the economic recession, but has since rebounded(Supporting Information(SI)Figure S1).An emerging insecticide in urban environments is the phenylpyrazoleﬁpronil,now used in applications previously reserved for pyrethroids and organophosphates before them. Though used in both agricultural and urban environments elsewhere,there are no approved agricultural uses in California so its presence in surface waters indicates input from landscape maintenance and structural pest control.There was essentially no use of the compound in California prior to2000,but use has been climbing since,albeit with the same macroeconomic-related decline from2006to2009(SI Figure S1).the environmental impact of these insecticides is challenge because the compounds have received regulatory approval and come into widespread use with signiﬁcant data gaps regarding their fate and eﬀects,or analytical diﬃculty in quantifying environmental concentrations.Diazinon caused frequent toxicity in some of California’s largest rivers in the 1990s.2Analytical quantiﬁcation of pyrethroids has not been possible until concentrations reach the threshold of acute mortality for sensitive species,and thresholds for chronic toxicity are probably below current detection limits.3A major challenge withﬁpronil is degradation into a desulﬁnyl by photolysis,degradation into an amide by hydrolysis under basic conditions,oxidation to a sulfone in aerobic environments,and reduction to a sulﬁde in anaerobic soils or sediments.4Little is known about the toxicity of these derivatives.A published2007 review provided degradate EC50or LC50data for twoﬁsh and one aquatic invertebrate.4The U.S.Environmental Protection Agency’s(EPA)2007risk assessment in support ofﬁpronil registration contained degradate aquatic toxicity data on two ﬁsh and four invertebrates,obtained almost entirely from publicly unavailable reports submitted by the registrants.5 Although recent work has shownﬁpronil to be commonly found in urban runoﬀ,6there are very few data on itsReceived:October13,2013Revised:December19,2013Accepted:December24,2013Published:December24,2013concentrations in waterbodies to which the runoﬀis discharged. The present study sampled16urban waterbodies for both toxicity testing and pesticide analysis(ﬁpronil,its degradates, and pyrethroids).Because degradate toxicity data are available for so few species,EC50s and LC50s forﬁpronil and two derivatives were determined for14freshwater invertebrates. Finally,since other urban-use pesticides have been found in municipal wastewater at toxic concentrations,7we collected preliminary data on the concentration ofﬁpronil and degradates in raw and treated wastewater.■MATERIALS AND METHODSDetermination of Fipronil Sensitivity.Fourteen macro-invertebrate species were tested for sensitivity toﬁpronil,ﬁpronil sulfone,and/orﬁpronil sulﬁde(Table1).Hyalellaazteca and Chironomus dilutus were obtained from cultures maintained at the University of California Berkeley.Hexagenia sp.(25−30mm long)wasﬁeld-collected from the Great Lakes region,and provided by a commercial supplier(Aquatic Research Organisms,Hampton,NH).All other species were obtained between February2012and April2013from northern California waterbodies in areas with minimal development.Leaf litter bags were placed in creeks for approximately two weeks, after which animals were sorted from the litter,and acclimated to laboratory water for24h.Although we generally conducted 96-h tests,preliminary tests with some species produced unacceptable mortality,so tests for those species were limited to48h.Tests were done using Milli-Q puriﬁed,deionized water made moderately hard by addition of salts.8Waters were spiked withﬁpronil,ﬁpronil sulﬁde,orﬁpronil sulfone(ChemService, West Chester,PA)dissolved in acetone.Acetone concen-trations were<36μL/L,and solvent controls never showed any toxicity.Test waters were distributed to three replicate vessels per concentration,with a control and4−7concentration steps separated by a factor of2(e.g.,2,4,8,16,and32ng/L).Glass exposure vessels ranged from100mL(H.azteca)to2000mL (Hexagenia sp.),depending on the species.Ten individuals per beaker were used for cultured species;4−6individuals per beaker were used forﬁeld-collected species for which availability was limited.Tests were done underﬂuoresent lights with a16-h light:8-h dark photoperiod.All test vessels contained a25-cm2nylon screen to which the animals could cling,except the H.azteca screen which was1cm2.C.dilutus received a thin layer of quartz sand for tube building,and Hexagenia sp.received glass tubes to mimic their burrows.H. azteca and C.dilutus were fed1mL of yeast/cerophyll/trout food or0.5mL of Tetraﬁnﬁsh food slurry,respectively,on the second day.After4−6h to allow for feeding,80%of the water was replaced with freshly prepared pesticide-spiked solutions. Water change procedures were identical forﬁeld-collected species,but they were not fed during tests since appropriate diets have not been established for these nontraditional test species.Water from a concentration step near the expected EC50based on preliminary tests was analyzed by methods described below for veriﬁcation of initial pesticide concen-tration,with compositing solutions prepared on days0and2. Actual concentrations were near nominal(median95%of nominal;range66−131%),but all data were adjusted to reﬂect actual initial concentrations.At test completion the number of survivors was recorded. Fipronil and degradates are neurotoxins,and many animals were alive but unable to move normally.A sublethal end point was also reported,which varied depending on the species’normal behavior.That end point was inability to swim for Ephemeroptera(that normally readily do so),or inability to cling to the nylon screen for Plecoptera(that typically do so tenaciously).The sublethal end point for Trichoptera was inability to thrash when gently prodded(Hydropsyche sp.), inability to cling to the screen(Helicopsyche sp.),or inability to crawl(Nectopsyche sp.).To minimize stress onﬁeld-collected animals,tests were conducted at in situ temperatures of each species’collection site,ranging from8to23°C(Table1).However,species comparisons could be confounded by a temperature depend-ence ofﬁpronil toxicity,much as there is for pyrethroids.9To assess this risk,ﬁpronil toxicity to laboratory-cultured C.dilutus was determined at13,18,and23°C.Probit analysis and CETIS software(Tidepool Scientiﬁc Software,McKinleyville,CA)were used to derive EC50and LC50values.Two independent tests were done with cultured species,butﬁeld-collected species were in suﬃcient numbers for only one test for each pesticide.Control survival is reported for all tests.The lowest survival was69%(Taenionema sp. tested withﬁpronil),although in two-thirds of the tests survival was90%or greater.While90%is often used as a threshold for acceptability when testing with standard species,8we usedTable1.Species Used to Determine Toxicity of Fipronil, Fipronil Sulﬁde,and Fipronil Sulfonespecies source a exposureduration(h)testtemp.(°C)sublethal endpointAmphipodaHyalella azteca UCB9623ability to swimDipteraChironomus dilutus UCB9623ability tothrash whenproddedEphemeropteraHexagenia sp.ARO9618ability to swim Baetis tricaudatus b WC,CC,AR4812−17c ability to swimDiphetor hageni WC4818ability to swim Fallceon quilleri CC4823ability to swim Serratella micheneri AR4823ability to swim Ephemeralla excrucians b AR4813ability to swimPlecopteraTaenionema sp.DC,AR968ability to clingIsoperla quinquepunctata CC9613ability to cling Tricorythodes 4818ability to clingTrichopteraHydropsyche 9612ability tothrash whenprodded Nectopsyche 9612−23d ability to crawl Helicopsyche 9613ability to cling a UCB=University of California Berkeley lab culture;ARO=Aquatic Research Organisms.WC=Walker Creek,Petaluma,CA;CC= Cache Creek,Esparto,CA;AR=American River,Auburn,CA;DC= Donner Creek,Concord,CA.b Considered a species group by the Southwest Association of Freshwater Invertebrate Taxonomists (SAFIT).c12°C forﬁpronil sulﬁde andﬁpronil sulfone,17°C for ﬁpronil.d12°C forﬁpronil,23°C forﬁpronil sulﬁde andﬁpronil sulfone.nonstandard species for which optimal testing conditions have not been established,and therefore believe some latitude is appropriate.Water quality parameters monitored included temperature,dissolved oxygen,conductivity,pH,alkalinity, hardness,and ammonia.Urban Stream Sampling.Sixteen waterbodies in ten northern California communities were sampled(SI Table S1). Most sites were urban streams,though one was an urban river, and two were tidal sloughs receiving urban runoﬀ.A few waterways(Mosher Slough,Ulatis and New Alamo Creeks) drained agricultural lands farther up their watersheds.While either urban or agricultural areas could have been the source of pyrethroids,ﬁpronil could only be of urban origin as it has no agricultural uses in the state.Each site was sampled1−3times throughout2012.Nearly all sampling was done in the rainy season(November−April) during or immediately after2−5cm of rainfall.Dry season conditions,with runoﬀthrough landscape irrigation,were not a study focus and were characterized by only three samples taken in September.Water samples were collected from the stream banks using glass jars precleaned for pesticide sampling(I-Chem200series; Thermo Fisher Scientiﬁc,Rockwood,TN).The bottles were ﬁlled just below the water surface,and held at4°C until toxicity testing within24h or extraction for pesticides within72 h.Samples for pesticide analysis received1mL of hexane as a keeper solvent upon collection.Municipal Wastewater Sampling.Samples were col-lected at two California municipal wastewater treatment plants. The Sacramento Regional County Sanitation District’s plant provides secondary treatment to an average dry weatherﬂow of 525600m3/d(samples collected prior to ongoing upgrade to tertiary treatment).The San Jose/Santa Clara Regional Wastewater Facility provides tertiary treatment to an average dry weatherﬂow of416400m3/d.Inﬂuent andﬁnal eﬄuent were collected using composite samplers:Teledyne Isco3700 or4700at Sacramento(Teledyne Isco,Lincoln,NE)or Hach Sigma900MAX at San Jose(Hach,Loveland,CO).Time-weighted samples over24h were taken,except the eﬄuent sample at Sacramento wasﬂow-weighted.The eﬄuent composite was delayed relative to the inﬂuent composite by the plant’s detention time(8−11h at Sacramento,8h at San Jose),to best sample water as it moved through the treatment process.Samples were collected in refrigerated7-L glass jars, and extracted within72h(no toxicity testing).Two sampling events at Sacramento were done during dry periods,and a third was done during rain with4cm accumulation.Four sampling events at San Jose were all in dry weather due to minimal rain during the period we had plant access.Toxicity Testing of Field Samples.Urban water samples were tested with the amphipod,H.azteca,using individuals7−14d in age.10Ten individuals were placed in each ofﬁve replicate100-mL beakers per sample,containing80mL of test water and1cm2nylon screen.Tests were conducted at23°C with a16-h light:8-h dark photoperiod.On the second day the animals were fed and the water was changed using fresh sample that had been held at4°C since collection.After4d exposure, the animals were scored both for death and paralysis(unable to demonstrate coordinated swimming;typically no movement except occasional twitching).All samples were tested with H.azteca,but samples from the last sampling event were also tested with third instar larvae of C.dilutus.11Water samples(600−700mL)were placed inﬁve replicate1-L beakers containing a thin layer of quartz sand.Ten individuals were added to each beaker,and tested at23°C with a16:8h photoperiod.On the second day0.5mL of a Tetraﬁn ﬁsh food slurry was added prior to water change.After4d animals were scored both for death and the ability to show the typical thrashing response when gently prodded(also described asﬁgure-eight movement12).All tests included controls using Milli-Q puriﬁed water made moderately hard.Water quality parameters measured were as described above.Diﬀerences between test waters and controls were determined by t test if parametric assumptions were met, and Wilcoxon rank-sum if they were not,using CETIS software. Chemical Analysis.Water samples were analyzed for eight pyrethroids(bifenthrin,cyﬂuthrin,cyhalothrin,cypermethrin, deltamethrin,esfenvalerate,fenpropathrin,permethrin),ﬁpro-nil,and three degradates(the sulﬁde,sulfone,and desulﬁnyl derivatives).Extraction and cleanup methods generally followed Wang et al.13Brieﬂy,the surrogates4,4′-dibromoocta-ﬂuorobiphenyl and decachlorobiphenyl(Supelco,Bellefonte, PA)were added to the samples.Liquid:liquid extraction(EPA Method3510C)used three additions of60mL of dichloro-methane.One aliquot was also used to extract any residues remaining adsorbed to the sample bottle.The combined extract was solvent exchanged to hexane and reduced in volume to1 mL.Other than the municipal wastewater inﬂuent samples,ﬁpronil and degradates could be quantiﬁed without further extract cleanup.For pyrethroids,the extract was put through a dual-layer cartridge containing600mg of primary and secondary amines and300mg of graphitized carbon black. After conditioning the cartridge with6mL of1:1hexane/ acetone(v/v),the extract was loaded,as well as two0.5mL hexane rinses of the tube previously containing the extract. Target analytes were eluted using10mL of a30%methylene chloride in hexane solution.The eluent was reduced in volume to near dryness and reconstituted to125μL in0.1%acetic acid in hexane for analysis.The municipal wastewater inﬂuent samples required additional cleanup.They wereﬁltered (Whatman GD/X,0.45um)into a volumetric test tube, volume was adjusted to4mL,and500μL extract was injected into a gel permeation chromatography(GPC)system.The GPC included a Rheodyne7225injector with0.5-mL sample loop(IDEX Health&Science,Oak Harbor,WA),a Waters300 mm×19mm Envirogel GPC column with a5mm×19mm precolumn(Waters,Milford,MA),and an Agilent1100high-performance liquid chromatograph equipped with an ultraviolet detector(Agilent Technologies,Palo Alto,CA).A Foxy Jr. fraction collector(Isco,Lincoln,NE)was used to collect the appropriate fraction,which included the surrogates,pyreth-roids,ﬁpronil,and degradates.The extract was evaporated to near dryness and solvent exchanged to1.0mL of hexane. Fipronil and degradates were quantiﬁed immediately and additional cleanup was provided for the pyrethroids as described earlier.Analyses were performed on an Agilent6850gas chromato-graph(GC)with a5975C mass selective(MS)detector in electron impact mode.An Agilent HP-5MS(30m×0.25mm ×0.25μmﬁlm thickness)column was used to separate the analytes.Helium was the carrier gas at aﬂow of1mL/min.A2-μL sample was injected in pulsed splitless mode and the injector was set at260°C.The oven was initially50°C,heated to200°C at20°C/min,heated to295°C at10°C/min,andT a b le 2.C o n t r o l S u r v i v al (C S )o f E a c h T e s t S p e c i e s ,E C 50,a n d L C 50P o i n t E s t i m at e s f o r F i p r o n i l ,F i p r o n il S u l ﬁd e ,a n d F i p r o n i l S u lf o n e a H y a l e l l a a z te ca (t e s t 2)100727(648−816)1725(1461−2037)100375(325−433)1398(1145−1716)100155(122−179)426(346−497)C h i r o n o m u s d i lu t us (t e s t 1)8335.0(21.1−41.5)>81.5779.3(7.6−11.4)>62.4877.5(5.3−9.2)>102C hi r o n o m u s d i l u t u s (t e s t 2)8730.0(23.3−36.0)>81.57010.5(6.7−13.4)>75.8857.9(5.0−10.3)>106H e x a g e n i a s p .93480(348−603)1231(769−1667)93163(51−223)257(109−362)B a e t i s t r i c a u d a t u s b (t e s t 1)8051.9(37.3−72.)105(76.0−146)8780.3(53.1−108)>717100>341>341B a e t i s t r i c a u d a t u s b (t e s t 2)87143(80.2−195)535(382−750)B a e t i s t r i c a u d a tu s b (t es t 3)9575.0(44.9−109)>684D iph e t o r h a g e n i 90163(107−208)347(196−568)8792.6(56.5−128)330(188−536)F a l l c e o n q u i l l e r i 7770.7(36.5−93.5)>1879034.2(14.0−48.8)103(58.7−142)9571.7(52.3−90.6)>196S er r a t e l l a m ic h e n e r i 100589(478−742)>72295159(106−214)331(257−426)E p h e m e r a l l ae x cr u c i a n s b 100>436>436T a e n i o n e m a s p .100>184>18410095.9(62.1−126)>261I so p e r l aq u i n qu e p unc ta ta 69101(84.6−119)113(94.2−135)10042.2(37.1−47.4)94.5(66.8−175)8547.4(40.2−55.9)50.0(43.1−58.1)T ricor y t h o de s s p .100>1229>1229H y d r o p s y c h es p .94602(417−788)2107(1218−2668)7572.9(56.5−94.0)>824N ec to p sy c h e s p .100634(531−756)>29479628.5(18.7−36.5)122(60.2−177)9631.3(23.0−40.1)51.5(37.0−69.1)H e l ic o p sy ch e s p.100267(210−338)>842100177(146−216)>55110073.8(38.6−140)>626a V a l u es i n p ar e n t h e s e s a re 95%c o n ﬁd en c e in t ervals .Empt ycellsindic ate test notdo n ebeca useofinsuﬃcientnu mbe rofindividuals.b Consid eredaspecie sgroupbytheSouthwestA sso cia tionofFreshwaterIn vert e b rateTaxo nomists(SAFIT).held at this temperature for 5min.The temperatures of the ion source and the interface were 230and 295°C,respectively.The MS was operated in selected ion monitoring (SIM)mode,and the characteristic ions for the target compounds are listed in SI Table S2.Quanti ﬁcation was performed using internal standard calibration (triphenyl phosphate)with a six-point curve.Recovery of the pesticides from matrix spikes ranged from 58to 147%(pyrethroids)and 52to 118%(ﬁpronil and degradates).The method detection limit (MDL)was determined by multiplying the standard deviation of seven replicate samples by the Student ’s t value for a 99%con ﬁdence level and (n −1)degrees of freedom.14Previous work using a similar analytical method reported MDLs for the target pyrethroids from 0.41to 1.59ng/L.13We conducted a similar study with ﬁpronil and its degradates using two urban creek waters and found MDL values ranging from 0.88to 1.49ng/L (unpublished data).We report data down to 1ng/L for all analytes when the analyst believed quanti ﬁcation was reliable.A method blank,laboratory control spike,matrix spike,and matrix spike duplicate were analyzed with every batch of samples.The GC-MS was calibrated daily and correlation coe ﬃcients were all >0.995.A midstandard was run every 10samples to ensure <20%variation from the calibration standards.■RESULTS AND DISCUSSION Toxicity of Fipronil and Its Degradates.To determine the extent to which di ﬀerences in apparent ﬁpronil sensitivity between species was merely a consequence of di ﬀerences in test temperatures,ﬁpronil toxicity to C.dilutus was determined over a range of temperatures.In two tests at 23°C,the 96-h EC50was 30.0ng/L (95%con ﬁdence interval =23.3−36.0)and 35.0ng/L (21.1−41.5).At 18and 13°C,EC50s were 53.3ng/L (48.4−58.7)and 39.7ng/L (33.0−48.5),respectively.Any e ﬀect of temperature on ﬁpronil toxicity was slight at best,and negligible in comparison to the interspeci ﬁc EC50di ﬀerences found.Whereas EC50estimates were obtained for nearly all species,the high frequency of “greater than ”LC50values (Table 2)indicates lethality was rare at the highest concentrations tested.In half of the 41tests,we failed to attain 50%mortality.Particularly for neurotoxins such as ﬁpronil,sublethal end points such as inability to cling to the substrate or move normally are necessary to increase test sensitivity,as well as being ecologically relevant.Among the 14species tested, C.dilutus was the most sensitive to ﬁpronil and its degradates.The species ’96-h EC50was 30−35ng/L for the parent compound,and 7−10ng/L for the sul ﬁde and sulfone derivatives.The next-most sensitive species had EC50s approximately 2-to 3-fold higher (ﬁpronil EC50of 70.7ng/L for Fallceon quilleri ;ﬁpronil sul ﬁde EC50of 28.5ng/L for Nectopsyche sp.;ﬁpronil sulfone EC50of 31.3ng/L for Nectopsyche sp.).The least sensitive species were the stone ﬂy,Tricorythodes sp.,and the amphipod,H.azteca .The H.azteca EC50s were >727ng/L for the parent compound,and 155−540ng/L for the degradates.H.azteca is extremely sensitive to pyrethroids and valuablefor monitoring water-bodies containing these compounds.3However,it is a poor choice when ﬁpronil toxicity is of concern.Multispecies testing may be necessary where both pesticides are anticipated.There was high variability among three independent tests of the sulfone derivative with the may ﬂy,Baetis tricaudatus .For other species within Table 2,as well as our previous work,variation of EC50or LC50estimates between tests is typically less than a factor of 2.15,16The larger di ﬀerence among B.tricaudatus tests may be due to the fact that test animals were obtained from di ﬀerent locations (American River for test 1,Cache Creek for tests 2and 3).Taxonomically,the “species ”is believed to be a group,thus each location may have contained di ﬀerent members with varying ﬁpronil sensitivity.Even a single species can have dramatically varying pesticide sensitivity if resistance has been acquired by some populations.16The degradates were more toxic than ﬁpronil for the majority of species tested.The sulfone was more toxic than ﬁpronil to 9of the 12species evaluated,often by a factor of 2−4.The data for the sul ﬁde is not as extensive,but suggests its toxicity is comparable to the sulfone.Fipronil degradates have beenreported to be more toxic than the parent compound to several vertebrates and invertebrates,though this relationship is not always the case.17−19Fipronil in Municipal Wastewater.Untreated municipal wastewater at both treatment plants contained ﬁpronil on at least one occasion (SI Table S3).The Sacramento plant ’s inﬂuent was sampled three times,and contained measurable ﬁpronil only on one occasion when it rained,though this relationship may have been coincidental.The San Jose plant ’s in ﬂuent contained ﬁpronil in at least two of the four sampling events.On the other two occasions it could not be quanti ﬁed in the in ﬂuent due to interfering substances,but was likely present given its appearance in the e ﬄuent.Measurable ﬁpronil in ﬂuent concentrations ranged from 39to 119ng/L.Neither the sul ﬁde or sulfone derivative were found,though untreated wastewater contained <6ng/L of the desul ﬁnyl product.On the one occasion that ﬁpronil was found at the Sacramento plant,approximately half remained after treatment.At the San Jose plant there was little,if any,removal during treatment.The limited data suggest 30−60ng/L ﬁpronil can be expected in treated municipal paring these values to the species sensitivity data,these concentrations are likely to cause toxicity to the most sensitive species,C.dilutus ,unless diluted in receiving waters,and assuming other wastewater constituents,such as dissolved organic carbon,do not alter ﬁpronil bioavailability.Fipronil in Urban Streams.Twenty-four samples from sixteen urban waterbodies were collected during orimmediatelyTable 3.Frequency of Detection and Median and Maximum Concentrations of Pesticide Analytes in Urban Waterbodies during Rain Events (n =24)amedian concentration (ng/L)21.2 5.1<110.210.8<1<1<1maximum concentration (ng/L)49.111.514.836.041.120.39.036.6a The pyrethroids cy ﬂuthrin,deltamethrin,esfenvalerate,and fenpropathrin were not detected (<1ng/L).after rain events.Among the pyrethroids,bifenthrin was found in nearly all(92%)wet-weather samples with a median concentration of10.8ng/L and a maximum of41.1ng/L (Table3;SI Table S1).There were scattered detections of cypermethrin,cyhalothrin,and permethrin.The relative dominance of bifenthrin over the other pyrethroids in urban areas has been frequently reported in California and many other states.3,20−22Fipronil was measurable in88%of the wet-weather samples with a maximum concentration of49.1ng/L,and a median concentration of21.2ng/L.Fipronil sulfone was equally common(88%detection)with a median concentration of10.2 ng/L.The desulﬁnyl photoproduct was present in most samples though at a lower median concentration of5.1ng/L. The sulﬁde was the least commonly detected degradate,found in42%of the samples.Its rarity in urban runoﬀhas been attributed to photolysis or aerobic degradation,rather than anaerobic processes,being the primary routes of transformation of urban-useﬁpronil.6Only three dry season samples were collected,but they suggestﬁpronil in urban surface waters is primarily associated with storm events.Noﬁpronil or degradates were found in two of the samples,and only3.1ng/L ofﬁpronil was in the third sample.One of the three samples was taken from the AmericanRiver,which typically had lowﬁpronil concentrations even during rain events,but the other two creeks(Morrison and Ulatis Creeks)commonly had10−30ng/L totalﬁpronil and degradates when carrying stormwater runoﬀ.These dry season results,while tentative due to the few samples,are consistent with the reports that Sacramento dry-season irrigation runoﬀcontains low median concentrations of3.1−5.6ng/Lﬁpronil and4.7−8.0ng/Lﬁpronil sulfone.6As these other data were collected directly from storm drains,it is reasonable to expect the waterways into which they discharge to contain undetectable amounts,such as found in the present study during dry weather.It is diﬃcult to compare theﬁpronil concentrations found in California to those elsewhere since the few published urban data are limited to summary statistics,such as state-wide averages from U.S.Geological Survey databases.4,23They indicateﬁpronil and degradate residues can be found in streams in many other states,even in spite of the fact that much of the data were collected early in the2000s whenﬁpronil use was lower.When observed wet-weather concentrations are compared to the EC50s of macroinvertebrates reported in the present study (Figure1)it is clear that several species are at risk given existing concentrations.The EC50s forﬁpronil,the sulﬁde,and sulfone for the most sensitive species(C.dilutus)are exceeded at many locations.Those sites with the highest concentrations also approach or exceed the tolerance of the second-most sensitive species(B.tricaudatus forﬁpronil,Nectopsyche sp.for the sulfone).Furthermore,a surface water concentration merely less than the EC50does not eliminate risk,since an eﬀect on 50%of the individuals is not a protective benchmark.Allowing a margin of safety between pesticide concentrations and EC50 estimates,it is apparent thatﬁpronil and its sulfone derivative in many California urban streams may currently represent a threat to several benthic macroinvertebrate species common in the region,not by lethality but by impairment to swimming, crawling,clinging,and other normal movements.An analysis such as Figure1may underestimate potential risks sinceﬁpronil and the degradates cause toxicity by the same mode of action,speciﬁcally,acting on theγ-aminobutyric acid(GABA)receptor to block the chloride channel.17Though interactive toxicity ofﬁpronil and its degradates to aquatic life has not been studied,additivity can be expected given the common mode of action.The median wet-weather concen-trations from all samples were21.2ng/Lﬁpronil,10.2ng/L ﬁpronil sulfone,5.1ng/Lﬁpronil desulﬁnyl,and undetected ﬁpronil sulﬁde.Excluding the desulﬁnyl because of lack of EC50estimates,concentrations can be converted to additive toxic units(TU).For any given species,the total TU can be calculated as=‐+‐+‐TUobserved fipronil concn.species specific fipronil EC50observed sulfone concn.species specific sulfone EC50observed sulfide concn.species specific sulfide EC50Using median concentration values to approximate a “typical”California urban stream,and excluding the sulﬁde because of its undetected median concentration,such a stream would contain nearly two TU for the chironomid,C.dilutus (Figure2).The same concentrations would represent over one-third of a TU for four more species,the stoneﬂy Isoperla quinquepunctata,the mayﬂies F.quilleri and B.tricaudatus,and the caddisﬂy Nectopsyche sp.A similar analysis using data from higher concentration samples(e.g.,Hinkle,Carmichael,and New Alamo Creeks),indicates these streams contained approximatelyﬁve TU for C.dilutus and one TU for the other four species.Existing concentrations ofﬁpronil and its degradates in urban streams appear suﬃcient to cause toxicity not only in a very sensitive species such as C.dilutus,but in several species across diverse macroinvertebrate groups.It is unclear if the impaired movement predicted,such as inability to swim,crawl,or cling to a substrate after48−96h of exposure,is manifested by population declines for these species,but the potential merits furtherinvestigation.parison of measured EC50concentrations from the present study with the concentrations ofﬁpronil and its degradates observed in urban waterways during storm events.Fipronil desulﬁnyl is not shown as no EC50estimates were derived.Four of the33EC50 data points are“greater than”estimates for which a precise EC50is unknown,but they are plotted at their“greater than”value so as not to distort the presentation by excluding some of the least sensitive species.。

A generalized friction correlation for louver fin geometry

Yu-Juei Chang a, Kuei-Chang Hsu b, Yur-Tsai Lin b, Chi-Chuan Wang a,*
a
Energy and Resources Laboratories, Industrial Technology Research Institute, D500 ERL/ITRI, Bldg. 64, 195 H 6 Section 4, Chung Hsing Road, Chutung 310, Hsinchu, Taiwan b Department of Mechanical Engineering, Yuan-Ze University, Tauyuan, Taiwan Received 6 June 1999; received in revised form 14 September 1999
2238
Y.-J. Chang et al. / Int. J. Heat Mass Transfer 43 (2000) 2237±2243
Nomenclature A Ac Dh Dm f f 1, f 2, f 3 Fd Fl Fp Ft Gc Kc Ke Lh Ll Lp M total surface area [m2] minimum ¯ow area [m2] hydraulic diameter of ®n array [mm] major tube diameter [mm] Fanning friction factor dimensionless correlation parameter ®n depth [mm] ®n length [mm] ®n pitch [mm] ®n thickness [mm] rmVc, mass ¯ux at minimum ¯ow area [kg mÀ2] abrupt contraction coecient, dimensionless abrupt expansion coecient, dimensionless louver height [mm] louver length [mm] louver pitch [mm] number of test data point ReDh ReLp Th Tp Td Vc DP r1 r2 rm s y Reynolds number based on hydraulic diameter, dimensionless Reynolds number based on louver pitch, dimensionless TpÀDm [mm] tube pitch [mm] tube depth [mm] maximum velocity [m sÀ1] pressure drop [Pa] inlet air density [kg mÀ3] outlet air density [kg mÀ3] mean density [kg mÀ3] contraction ratio, dimensionless louver angle [deg]

A Competitive Infection Model of Hematogenously Disseminated

A Competitive Infection Model of Hematogenously Disseminated Candidiasis in Mice Redeﬁnes the Role of Candida albicans IRS4in PathogenesisSuresh B.Raman,a M.Hong Nguyen,a Shaoji Cheng,a Hassan Badrane,a Kenneth A.Iczkowski,c Marilyn Wegener,b Sarah L.Gaffen,a Aaron P.Mitchell,d Cornelius J.Clancy a,bDepartment of Medicine,University of Pittsburgh,Pittsburgh,Pennsylvania,USA a;VA Pittsburgh Healthcare System,Pittsburgh,Pennsylvania,USA b;Department of Pathology,University of Colorado,Denver,Colorado,USA c;Department of Biological Sciences,Carnegie Mellon University,Pittsburgh,Pennsylvania,USA dCandida albicans IRS4encodes a protein that regulates phosphatidylinositol-(4,5)-bisphosphate,which was shown to contrib-ute to hematogenously disseminated candidiasis(DC)after several days in the standard mouse model.Our objective was to more accurately deﬁne the temporal contributions of IRS4to pathogenesis.During competition assays in vitro,an irs4-null(⌬irs4) mutant exhibited wild-typeﬁtness.In DC experiments,mice were infected intravenously with the⌬irs4mutant,strain CAI-12 (1؋105CFU),or a mixture of the strains(0.5؋105CFU each).In single-strain infections,quantitative PCR revealed reduced ⌬irs4mutant burdens within kidneys at days1,4,and7but not6h.In competitive infections,the⌬irs4mutant was outcom-peted by CAI-12in each mouse at>6h(competitive indices,P<0.0001).At4and7days,the⌬irs4mutant burdens during com-petitive infections were signiﬁcantly lower than those during single-strain infections(P؍0.01and P<0.001,respectively),sug-gesting increased susceptibility to inﬂammatory responses.Phagocytic inﬁltration of kidneys in response to CAI-12or competitive infections was signiﬁcantly greater than that in response to⌬irs4mutant infection at days1and4(P<0.001),and the⌬irs4mutant was more susceptible to phagocytosis and killing by human polymorphonuclear cells(P؍0.01and P؍0.006, respectively)and mouse macrophages in vitro(P؍0.04and P؍0.01,respectively).Therefore,IRS4contributes to tissue inva-sion at early stages of DC and mediates resistance to phagocytosis as DC progresses.Microarray analysis revealed remarkably similar gene expression by the⌬irs4mutant and reference strain CAI-12within blood,suggesting that IRS4is not signiﬁcantly involved in the hematogenous stage of disease.A competitive DC model detects attenuated virulence that is not evident with the standard model.C andida albicans is the major fungal pathogen among hospital-ized patients in the developed world,causing a wide range of superﬁcial,mucosal,and invasive infections(1).Candidemia is the fourth most common bloodstream infection in the United States and is associated with mortality rates approaching or ex-ceeding40%,despite antifungal therapy(2,3).The development of new treatment,prevention,and diagnostic strategies against candidemia and disseminated candidiasis depends upon better understanding of the pathogenesis of C.albicans infections.In previous studies,we demonstrated that C.albicans IRS4was essential for full virulence during hematogenously disseminated candidiasis in mice.IRS4encodes an Eps15homology(EH)do-main protein that physically interacts with the5=-phosphatase Inp51to regulate the levels and plasma membrane distribution of phosphatidylinositol-(4,5)-bisphosphate[PI(4,5)P2](4–6).Dis-ruption of IRS4resulted in cell wall derangements and impaired hyphal formation within solid agar and mouse kidneys.The tissue burdens of the irs4-null(⌬irs4)mutant within mouse kidneys were similar to those of wild-type C.albicans after20h.However, the⌬irs4mutant was unable to form dense mats of hyphae,and tissue burdens at4days were signiﬁcantly attenuated(4,5).The data suggested that PI(4,5)P2regulation is required for the pro-gression but not initiation of kidney infection.We hypothesized that the attenuated virulence of the⌬irs4mutant results from a failure to maintain cell wall integrity in the face of ongoing cell wall stress during tissue invasion.At the same time,we recognized that IRS4may make contributions to pathogenesis within the kid-neys that were not apparent in the mouse model.Indeed,mutants were signiﬁcantly impaired in adherence to various epithelial cell lines in vitro,suggesting that IRS4is relevant at the early stages of tissue invasion.Furthermore,if the impaired cell wall integrity of the⌬irs4mutant was contributing to attenuated virulence at later time points,it was not clear why such defects would not also be important at early time points.In assessing the contribution of IRS4to virulence,we used the standard mouse model of hematogenously disseminated candidi-asis(7).In this model,C.albicans strains are inoculated via the lateral tail vein,and the kidneys are the primary target organ. Groups of mice are typically infected with a wild-type,isogenic gene disruption,or gene reinsertion strain,and endpoints like mortality and tissue burdens are compared between groups at serial time points(7).The model has advantages of simplicity and reproducibility,but it is limited by relatively insensitive end-points.Moreover,large numbers of mice are typically required toReceived26July2012Returned for modiﬁcation13August2012Accepted12February2013Published ahead of print19February2013Editor:G.S.Deepe,Jr.Address correspondence to Cornelius J.Clancy,cjc76@.Supplemental material for this article may be found at /10.1128/IAI.00743-12.Copyright©2013,American Society for Microbiology.All Rights Reserved.doi:10.1128/IAI.00743-12 Infection and Immunity p.1430–1438May2013Volume81Number5 on January 18, 2015 by guest / Downloaded fromdemonstrate that virulence is attenuated in mutant strains,al-though expenditure of lives may be reduced by the technical skill of investigators and in cases of large differences between strains. Competitive infection models,in which individual hosts are in-fected with a mixture of microbial strains,are powerful tools for identifying small differences inﬁtness(i.e.,relative virulence)be-tween strains(8–16).By eliminating interhost variability,they achieve high levels of sensitivity and spare animals.In recent years, investigators have begun to use competitive models of oral,gas-trointestinal,and disseminated candidiasis in lieu of conventional mouse models to compare the virulence of wild-type and mutant C.albicans strains(8,10,11,13,15,16).These studies have con-clusively identiﬁed C.albicans genes that encode virulence deter-minants,but they have not tapped the full potential of competitive models to characterize the pathogen-host interaction.In particu-lar,the studies have not compared C.albicans strain behavior and host responses during both competitive and single-strain infec-tions,which is a particularly useful strategy for deﬁning the con-tribution of a speciﬁc gene to the pathogenic process over the time course of disease.The primary objective of the present study was to deﬁne more accurately the temporal contribution of C.albicans IRS4to the pathogenesis of disseminated candidiasis.Toward this end,we developed a competitive infection model of hematogenously dis-seminated candidiasis in mice and characterized gene expression by the⌬irs4mutant within blood.We hypothesized that these methods would demonstrate that the virulence of the⌬irs4mu-tant is attenuated at the early stages of kidney invasion,rather than after several days,as suggested in our prior studies. MATERIALS AND METHODSC.albicans strains and growth conditions.C.albicans⌬irs4and an IRS4 reinsertion strain were created and characterized as described in our pre-vious publications(4,5)(Table1).C.albicans CAI-12was the IRS4-intact, isogenic strain used for comparisons throughout the study.Growth rates in vitro were determined in yeast peptone dextrose(YPD)and Sabouraud dextrose(SD)media at30and37°C in microtiter plates,as described previously(19).Extended growth curves in vitro were assessed through four dilution and regrowth cycles of24h each.To induce hyphal forma-tion in liquid media,C.albicans strains grown overnight on YPD agar were subcultured into liquid YPD supplemented with5%fetal calf serum (FCS)and liquid RPMI1640at37°C(4,5).In preparation for intravenous challenge of mice,all strains were grown to stationary phase overnight in YPD medium at35°C.C.albicans cells were washed in sterile saline,and inocula were prepared at the desired concentration in sterile saline.Quantitative real-time PCR.For the standard curves relating changes in threshold cycle(⌬C T)values to C.albicans cell numbers,DNA samples were prepared from sterile saline or naive kidneys spiked with cells from a mid-logarithmic-phase culture of C.albicans CAI-12grown at30°C in YPD medium.The cell density of the undiluted culture was determined by direct counting with a hemocytometer and veriﬁed by plating.The culture was serially diluted in YPD medium.In both spiking and disseminated candidiasis experiments,kidneys were homogenized and subjected to lyti-case treatment(30min at37°C).Genomic DNA was isolated using aDNeasy blood and tissue kit(Qiagen).Our analyses included at least threebiological replicates for all samples,each from independent collections.The quantitative real-time PCR was performed using PerfeCTa SYBRgreen Fast Mix(Quanta Biosciences Inc.)with an ABI7500sequencedetection system(Applied Biosystems).For each20-␮l PCR mixture,2␮lgenomic DNA was used with6␮l nuclease-free water,10␮l PerfeCTaSYBR green Fast Mix,and1␮l of each primer.Gene-speciﬁc primers forIRS4detection in CAI-12(primers IRS-qPCR-For[5=-ACC AGC AATCTT CCA CTG AGA TCA ACA-3=]and IRS-qPCR-Rev[5=-CTT CCATGG CTT CAA CTC ATT AAA CCT TGA-3=],yielding a138-bp product)and primers speciﬁc to the⌬irs4mutant(primers⌬irs-qPCR For[5=-CTG AGG TGG AAG TTG GAG AAA CAA CC-3=]and⌬irs-qPCR-Rev[5=-CCC TGA TTG ACT GGA ACA GGA TCC TC-3=],yielding a122-bpproduct)were designed.The thermocycler program was95°C for10minand40cycles of95°C for30s and58°C for45s.Quantitative PCR(qPCR)assays were run according to the manufacturer’s directions,and resultswere analyzed with sequence detection system software(v2.0.1;AppliedBiosystems).Each sample was assigned a C T value,which identiﬁes thecycle number during PCR whenﬂuorescence exceeds a threshold valuedetermined by the software.Differences in DNA recovery between sam-ples were normalized by determining the total DNA concentration of eachinfected kidney sample([DNA]sample),comparing that concentration tothe DNA concentration values from uninfected kidney([DNA]uninfected),and adjusting each sample C T value according to the formula C T ad-justedϭC Tϩx,where x is log2([DNA]sample/[DNA]uninfected)(20).⌬C T values were used to calculate genomic equivalents(GE)from a standardcurve generated from DNA samples prepared from uninfected kidneysspiked individually with CAI-12,the⌬irs4mutant,and a reinsertionstrain yeast.All qPCR results for samples from infected tissues are ex-pressed as GE per gram equivalent of tissue.Since a full copy of IRS4wasreintroduced into a disrupted native locus in the reinsertion strain,bothwild-type(IRS4)-speciﬁc and⌬irs4mutant-speciﬁc primers ampliﬁedsignals.Therefore,in the mixed infection(infection with CAI-12plus thereinsertion strain),the IRS4primer set ampliﬁed both wild-type allelesfrom CAI-12and one wild-type allele from the reinsertion strain(wild-type total GE),and the⌬irs4mutant primer set ampliﬁed one allele fromthe reinsertion strain(reinsertion strain GE).The actual number of wild-type GE in the mixed infection was calculated using the formula wild-type total GEϪreinsertion strain GE.No signal was detected when thefollowing templates were tested as negative controls:genomic DNA pre-pared from kidneys of uninfected mice,mutant primers for kidneysspiked or infected with CAI-12,and CAI-12primers for kidneys spiked orinfected with the⌬irs4mutant.Competitive infection model.Mouse experiments were approved bythe Institutional Animal Care and Use Committee(IACUC)at the Uni-versity of Pittsburgh.Groups of7-week-old male ICR mice(8to10miceper group;Harlan-Sprague)were individually inoculated by single intra-venous injections of the lateral tail vein with200␮l of sterile saline con-taining(i)1ϫ105CFU of CAI-12,the⌬irs4mutant,or the IRS4reinser-tion strain;(ii)0.5ϫ105CFU each of CAI12and the⌬irs4mutant,or(iii)0.5ϫ105CFU each of CAI-12and the reinsertion strain.Mice wererandomly selected to be included in a given group.Strain concentrationswere conﬁrmed by serial dilution of inocula and enumeration of CFU.Mice in the CAI-12,the⌬irs4mutant,and the CAI-12–⌬irs4mutantgroups were sacriﬁced at6h,1day,4days,and7days after intravenousinoculation,and their kidneys were aseptically removed.Reinsertionstrain-infected groups were sacriﬁced on days1and4after challenge.C.albicans CAI-12does not kill mice at this inoculum over7days,whichensured that interpretation of tissue burdens would not be obscured bydeath.Kidneys were sectioned such that one-half of the left kidney andone-half of the right kidney were combined and used for CFU determi-nation,while the other halves were combined and used for qPCR analysis.The organs were weighed and homogenized in2ml sterile phosphate-TABLE1C.albicans strainsStrain Description ReferenceSC5314Clinical isolate17CAI-12a⌬ura3::imm434/URA318⌬irs4mutant a⌬irs::hisG/⌬irs::hisG⌬ura3::imm434/URA34IRS4reinsertion a⌬irs::hisG/IRS::URA3-hisG⌬ura3::imm434/⌬ura3::imm4344a Strains did not differ in activity of orotidine5=-monophosphate decarboxylase.Competitive C.albicans Infections in Mice1431 on January 18, 2015 by guest / Downloaded frombuffered saline(PBS).For CFU enumeration,serial dilutions were plated onto SD plates containing piperacillin(60mg mlϪ1)and amikacin(60mg mlϪ1).The plates were incubated at30°C for48h.Values were expressed as the log number of CFU per gram kidney.Differences in tissue burdens (numbers of GE or CFU)between strains were compared using analysis of variance(ANOVA).Pairwise comparisons were calculated using a Bon-ferroni adjustment.Within individual mice during competitive infec-tions,results were expressed as the competitive index(CI).CI was deﬁned as[(log10GE/g kidney for mutant strain/log10GE/g kidney for CAI-12)/ (log10GE/ml inoculum for mutant strain/log10GE/ml inoculum for CAI-12)](9).Results were compared using Student’s t test.Histopathology.Histopathology was performed by a pathologist blinded to the experimental design.Kidneys wereﬁxed with formalin and embedded in parafﬁn,after which thin sections were prepared and stained with hematoxylin-eosin(H&E)or periodic acid-Schiff(PAS)(21).For each strain,kidneys from three mice were chosen for image analysis.TIFF images of all the tissue on each of the slides were captured.The images were analyzed on a Windows XP personal computer using the public domain National Institutes of Health(NIH)image program ImageJ(http: ///nih-image/).For each image,outline splines were traced around the total area(s)of tissues,and another series of outline splines was traced around the area(s)involved in acute inﬂammation(5). At least20images for each kidney were analyzed.The percentage of the total area with inﬂammation was calculated and expressed as the meanϮstandard deviation.Phagocytosis and killing assays.Phagocytosis and killing assays were performed as previously described(19,22),with slight modiﬁcations. Polymorphonuclear cells(PMNs)were isolated from heparinized blood by dextran sedimentation,followed by centrifugation through Ficoll-Hypaque.After removing contaminating erythrocytes by hypotonic lysis, the PMNs were resuspended in RPMI1640.Prior to phagocytosis and killing assays,C.albicans strains were opsonized with50%normal human serum at30°C for30min.For phagocytosis assay,0.5ml of opsonized C. albicans cells was incubated with0.5ml of PMNs at a PMN/C.albicans ratio of1:1in1ml RPMI1640at37°C for15min on a shaker.Three drops of the sample were then cytospun and Gram stained.Percent phagocytosis was calculated as the proportion of PMNs containing one or more yeast cells after counting of100PMNs.For the killing assay,opsonized C.albi-cans strains were incubated with106PMNs at a PMN/C.albicans ratio of 50:1in1ml of RPMI1640containing5%human serum at37°C for2h with gentle shaking.After complete lysis of PMNs with sterile water,serial 10-fold dilutions were made and colony counts were enumerated.For mouse macrophage(J774A.1)studies,cells procured from ATCC were generated in a96-well plate(5ϫ105cells/well)by incubation for30min at37°C in5%CO2.Prior to phagocytosis and killing assays,C.albicans strains suspended in modiﬁed Eagle’s medium(MEM)were opsonized with5%normal human serum at room temperature for30min.For the phagocytosis assay,200␮l opsonized C.albicans cells was added to wells containing the monolayer(effector/target cell ratio,ϳ10:1)and the cells were incubated for15min at37°C in5%CO2.After incubation,the supernatant was aspirated and washed with prewarmed MEM.Serial10-fold dilutions were made,and colony counts were enumerated.For the killing assay,200␮l MEM-Sabouraud broth(1:1)was added to wells containing monolayers with phagocytosed C.albicans cells,and the plates were incubated for3h at37°C in 5%CO2.The monolayers were scraped,and after complete lysis of macro-phages with sterile water,serial10-fold dilutions were made and colony counts were enumerated.The phagocytosis and killing experiments were per-formed in triplicate and repeated at least twice.The percentage of phagocy-tized C.albicans cells was deﬁned as[1Ϫ(number of uningested CFU/num-ber of CFU at the start of incubation)]ϫ100.The percentage of C.albicans cells killed by the phagocytes was deﬁned as[1Ϫ(number of CFU after incubation in the presence of phagocytes/number of CFU phagocytosed)]ϫ100.Thefungicidalactivitywascalculatedasthepercentsurvivalof C.albicans after2h of incubation with PMNs.Assessment of gene expression in whole blood using whole-genome microarrays.C.albicans Genome Oligo Set v1.1(Qiagen,Valencia,CA)was resuspended at30␮M in ArrayIt Micro Spotting Solution Plus(Telechem,San Jose,CA)and printed on UltraGAPS slides(Corning,Corning,NY)using a Genemachine Omnigrid arrayer(Genomic Instru-mentation Services,San Carlos,CA).Each slide had two replicated arrayswith6,936features,including70-mer oligonucleotides representing5,948predicted open reading frames(ORFs),318cloned genes,and random70-mer controls.Transcriptional proﬁling experiments in human bloodwere performed as previously described(23).In brief,three biologicalreplicates of C.albicans CAI-12and the⌬irs4mutant were grown tomid-exponential phase in YPD medium at30°C,washed once in0.1MPBS,pH7.0,and resuspended in this buffer at a density of37.5ϫ108cells/ml.After incubation for30min at37°C,100␮l of the suspension wasinoculated into7.5ml of fresh whole human blood from one of the inves-tigators(C.J.C.),which was collected in a heparinized tube.After incuba-tion of the tube with gentle shaking for2h at37°C,cells were harvestedand mixed with1g of425-to600-␮m-diameter glass beads(SigmaChemical Co.,St.Louis,MO).Total RNA was extracted using an RNeasyminikit(Qiagen)and a Mini-BeadBeater(Biospec Products,Bartlesville,OK),and on-column DNase digestion with an RNase-free DNase set(Qiagen)was systematically performed.Twenty to25␮g of total RNA wasused to generate labeled cDNA with a SuperScript indirect cDNA labelingsystem(Invitrogen,Carlsbad,CA).Two cDNAs from different sampleslabeled with different dyes(Cy3and Cy5)were mixed,denatured in3.4ϫSSC(1ϫSSC is0.15M NaCl plus0.015M sodium citrate),0.3%SDS,0.5␮g/␮l salmon sperm DNA,and hybridized overnight(14to16h)to mi-croarray slides.A dye-ﬂip strategy was employed.After washing and dry-ing,the slides were scanned in a GenePix4000B scanner(Axon Instru-ments,Union City,CA).GenePix Pro v3.0software(Axon)was used toextract the spot intensities for bothﬂuorescent dyes,and data were ar-chived in Microsoft Access software.For individual slides,the thresholdintensity for eachﬂuorescent channel was established as the average in-tensity of spots corresponding to negative-control70-mers plus1.5timesthe standard deviation.Only intensities higher than the threshold wereconsidered.Replicated spots in each slide had to have a coefﬁcient ofvariation in intensities ofϽ65%.The intensities of the replicated spots ineach slide that passed the criterion were averaged and used for normaliza-tion.We performed a global normalization among all slides so that thetotal slide intensity for each channel was identical among slides and chan-nels.The ratio of the intensity of the⌬irs4mutant to that of strain CAI-12was used for further analysis.We applied signiﬁcance analysis of microarrays(SAM)to detect dif-ferentially expressed ORFs(24).SAM assigns a score to each gene on thebasis of the change in gene expression relative to the standard deviation ofrepeated measurements.For genes with scores greater than an adjustablethreshold,SAM uses permutations of the repeated measurements to esti-mate the percentage of genes identiﬁed by chance,the false discovery rate(FDR).SAM has been shown to be superior to conventional microarrayanalysis based on pairwise fold change methods.Cluster software andTreeview software were used for cluster analysis and display of genome-wide expression data(25).Differentially expressed ORFs were assignedgene names,descriptions,and pathways on the basis of assignments in theCandida Genome Database(/).Gene expression by the strains was conﬁrmed for eight genes by re-verse transcription-PTR(GCA2,ERG16,RNR22,CWH8,SKN1,CHT2,RHO1,and ERK1),using primers designed and synthesized by Invitrogen(see Table S1in the supplemental material).cDNA was synthesized using1␮g total RNA,2.5␮M reverse primers,500␮M deoxynucleosidetriphosphate(dNTP)mix,5mM dithiothreitol,40U RNaseOUT,1ϫﬁrst-strand buffer,and100U SuperScript III reverse transcriptase(Invit-rogen)at46°C for2h.Ampliﬁcation was performed for30cycles,whichgave band intensities linearly proportional to the amount of cDNA.Thereaction mixtures included1␮l a1/10dilution of cDNA in0.2mM dNTPmix,1.5mM MgCl2,0.2␮M each primer,1ϫPCR buffer,and1.5UPlatinum Taq DNA polymerase(Invitrogen).Ten microliters of each PCRRaman et al. Infection and Immunity on January 18, 2015 by guest / Downloaded fromproduct was run in a1.5%agarose gel,and the gel was stained with ethidium bromide.Images were digitalized using a transilluminator con-nected to a camera and Quantity One software(Bio-Rad).The software esti-mated the band intensity for each PCR product,which was used to calculate the ratio of⌬irs4mutant expression/CAI-12expression for each ORF.Statistics.For comparisons across groups(see Fig.1,4,and5), ANOVA was used.Pairwise comparisons were then made using a Bonfer-roni adjustment.Otherwise,comparisons were made using t test.All anal-yses were performed using STATA v11software(College Station,TX).P values ofϽ0.05were considered signiﬁcant.Microarray data accession numbers.Microarray data were deposited in the NCBI GEO database under accession number GSM967204(https: ///geo/query/acc.cgi?accϭGSM967204). RESULTSFitness of⌬irs4mutant in vitro.To distinguish between C.albi-cans strains in competition assays,we designed qPCR primers that targeted speciﬁc segments of IRS4and the hisG cassette used for gene disruption.In titration experiments,serial dilutions of yeasts of strain CAI-12,the⌬irs4mutant,or the IRS4reinsertion strain were suspended in sterile saline and spiked into explanted,unin-fected mouse kidneys.Genomic DNA extracted from suspensions and spiked kidneys was used as a template for qPCR assays that established standard curves(see Fig.S1in the supplemental ma-terial).The signals from these reactions were linear over at least7 orders of magnitude for each strain(all R2values wereՆ0.96). The limits of detection in suspension and spiked kidneys were at or near1and10yeast cells,respectively.As we showed previously,growth rates of CAI-12and the⌬irs4 mutant were indistinguishable after the strains were indepen-dently inoculated into YPD or SD medium at30°C(see Fig.S2in the supplemental material).The growth curves for a mixed culture of CAI-12and the⌬irs4mutant were similar to those of the indi-vidual strains.At all time points,qPCR conﬁrmed that the con-centrations of the strains were the same whether they were grown alone or in mixed cultures.Similar results were obtained in SD medium at30°C and at37°C(not shown).In addition,there were no signiﬁcant differences in the growth curves of the strains through four dilution and regrowth cycles of24h each.The for-mation of hyphae by the strains in liquid medium was compara-ble,consistent with our previous reports(10,11).Relative virulence of the⌬irs4mutant during hematog-enously disseminated candidiasis.To establish our competitive infection model,we infected groups of mice intravenously with CAI-12alone(1ϫ105CFU/mouse),the⌬irs4mutant alone(1ϫ105CFU/mouse),or a1:1mixture of CAI-12and the⌬irs4mutant (0.5ϫ105CFU/strain/mouse).Using conventional methods for enumerating the CFU within the kidneys,we corroborated our earlierﬁndings for single-strain infections:tissue burdens of the the⌬irs4mutant were diminished compared to those of CAI-12at 4and7days but comparable to those of CAI-12at6h or1day(Fig. 1A).The qPCR method of quantitating genome equivalents(GE) detected absolute candidal burdens during single-strain infections that were approximately1log unit higher than those suggested by the numbers of CFU(Fig.1B).By qPCR,the⌬irs4mutant inoc-ulated alone caused signiﬁcantly lower tissue burdens than CAI-12at1day,as well as4and7days.At6h,there were still no differences in tissue burdens between mice infected with the⌬irs4 mutant and mice infected with CAI-12.In the competitive model,the tissue burdens of the⌬irs4mu-tant and strain CAI-12at6h and1day,as determined by qPCR,were identical to those observed when the strains were given alone (Fig.1B).At days4and7,however,the tissue burdens of the⌬irs4 mutant in the competitive model were signiﬁcantly attenuated compared to those from the⌬irs4mutant single-strain infection. The burdens of CAI-12at each time point were identical whether the strain was given alone or with the mutant.Of note,the tissue burdens of an IRS4reinsertion strain were identical to those of CAI-12in both single-strain and competitive infections,indicat-ing that the attenuated virulence of the⌬irs4mutant was a result of gene disruption(see Fig.S3in the supplemental material).Of course,the real power of competitive infection models comes from comparing strains within individual animals,rather than across groups.In these intrahost analyses,results are ex-pressed as the competitive index,which is the ratio of the mutant to a reference ing the competitive index,the⌬irs4mu-tant was signiﬁcantly attenuated at each time point,including6h, FIG1Recovery of CAI-12and the⌬irs4mutant from mouse kidneys.(A) Tissue burdens for groups of mice were determined by enumerating the CFU.(B)Relative tissue burdens were determined by qPCR measurement of ge-nome equivalents per gram of tissue.In a three-way comparison of the number of CFU of tissue burdens(A)at6h,1day,4days,and7days,P values were0.39, 0.03,0.0001,and0.007,respectively(ANOVA).In a four-way comparison of the number of GE relative to tissue burdens(B)at6h,1day,4days,and7days, P values were0.17,Ͻ0.0001,Ͻ0.0001,andϽ0.0001,respectively(ANOVA). *,PϽ0.001for CAI-12versus the⌬irs4mutant at day4;**,Pϭ0.03for CAI-12versus the⌬irs4mutant at day7;***,PՅ0.001for CAI-12versus the ⌬irs4mutant,in single-strain and mixed infections;****,Pϭ0.01for the⌬irs4 mutant in single-strain infection versus the⌬irs4mutant in mixed infections at day4;*****,PϽ0.001for the⌬irs4mutant in single-strain infection versus the⌬irs4mutant in mixed infections at day7.Mix,tissue burdens(number of CFU)during mixed infections,not distinguishing between strains(A);Mix–CAI-12,relative tissue burdens(GE)of CAI-12during mixed infections;Mix-⌬irs4,relative tissue burdens(GE)of the⌬irs4mutant during mixed infec-tions.Data are means of results for8to10mice per group per time point.Error bars represent standard deviations.Competitive C.albicans Infections in Mice1433 on January 18, 2015 by guest / Downloaded from。

Small Interfering RNA for Effective Cancer Therapies

See discussions, stats, and author profiles for this publication at: /publication/49744075 Small Interfering RNA for Effective Cancer TherapiesARTICLE in MINI REVIEWS IN MEDICINAL CHEMISTRY · FEBRUARY 2011Impact Factor: 3.19 · DOI: 10.2174/138955711794519528 · Source: PubMedCITATIONS 7DOWNLOADS9VIEWS1084 AUTHORS, INCLUDING:Xiaohui WangChinese Academy of Sciences 39 PUBLICATIONS 695 CITATIONSSEE PROFILE Yanguo ChenTexas A&M University464 PUBLICATIONS 7,979 CITATIONSSEE PROFILEAvailable from: Xiaohui WangRetrieved on: 10 July 2015114 Mini-Reviews in Medicinal Chemistry, 2011, 11, 114-1241389-5575/11 $58.00+.00© 2011 Bentham Science Publishers Ltd.Small Interfering RNA for Effective Cancer TherapiesX. Wang 1, Y. Chen 2, J. Ren 1 and X. Qu*,11Laboratory of Chemical Biology, Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Changchun, Jilin 130022, China2College of Life Science, Jilin University, Changchun, Jilin, 130021, ChinaAbstract: Small interfering RNA (siRNA) has become a specific and powerful tool to turn off the expression of target genes, and has turned into a promising tool in molecular medicine. It can be targeted against cancer by several strategies. These include the suppression of overexpressed oncogenes, retarding cell division by interfering with cyclins and related genes or enhancing apoptosis by inhibiting anti-apoptotic genes. RNA interference (RNAi) against multidrug resistance (MDR) genes or chemo/radio-resistance and angiogenesis targets may also provide beneficial cancer treatments. Success-ful cancer therapy by siRNA in vitro and in vivo provides the enthusiasm for potential therapeutic applications of this technique. Here, we review RNAi in cancer therapy, highlighting recent progress and examining the hurdles that must be overcome before this promising technology is ready for clinical use.Keywords: Small interfering RNA, pharmacodynamics, cancer, oncogene, apoptosis, cell cycle, angiogenesis, multidrug resistance.1. INTRODUCTIONSmall interfering RNA (siRNA), sometimes known as short interfering RNA, is a class of 21-23 nucleotide-long double-stranded RNA (dsRNA) with 2-nt 3' overhangs on either end (Fig. 1) [1-3]. These siRNAs intermediates are involved in the RNA interference (RNAi) pathway where siRNAs bind to the RNA induced silencing complex (RISC) and then selectively degrade the complementary single-stranded target RNA in a sequence-specific manner. Since the discovery of RNAi activity in mammalian cells, not only is siRNA being adopted for the discovery and validation gene function through cell culture and animal model studies [3], the growing success as a research tool and the favorable outcomes of several early phase clinical trials have also stirred up tremendous interest in using siRNA as a potential therapeutic agents [4-10]. With the explosion in knowledge generated by a growing understanding of the human genome and the development of high-throughput gene expression profiling of cancer cells, more and more genes that contrib-ute to the tumor transformation and metastasis are discov-ered [11-15]. RNAi has been applied in this setting to sup-press the expression of dominant mutant oncogenes, gene amplifications, translocations and viral oncogenes as a novel cancer therapy strategy and improve the efficacy of existing chemotherapeutic agents by specifically targeting and silenc-ing resistance-associated genes.While traditional therapeutic approaches have been widely used for cancer therapy, there are limitations to these*Address correspondence to this author at the Laboratory of Chemical Biol-ogy, Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China; Tel: 86-431-8526-2656; Fax: 86-431-85262656; E-mail: xqu@ approaches. Most small molecule inhibitors are not specific with regard to their targets. For example, protein tyrosine kinase inhibitors, many of which are ATP competitive in-hibitors and have alternative targets that compromise their specificity and contribute to toxicity [16-17]. Additionally, the structures of some target proteins are unavailable, which excludes the possibility of rational design of corresponding small molecule inhibitors. The advantages of siRNA over other therapeutic modalities include its high specificity, ver-satility, and efficiency, which enable the use of RNAi to se-lectively knock-down expression of alleles carrying point mutations, insertions, or deletions, and thereby to produce cells with reduced expression of essential genes. The dura-tion of knockdown by siRNA can typically last for 7-10 days, and has been shown to transfer to daughter cells [18]. Of further note, siRNA is effective at quantities much lower than alternative gene silencing methodologies, including antisense and ribozyme based strategies and can be works in the nanomolar range. Furthermore, siRNA is less likely to interfere with gene regulation by endogenous microRNAs [19-20]. Therefore, siRNA provides a novel therapeutic ap-proach to silence cancer-causing genes otherwise not amena-ble to conventional therapeutics, such as small molecules, proteins, or monoclonal antibodies. 2. RNAi MECHANISMThe mechanism of RNAi has been well characterized and reviewed [1-3, 21-22]. Here we only provide a brief account of this process.The RNAi pathway is initiated by an RNase III–type en-zyme, Dicer, which cleaves long dsRNA molecules into short RNA of between 19 and 21bp which have symmetric 2–3 nucleotide overhangs and 5’ phosphate and 3’ hydroxyl groups, although examples of shorter and longer duplexesSmall Interfering RNA for Effective Cancer Therapies Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 2 115 Fig. (1). Schematic representation of a siRNA molecule : a ~ 19-21 base pair RNA core duplex that is followed by a 2 nucleotide 3' overhang on each strand.have been reported [23]. This process is ATP dependent and thought to be restricted to the cytoplasm. The generated siRNA associates with the RNA-induced silencing com-plexes (RISC) composed of four different subunits, a heli-case, exonuclease, endonuclease, and homology searching domains. The siRNA duplex is then unwound by the helicase in RISC and then one strand of duplex (the “passage strand”) is cleaved and discarded, while the other strand, the one with the lowest duplex stability at its 5’ end which is usually the antisense one, guides RISC to its complementary or near-complementary mRNA, with target cleavage occurring at a single site in the centre of the duplex region between the guide siRNA and the target mRNA, 10 nucleotides from the 5’ end of the siRNA. An RNA endonuclease within the complex, Argonaute 2 (Ago2), cleaves the target mRNA that loses its protection against RNases and gets degraded via its PIWI domain, which adopts an RNase H– like structure [24]. Importantly, the guide strand loaded by RISC is protected from degradation and can direct the cleavage of many mRNA molecules [25-26]. This cleavage does not require ATP, but does require magnesium ions. Exogenous synthetic siRNA can also be directly incorporated into RISC, thereby bypassing the requirement for dsRNA processing by Dicer, and modulate transcript levels by rapid, Ago-2 mediated transcript cleavage (Fig. 2). SiRNA can also induce moder-ate level of gene knockdown through translational repression due to partial complementarity to 3’ un-translated region (3’ UTR) of the mRNA [27-29]. This silencing mechanism may be responsible for the ‘‘off-target’’ effects observable during RNAi experiments which can be minimized by including design parameters that select siRNA with low 3’UTR seed complement frequencies.3. PHARMACODYNAMICSSynthetic siRNA is usually used as effective therapeutics for oncology applications without inducing an interferon response. Unmodified naked siRNA has a half-life of less than 1 hour in human plasma and is cleared rapidly through the liver and kidneys due to their small size. Stabilizing chemical modifications (e.g., phosphorothioate, 4-thio, cho-lesterol) without affecting RNA-interference effect increase binding to serum proteins and the circulating half-life in vivo [30]. This allows more opportunity for the siRNA to enter target tissues. Morrissey et al. reported that the plasma half-life of an unmodified siRNA duplex in mice was 0.03 h, the half-life of a modified duplex was 0.8 h, and the half-life of a modified duplex packaged in a stabilized nucleic acid lipid particles (SNALP) was 6.5 h [31]. Similarly, Soutschek et al. reported studies using modified siRNAs in rats in which an unconjugated duplex had an elimination half-life of 0.1 h, while a cholesterol-conjugated duplex had a half-life of 1.5 h [32].Even when chemical modifications are employed to sta-bilize a duplex from degradation, plasma clearance may still be rapid. Incorporation siRNA with particulate carriers ap-pears to be a promising approach to improve pharmacokinet-ics because they are prevented from being excreted from the kidney due to their size. Furthermore, surface modifications of particulate vectors with aptamer/ targeted ligand /antibody leads to enhanced potency relative to non-targeted formula-tions and enables the cell-selective targeting of siRNAs in animals [33-36]. Bartlett et al. reported that transferrin-targeted siRNA cyclodextrin nanoparticles reduced tumor luciferase activity by approximately 50% relative to non-targeted siRNA nanoparticles and demonstrated that primary advantage of targeted nanoparticles is associated with proc-esses involved in cellular uptake in tumor cells rather than overall tumor localization [37]. Landen et al. investigated the uptake of siRNA against EphA2 using tail vein injection of siRNA complexed with DOTAP liposomes [38]. They showed successful application of siRNA delivered by DOPC liposomes to reach and treat intra peritoneally growing ovar-ian cancer in mice and found that 30% of tumor cells can be reached within 1 hour of injection. Even ten days after the administration, fluorescent siRNA molecules were still found in tumor cells.Recently, Svensson et al. have described a facile assay that facilitates the evaluation of the pharmacodynamics of a systemically delivered siRNA formulation using a mouse strain containing robust ubiquitous expression of firefly lu-ciferase [39]. The primary advantage of this approach is that siRNA efficacy against a nonessential target can be easily evaluated in any tissue. This method can enhance the ability to rapidly screen, compare and optimize various siRNA for-mulations for tissue-targeted systemic delivery in a preclini-cal development setting.4. RNAi AND CANCERMost cancers are characterized by abnormal gene expres-sion, which is thought to contribute to the tumor tansforma-tion and metastasis. Silencing such genes by siRNA would appear to be a rational approach to the therapy of cancer. Indeed, in vitro and in vivo findings have demonstrated the efficacy of siRNA knocking down of genes that are pivotal for carcinogenesis, tumor growth, metastasis, angiogenesis, and chemo-resistance, leading to tumor growth inhibition. Molecules that indirectly contribute to tumor growth are also potential candidates for RNAi intervention. In most cases, suppression of a single gene is not enough to cure the disease and it could be necessary to inhibit multiple targets simulta-neously or to combine gene silencing with other therapiessuch as chemotherapy and radiotherapy.116 Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 2 Wang et al.4.1. Carcinogenesis PathwaysViral oncogenes, Ras, p53, the receptor protein tyrosine kinases (PTKs) and p53 are the main components contribut-ing to the cancer development and progression. Herein we first try to review the recent progress in RNAi based cancer therapy targeting against these pathways. 4.1.1. Viral OncogenesInfection with viruses has been detected in several human cancers, and the functions of virally-encoded genes have been implicated in the development and maintenance of those malignancies. For example, More than 90% of cervical carcinomas contain genome sequences derived from humanFig. (2). Proposed mechanism of RNAi. The initial dsRNA is cleaved into siRNA by a nuclease called Dicer in an ATP-dependent reaction. SiRNA is then incorporated into RISC, which contains a helicase that unwinds the duplex. The antisense strand of the duplex guides the ac-tive RISC to the complementary mRNA. In the case of perfect complementarity, the target is cleaved by an Ago2-mediated mechanism. The cleaved mRNA is degraded rapidly and the protein that it encodes is not produced. SiRNA can also induce moderate levels of gene knock-down by seed-mediated translation attenuation. This process of off-targeting can be minimized by optimization of the siRNA design parame-ters. (This figure is adapted from the reference [28] with the permission of Nature Publishing Group).Small Interfering RNA for Effective Cancer Therapies Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 2 117papillomavirus (HPV), and the viral E6 and E7 genes are supposed to co-operate in mediating deregulated cell prolif-eration and survival [40]. Targeting of these genes with siR-NAs resulted in attenuation of cell proliferation, increased apoptosis, and reduced tumor size in a mouse model and these phenotypic outcomes were not manifested in HPV-negative cells [40-44]. The development of several other types of cancer are also associated with viral infection, for example Epstein-Barr Virus (EBV) in nasopharyngeal carci-noma, Kaposi’s sarcoma-associated herpesvirus (KSHV) in primary effusion lymphoma, and human T-cell leukemia virus type 1 (HTLV-1) in adult T-cell leukaemia, and in models of each of these diseases RNAi targeting of virally-encoded genes resulted in changes in invasive behavior, tu-mor cell apoptosis or reduced tumorigenicity in a mouse model [45-49].4.1.2. Ras PathwaysRas is one of the most commonly mutated oncogenes in human malignancies (about 30% of all human tumors) [50], making it difficult to target. One major obstacle regarding designing a sequence-specific anti-Ras therapy simply lies in the nature of its activation: a single point mutation suffices to turn the protein on and convert it to a dominant oncoprotein [51]. Depletion of K-rasV12 using RNAi resulted in almost completely abrogated anchorage-independent growth and failure to produce tumors not only in pancreatic cancer cell culture but, encouragingly, in an animal in vivo model as well. Importantly, expression of the wild-type K-Ras gene was unaffected [52]. Zhou et al. reported that silencing of N-Ras gene expression using RNAi decreased transformation efficiency and tumor growth in transformed cells [53]. A major advantage of RNAi-mediated anti-Ras cancer therapy, in contrast to other Ras-based therapies, is the specific tar-geting of oncogenic Ras. This might be crucial, since inhibi-tion of Ras function in cells could interfere with normal cell viability.4.1.3. PTKs PathwaysPTKs are key molecules involved in cellular signal trans-duction and are frequently mutated in human cancers. When mutated or altered structurally, PTKs become potent onco-proteins, leading to cellular transformation from constitu-tive/enhenced kinase activity [54]. Such transformation can occur due to genomic rearrangements such as chromosomal translocations, which produce oncogenic fusion proteins that include a PTK catalytic domain and an unrelated protein that provides constitutive activity to the tyrosine kinase [55]. One example is the Bcr-Abl fusion protein, which is commonly observed in chronic myeloid leukemia. Transfection of leu-kemia cells with siRNA targeting a Bcr-Abl fusion transcript induced apoptosis [56-58]. Noticeably, leukemic cells with-out Bcr-Abl rearrangement were not killed by anti-Bcr-Abl siRNA. Moreover, the introduction of Bcr-Abl siRNA in-creases the sensitivity of Bcr-Abl overexpressing cells to the chemotherapeutic agent imatinib [56-57, 59]. Wohlbold et al. found that treatment of cells with imatinib mesylate and a suboptimal dose of BCR/ABL siRNA resulted in regained sensitivity to imatinib in formerly resistant leukemic cells as well as increased sensitivity to gamma irradiation [60].Non-small-cell lung cancers (NSCLC) often have muta-tions within the epidermal growth factor receptor’s (EGFR) catalytic protein tyrosine kinase domain, resulting in consti-tutive activation. The oncogenic activity of EGFR promotes cell proliferation and survival. RNAi mediated knocking-down the mutant EGFR in vitro results in apoptosis [61-63]. Sequence-specific siRNAs target specifically the mRNA of the mutant EGFR allele and have no effect on the expression of the wild-type EGFR. These studies indicate that the ex-pression of the mutant EGFR is essential for the oncogenic-ity in lung cancers harboring these mutations, and that the RNAi-mediated down-regulation of the mutant EGFR results in specific and extensive apoptosis of NSCLC cells.4.1.4. p53 PathwaysThe tumor suppressor protein p53 is a frequent target for point mutations associated with oncogenesis. Since mutated p53 forms have dominant negative effects on wild type p53 in addition to intrinsic properties such as an altered binding partner repertoire, a specific inhibition of the mutant forms might restore wild type p53 functions [53]. Indeed, it has been demonstrated that transfected siRNAs suppress a mu-tant p53 carrying a point mutation without affecting the ex-pression of its wild type counterpart [64].As a consequence, p53-dependent apoptosis can be induced.p53 pathway interacts with several other signal transduc-tion pathways, and a number of positive and negative auto-regulatory feedback loops act upon the p53 response. Several molecules participated in the p53 pathway have been inves-tigated using RNAi technology and some of these may serve as potential targets for RNAi based cancer therapy. Hdm2, which is a key p53 negative regulator, has been found to be overexpressed in many human cancers [65-67]. RNAi-mediated silencing of Hdm2 significantly inhibits the growth potential of human breast cancer cells in a p53- dependent manner [65, 68]. Similarly, knocking-down the expression of Notch-1[69], Delta-like-1 or Jagged-1 within the p53 path-way by RNAi also induces apoptosis and suppresses cell proliferation in multiple glioma cells transfected with siR-NAs [70].PPM1D, a negative regulator of the p38 MAP kinase-p53 pathway, is an oncogene that is overexpressed in many can-cers and is also proposed to participate in other critical cell survival pathways [71]. Down-regulation of PPM1D by siRNA induced reduced cell proliferation and apoptosis in MCF7 and ZR-75-1 cells carrying wild-type p53 but not in BT-474 carrying mutant p53 [71]. Thus, targeting PPM1D by siRNA may be another useful strategy for tumors with normal p53.4.1.5. Other Oncogenesis PathwaysOther cellular pathways altered in cancer include glioma-associated oncogene (GLI) pathway, adenomatous polyposis coli (APC) pathway, hypoxia-inducible transcription factor (HIF) pathway, and so on. For example, more than 60 puta-tive direct HIF-1 target genes have been identified to play important roles in tumor progression [72].Knocking-down the expression of HIF-1a using RNAi enhanced hypoxiame-diated tumor cell apoptosis in vitro, prevented tumor growth and resulted in increase of chemosensitivity [73-75]. There-118 Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 2 Wang et al.fore, targeting the HIF-1alpha may be useful treatment mo-dality for cancers.-Catenin is a member of the APC signaling pathway which is crucially involved in the regulation of cellular pro-liferation. Mutation of this gene results in increased -catenin protein level which enhances cellular proliferation and support colon cancer development. Knocking-down of the overexpressed -catenin by siRNAs decreased the ex-pression of -catenin-dependent genes and reduced the pro-liferation rates of colon cancer cells [76-77].Another poten-tial target of cancer therapy includes inhibiting gene amplifi-cation exemplified by N-MYC (neuroblastoma-myelocyto-matosis viral oncogene) expressed at late stages [78] or in aggressive forms of neuroblastoma and erbB-2 (verbB-2 avian erythroblastic leukemia viral oncogene homolog 2) [79-81]. In conclusion, although a thorough discussion of each gene involved in carcinogenesis pathways is almost impossible, many reports have showed silencing of various genes results in the suppression of proliferation and/or en-hanced apoptosis of cancer cells, which emphasizes the po-tential of RNAi as a therapeutic method to treat malignant tumors.4.2. Cell CycleRNAi can be directed against cell-cycle control genes to block cell division and promote apoptosis. Polo-like kinase-1 (PLK1) is a key cell-cycle regulator that is overexpressed in various human tumors [82-83]. Spänkuch-Schmitt et al. used RNA interference to silence the PLK1 in breast cancer, cer-vical cancer, colon cancer and lung cancer cell lines [82]. Several cells manifest abrogation of spindle formation and mitosis was arrested at various time points dependent on the cell line Treatment with siRNA also led to a statistically sig-nificant anti-proliferative activity. Additionally, Nogawa et al. showed that intravesical administration of PLK1-targeted siRNA carried by cationic liposomes prevented tumor growth in an animal model of bladder carcinoma [84]. More importantly, RNA interference-mediated silencing of the Polo-like Kinase 1 enhanced cancer cell chemo- and radia-tion sensitivity [85-87].An increase of Skp-2, which is involved in the degrada-tion of cell-cycle regulators, is thought to be one of the po-tential mechanisms underlying cell-cycle derangement in various types of cancer. Anti-Skp-2 RNAi was delivered to a human small-cell lung carcinoma cells with increased Skp-2 and knocking-down the expression of Skp2 resulted in effi-cient inhibition of the in vitro cell growth [88]. However, no significant effect on the growth of cells with low Skp-2 lev-els was observed upon administration with Skp-2 siRNA [88]. Overexpression of p28GANK increases the phosphory-lation of pRB and inhibits p16INK4a activity, thus accelerat-ing cell-cycle progression. RNAi was used to inhibit p28GANK expression in HCC [89]. The results showed that the depletion of p28GANK led to the decrease of pRB phos-phorylation, the enhancement of caspase 8- and 9-mediated apoptosis, and the suppression of HCC tumor growth in nude mice.Other molecules targeted by siRNA are the cyclins that are important for phase specific progression. Li et al. showed that depletion of cyclin E by RNAi promoted apoptosis of hepatocellular carcinoma cells, blocked cell proliferation and inhibited hepatocellular carcinoma tumor growth in nude mice [90]. Cyclin A2 is critically involved in two steps in the cell cycle, both in the entry of G1 cells into S phase and in the G2 - M transition [91-92]. Increased expression of cyclin A2 has been detected in many types of cancers [93]. Wang et al. indicated that transient transfection of chronic myeloge-nous leukemia cell lines with siRNA suppressed cyclin A2 overexpression, thereby inhibiting cell growth and promot-ing apoptosis [91]. The role of cyclin B1 in progression through the G2/M phase of the cell cycle is well character-ized, and its expression is also known to be aberrant in a number of malignant tumors [93]. The reduction of cyclin B1 levels in HeLa cells by siRNA caused inhibition of prolifera-tion and apoptosis [94]. Cells with reduced cyclin B1 were more susceptible to the administration of taxol and showed inhibited tumor growth in nude mice [94].4.3. Cell SenescenceIn contrast to normal somatic cells, cancer cells over-express telomerase which maintains the length of telomeres, preserving genome stability [95]. Silencing the human te-lomerase reverse transcriptase (hTER) by siRNA can results in cancer cell growth impairment [96-97]. Inhibition of hTERT by RNAi in a model of bladder cancer resulted in decreased cell number of S phase, increased cell population of G0/G1 phase both in vitro and in vivo, and attenuated tu-mor growth of xenograft mice model compared with controls [98]. Moreover, Nakamura et al. showed that knocking down the expression of hTER by siRNA sensitized cancer cells to ionizing radiation and chemotherapy [99].Other molecules associated with cellular senescence may also serve as targets for RNAi cancer therapy. The mammal-ian heterogeneous nuclear ribonucleoparticule A1 and A2 proteins bind to the G-tails of telomeres with high affinity. siRNA mediated down-regulation of A1/A2 protein expres-sion in various human cancer cells, such as those derived from cervical, colon, breast, ovarian and brain cancers, in-duces specific and rapid apoptosis but not in normal cells [100-101].4.4. ApoptosisOver-expression of anti-apoptotic factors characterizes cancer cells. These anti-apoptotic proteins include Bcl-2, Bcl-xL, surviving, X chromosome-linked IAP (XIAP) and Fas-associated death domain-like interleukin-1b-converting enzyme-like inhibitory protein (FLIP). This property enables them to survive under abnormal growth stimuli and confers resistance to different apoptotic triggers, like oxidative stress, chemo- and radio-therapeutic agents. Therefore, the restoration of apoptosis by using RNAi to target key anti-apoptotic proteins expressed by cancer cells would have im-portant therapeutic implications. SiRNA against Bcl-2 had a strong ant-tumor activity in a mouse model of liver metasta-sis and in a xenograft model of human prostate cancer [102]. Specific suppression of Bcl-2 and c-Raf by RNAi induced apoptosis and enhanced the efficacy of the chemo-therapeutic agent Etoposide on myeloid leukemia cells [103]. Remarkably, synergistic anti-tumor effects have also beenSmall Interfering RNA for Effective Cancer Therapies Mini-Reviews in Medicinal Chemistry, 2011, Vol. 11, No. 2 119obtained in vivo by co-administering cisplatin and Rad51-targeted siRNA [104] . Down-regulation of survivin by RNAi inhibited tumor growth in vitro and in vivo through induction of apoptosis [105-106]. RNA interference target-ing the XIAP, leading to an elevation of caspase-3 activity, promotes radiation-induced apoptosis [107-108]. Other molecules with anti-apoptotic function, though not directly related to the apoptosis pathway, may also serve as targets for RNAi technology.4.5. AngiogenesisFor tumors to become prolific and metastasize, they must be well vascularized. The process of tumor angiogenesis has been investigated as an important target for siRNA against cancer. VEGF plays a critical role in the pathological angio-genesis that occurs in a number of cancers. Takei et al. have shown that siRNA against VEGF mixed with atelocollagen suppressed the in vivo growth of PC-3 prostate cancer cells [109]. Suppression of VEGF by RNAi in colorectal carci-noma, osteosarcoma, hepatocellular carcinoma and retino-blastoma can also lead to the inhibition of tumor growth and metastasis [110-113].CD31/PECAM-1 (Platelet endothelial cell adhesion molecule-1) is also a major constituent of the endothelial cell intercellular junction. Santel et al. systematically adminis-tered lipoplexed siRNA anti-CD31 to mice with establishes tumors grown subcutaneously and found a decrease in the total amount of CD31 positive vessels and in the vessels length [114]. Notably, tumor growth inhibition as well as reduction in the volume of lymph node metastases was ob-served even when tumors were grown orthotopically.4.6. Cellular Adhesion and InvasionRNAi technology can be used to target the molecules involved in tumor invasion and metastasis. Urokinase plas-minogen activator receptor (uPAR) and cathepsin B are pro-teases implicated in the extracellular matrix degradation, a characteristic feature of tumor progression. Some investiga-tors used the RNAi approach to silence uPAR and cathepsin B. They found that RNAi of uPAR and cathepsin B reduces glioma cell invasion and angiogenesis both in vitro and in vivo [115-119].The metalloproteinase family of proteins is involved in cellular migration and invasion [120-121]. Jia et al. reported that down-regulation of metalloproteinase 11 by siRNA in-hibited the metastatic capability of murine hepatocarcinoma cell Hca-F to lymph nodes [122]. Blackburn et al. showed that RNA interference inhibition of matrix metalloprotein-ase-1 prevents melanoma metastasis by reducing tumor col-lagenase activity and angiogenesis [123]. Sanceau et al. em-ployed siRNA to inhibit matrix metalloproteinase- 9 and demonstrated that suppression of this protein led to a change in phenotype for Ewing’s sarcoma COH cells and the treated cells became less migration, aggregated in clusters, and ex-pressed increased levels of E-cadherin and other cytoskeletal proteins associated with cell-cell adhesion [124].The elevated glycolysis and proton secretion in tumors is also supposed to contribute to the transformation, prolifera-tion and metastasis of cancer cells. Proton pump V-ATPase is the enzyme that can pump protons into extracellular envi-ronment and is important in solid tumors for maintaining neutral cytosolic pH and acidic extracellular pH of cancer cells [125]. Inhibition of V-ATPase activity by down-regulation of ATP6L by RNAi has been shown to effectively retard cancer growth and suppressed cancer metastasis [125].CXC chemokine receptor-4 (CXCR4) plays an active role in the adhesion/metastatic process. SiRNA against CXCR4 effectively suppressed CXCR4 expression in sali-vary gland mucoepidermoid carcinoma cells [126] and hu-man breast carcinoma cells[127], leading to significant de-crease in malignant cell invasiveness in vitro. More impor-tantly, direct injection of a pool of naked siRNA against CXCR4 has been found to prevent tumorigenesis of a breast cancer cells in SCID mice [127]. Thus, siRNA targeting CXCR4 may represent a potentially novel preventive and therapeutic strategy for cancer treatment.Over-expression of RhoA and RhoC is associated with poor prognosis of breast cancer, due to increased tumor cell proliferation and invasion. In vitro, RhoA and RhoC siRNAs inhibited human breast cancer cells proliferation and inva-sion more effectively than conventional blockers of Rho cell signaling [128]. Moreover, in a nude mouse model, intratu-moral injections of anti-RhoA siRNA almost totally inhib-ited the growth and angiogenesis of xenografted human breast tumors [128].Overexpression of receptor tyrosine-kinase RON medi-ates the transformed phenotypes in immortalized colon epithelial cells. Knocking down the expression of RON by siRNA significantly affected cancer cell proliferation and motility and led to increased apoptotic cell death [129]. Fo-cus-forming activities and anchorage-independent growth of colon cancer cells were also dramatically reduced [129].Heparanase is an endoglycosidase which cleaves heparan sulfate and participates in degradation and remodeling of the extracellular matrix (ECM) [130]. It is preferentially ex-pressed in human cancers. Administration of siRNA against heparanase into breast carcinoma and hepatocellular carci-noma cells led to a profoundly reduced angiogenesis, inva-sion and adhesion ability both in vitro and in vivo [131-133].The a6b4 integrin has been implicated to play an impor-tant role in the invasive phenotype of many carcinomas. SiRNA targeting the a6b4 integrin reduced the expression of this integrin and resulted in decreased invasiveness of breast carcinoma cells [134]. Osteopontin, a ligand for integrin- V 3 and CD44 receptors, is also involved with adhe-sion/metastasis in several cancers. Inhibition the expression of osteopontin by siRNA suppressed the metastasis of colon cancer, gastric cancer and pancreatic cancer [135-137].4.7. Radiotherapy/Chemotherapy ResistanceImproving the efficacy of radiotherapy and chemother-apy in cancer has been another potential application of RNAi. Inhibition the expression of HIF-1 by RNAi led to the increased sensitivity of tumor cell to ionizing radiation [138-139]. It is known that defects in repair of DNA double-strand breaks makes cells hypersensitive to ionizing radiation. Peng et al. used RNAi to target Prkdc that encodes for the cata-。

2012Prognostic value of novel biomarkers in astrocytic brain tumors nuclear

LABORATORY INVESTIGATION -HUMAN/ANIMAL TISSUEPrognostic value of novel biomarkers in astrocytic brain tumors:nuclear receptor co-regulators AIB1,TIF2,and PELP1are associated with high tumor grade and worse patient prognosisZinovia Kefalopoulou •Vassiliki Tzelepi •Vassiliki Zolota •Petros D.Grivas •Christos Christopoulos •Haralabos Kalofonos •Theodoros Maraziotis •Georgia Sotiropoulou-BonikouReceived:4March 2011/Accepted:17June 2011/Published online:7July 2011ÓSpringer Science+Business Media,LLC.2011Abstract Estrogen receptors alpha (ER a )and beta (ER b )and their co-regulatory proteins are key components of complex signaling networks that speciﬁcally regulate the growth and development of various tissues and tumors.Still,their protein expression proﬁles and possible role in the pathogenesis of astrocytic tumors remain largely unknown.The purpose of the present study is to evaluate the differ-ential protein expression of E R a ,ER b ,and their co-activa-tors,AIB1,TIF2,and PELP1in astrocytic tumors of World Health Organization (WHO)grade II–IV,using immuno-histochemistry.Potential correlations with clinicopatho-logical parameters and patient prognosis were also explored.ER a protein expression was undetectable while ER b levels were signiﬁcantly decreased with progression of tumor grade (P \0.001).High expression of ER b was an inde-pendent favorable prognostic factor on multivariate analysis (P =0.003).Expression of AIB1,TIF2,and PELP1was not correlated with ER b expression and followed an opposite trend,with increasing levels in high-grade relative to low-grade tumors (P \0.001).Univariate survival analysis revealed that high AIB1,TIF2,and PELP1expression was associated with worse prognosis (P =0.049,P =0.033,and P =0.020,respectively).ER b and ER co-activators AIB1,TIF2,and PELP1appear to play an important role in the pathogenesis and progression of astrocytic tumors and might have prognostic signiﬁcance.The mechanisms underlying their involvement in astrocytic tumorigenesis,as well as their utility for prognostic and therapeutic purposes merit further investigation.Keywords Astrocytic tumor ÁER b ÁAIB1ÁTIF2ÁPELP1ÁCo-activatorIntroductionAstrocytic tumors are the most common primary neo-plasms of the central nervous system (CNS).Traditionally these tumors have been associated with disappointing clinical outcomes [1–3].Glioblastoma multiforme (GBM),the most prevalent and aggressive astrocytoma variant,grade IV according to the World Health Organization (WHO)classiﬁcation system,has median survival of 12–15months,despite multimodal treatment including image-based resections,radiotherapy,and chemotherapy [1–4].The current challenge is to develop more efﬁcacious and targeted therapeutic paradigms exploiting knowledgeZ.Kefalopoulou ÁV.Tzelepi ÁV.ZolotaDepartment of Pathology,Medical School,University of Patras,26500Patras,GreeceP.D.GrivasDivision of Hematology/Oncology,Department of Internal Medicine,University of Michigan,Ann Arbor,MI,USA C.ChristopoulosDivision of Radiological Oncology,Department of Radiology,Medical School,University of Patras,26500Patras,Greece H.KalofonosDivision of Oncology and Clinical Oncology Laboratory,Department of Internal Medicine,Medical School,University of Patras,26500Patras,GreeceT.MaraziotisDepartment of Neurosurgery,Medical School,University of Patras,26500Patras,GreeceG.Sotiropoulou-Bonikou (&)Department of Anatomy and Histology-Embryology,Medical School,University of Patras,26500Patras,Greecee-mail:sotiropouloug@;gsot@med.upatras.grJ Neurooncol (2012)106:23–31DOI 10.1007/s11060-011-0637-yderived from molecular,cellular,and systems biology studies of tumor formation and progression[5,6].Epidemiological and experimental data indicate a pos-sible role of estrogen signaling in glial tumorigenesis[1,2, 7].Estrogens’pleiotropic effects are mediated by two balancing receptors,estrogen receptors alpha(ER a)and beta(ER b),which belong to the nuclear receptor(NR) superfamily of ligand-regulated transcription factors[8]. ER a and ER b are key components of complex,genomic and nongenomic signal transduction pathways that specif-ically regulate the growth and development of target tissues and tumors[9–11].It appears quite clear today that ER b has biological roles that are distinct from those of ER a[8, 12].Overall,in contrast to ER a,a gradual decline of ER b expression has been reported in the progression of various malignancies including,among others,breast,ovarian,and prostate cancer[13–15].Correspondingly,ER b appears to play a protective role in glial tumorigenesis[16,17].Since ER a is reported to be minimally expressed in human astrocytic neoplasms,ER b should probably be considered as the predominant mediator of estrogen signaling in these tumors[18,19].ER-mediated transcriptional activity requires a number of co-regulatory proteins whose cell-speciﬁc expression explains some of the distinct cellular actions of estrogens [20,21].Co-regulators,by integrating diverse signaling afferents with transcription programs,serve as master regulators of estrogen-mediated transcriptional activity in various physiological cellular processes and a variety of disease states including cancer[22,23].Ampliﬁed in breast cancer1(AIB1)and transcriptional intermediary factor2 (TIF2),also known as steroid receptor co-activator3 (SRC-3)and2(SRC-2),respectively,belong to the p160/ SRC family of nuclear receptor co-activators[23–25]. AIB1and TIF2overexpression has been associated with initiation,progression,and metastatic potential in a variety of distinct tumor types including breast,gynecologic,and prostate cancers,as well as neoplasms of the digestive system[26,27].However,their role in the pathogenesis and progression of brain glial tumors remains unknown.Proline-,glutamic acid-,and leucine-rich protein1 (PELP1),also referred to as modulator of nongenomic actions of the estrogen receptor(MNAR),serves as a scaffolding protein that couples various signaling complexes with ERs and participates in genomic and nongenomic functions[28–31].Emerging evidence sug-gests that PELP1has tumorigenic potential,by interacting with several oncoproteins[32].PELP1expression is deregulated in a number of malignancies,including breast, endometrial,prostate,and ovarian cancer[33].However, the expression and potential role of PELP1in glial tumorigenesis have not been elucidated.In the present study,we hypothesized that ER b,and its co-activators,AIB1,TIF2,and PELP1,protein levels ﬂuctuate as tumor grade increases in astrocytomas and correlate with clinical and histopathological parameters.To the best of our knowledge,this is theﬁrst study that has explored via an integrated approach the expression of ER b pathway components in astrocytic tumors of different grades and associated them with clinicopathological parameters and patient prognosis.Materials and methodsTissue samples and patientsA total of86formalin-ﬁxed,parafﬁn-embedded(FFPE) serially collected specimens of astrocytic tumors,from an equal number of patients,retrieved from the Pathologyﬁles of the University Hospital of Patras(Patras,Greece),were investigated.Hematoxylin and eosin(H&E)-stained sec-tions were reviewed,and tumors were histopathologically classiﬁed according to the WHO grading system[2].The protocol of our study was approved by the Institutional Review Board of the University of Patras,and the ethical considerations were fully consistent with the Declaration of Helsinki.The clinicopathological characteristics of the patients are presented in Table1.All patients had undergone extensive surgical resection of their brain tumor at the Department of Neurosurgery of the University Hospital of Patras(Patras, Greece).None of the patients had received radiotherapy prior to surgery.Of the86patients who were included in the study,20(23%)had diffuse astrocytomas(WHO grade II), 22(26%)were diagnosed with anaplastic astrocytomas (WHO grade III),and44(51%)had glioblastoma multi-forme(WHO grade IV).Mixed gliomas were not includedTable1Clinicopathological characteristics of patientsN(%)Male/female,N(%)Age(years),mean±SDOverall survival(months),mean±SDGrade II20(23)9/11(10/13)42.9±13.344±17.2Grade III22(26)15/7(17/8)50.6±15.732.7±15.4Grade IV44(51)26/18(30/21)60.5±11.614.9±11.4Total86(100)50/36(58/42)53.8±14.922.5±17.3in the present study.Patients with high-grade gliomas (WHO grade III and IV)received adjuvant radiotherapy, according to standard protocols[3].Overall survival [mean±standard deviation(SD)22.5±7.3months]was calculated as the period from date of surgery until cancer-related death or last follow-up(48months at least).ImmunohistochemistrySerial4-l m sections were cut from the FFPE blocks and subjected to immunohistochemical analysis,as previously described[34–37].Rabbit polyclonal antibodies against ER b(prediluted,AR385-5R;Biogenex,CA,USA),which recognized the ER b1(full-length wild-type)isoform[12], and PELP1(PELP1/MNAR,1:600;Novus Biologicals,CO, USA),and mouse monoclonal antibodies against ER a (1:30,NCL-L-6F11;Novocastra,UK),AIB1(1:50, clone34;BD Biosciences,CA,USA),and TIF2(1:50, clone29;BD Biosciences,CA,USA)were used as primary antibodies.Brieﬂy,sections were deparafﬁnized in xylene and rehydrated in a series of graded ethanol solutions. Endogenous peroxidase activity was blocked by incubating with0.3%hydrogen peroxide for15min at room temper-ature.For antigen retrieval,sections were heated in10mM citrate buffer,pH6(ER a,ER b)or1mM ethylenediamine tetraacetic acid(EDTA)-NaOH,pH8(AIB1,TIF2)for 15min in a microwave oven.According to manufacture instructions,no antigen retrieval was performed for PELP1. After cooling to room temperature,sections were incubated with blocking serum(1%bovine serum albumin fraction V; Serva Electrophoresis,Germany)for30min and then with the primary antibody for1h at room temperature.Slides were next incubated with Dako EnVision labeled polymer (Dako,CA,USA).Diaminobenzidine(Dako,CA,USA) was used as the chromogen.Nuclei were counterstained with Harris hematoxylin.Sections from breast carcinoma were used as positive controls.In negative control slides, the same method was performed and the primary antibody was substituted by1%Tris-buffered saline(TBS). Staining evaluationImmunohistochemically stained slides were individually reviewed and scored by two investigators blinded to all clinical data.Discrepancies in scoring between the observers were resolved by additional review of the specimens and discussion until consensus was achieved.Representative areas were selected at low(1009)magniﬁcation.Cell counts were performed at4009magniﬁcation.Both the number of positive stained cells and the total number of cells(at least 1,000)were determined by visual inspection of six different ﬁelds per section.For eachﬁeld,the percentage of positive cells was calculated,and the average of those was taken.Statistical analysisLevels of biomarkers expression were analyzed as contin-uously scaled measures,and the numerical values followed normal distribution.One-way analysis of variance (ANOVA)was used to assess the mean difference in bio-marker expression between groups.Because of multiple comparisons,these tests were followed by a post hoc Bonferroni test.Pearson’s rank correlation test was used to examine the possible associations between the expression levels of the biomarkers.Univariate analysis of overall survival was performed with Kaplan-Meier curves,and differences between groups were assessed using the log-rank test.For the purpose of this study,in survival analysis, we considered patients with grade III and IV astrocytomas to have advanced-grade tumors.For statistical purposes, expression levels were dichotomized into low and high expression using as a cut-off value50%positive cells for ER b,to facilitate comparison with a previous immuno-histochemical study of ER b in glial tumors[17],and the mean expression levels for AIB1,TIF2,and PELP1,as described before[34,35].Multivariate analysis taking into account patients’clinicopathological parameters and expression of the biomarkers was performed using Cox proportional-hazards method.Differences in the expression of all the biomarkers investigated were noted between grade II and III/IV astrocytomas,hence they are collec-tively discussed as low-grade(grade II)and high-grade (grade III and IV)astrocytomas.All data were analyzed using the SPSS statistical package(SPSSÓ,release17.0; SPSS,Chicago,IL,USA).All reported P values are two-sided at signiﬁcance level of0.05.The design and conduction of the study,methodological and technical considerations,as well as data presentation were based on the reporting recommendations for tumor marker prognostic studies(REMARK)criteria[38]. ResultsEighty-six astrocytic tumors,WHO grade II–IV,were examined for expression of ER a,ER b,AIB1,TIF2,and PELP1by immunohistochemistry.ER a protein expression was undetectable,and thus no further analysis could be performed for this biomarker.All other proteins were consistently detected in the nuclei of tumor cells,whereas expression within the cytoplasm was negligible.Addi-tionally,positive staining for all proteins was observed in endothelial and inﬂammatory cells,as has been described before[34–37].In the present study,only tumor cell staining was evaluated.The mean percentage and standard deviations(SD)of positive cells for each protein relative to tumor grade are displayed in Table2and Fig.1.Representative images of ER b,AIB1,TIF2,and PELP1 immunostains in relation to tumor grade are shown in Fig.2.ER bER b was expressed in71±17%of tumor cells of grade II astrocytomas,43±22%of tumor cells of grade III,and 41±25%of tumor cells of grade IV astrocytomas.Sta-tistical analysis showed that ER b expression was signiﬁ-cantly higher in grade II astrocytomas than in grade III (P\0.001)and grade IV astrocytomas(P\0.001). However,it did not differ signiﬁcantly between grade III and grade IV astrocytomas(P=0.8).AIB1Regarding AIB1,the mean percentage of positive tumor cells was11±12%,41±20%,and53±23%for gra-de II,grade III,and grade IV astrocytomas,respectively. Statistical analysis revealed that AIB1expression was less frequently noted in grade II compared with grade III (P\0.001)and grade IV astrocytomas(P\0.001).No difference was noted between grade III and grade IV astrocytomas(P=0.1).TIF2TIF2expression was signiﬁcantly higher in grade III and grade IV astrocytomas(mean percentage of positive tumor cells±SD:58±17%and69±19%,respectively)rela-tive to that observed in grade II astrocytomas(23±16%, P\0.001).Furthermore,a trend for a marginal statisti-cally signiﬁcant difference in its expression levels was observed between grade III and grade IV astrocytomas (P=0.055).PELP1Expression of PELP1was less frequently noted in grade II astrocytomas(mean percentage of positive tumor cells±SD:44±24%)in comparison with grade III(74±14%,Table2Mean protein expression levels(%)and standard deviations(SD)of positive tumor cells by tumor gradeER b(mean±SD)AIB1(mean±SD)TIF2(mean±SD)PELP1(mean±SD)Grade II71±1711±1223±1644±24Grade III43±2241±2058±1774±14Grade IV41±2553±2369±1980±14Total48±2540±2655±2670±22Fig.1Expression levels ofa ERb decrease,while b AIB1,c TIF2,and d PELP1expression increases during progression from low-to high-grade tumorsP \0.001)and grade IV astrocytomas (80±14%,P \0.001).Statistical analysis failed to reveal any signiﬁ-cant difference between grade III and IV astrocytomas regarding PELP1expression levels (P =0.5).None of the examined protein expression levels were correlated with patient age or gender.This was also true when grade II,grade III,and grade IV astrocytomas were examined separately.Correlation between biomarkersConsidering that AIB1,TIF2,and PELP1are ER tran-scriptional co-regulators,their correlative expression was also explored.Pearson’s correlation test revealed a positive correlation between AIB1and TIF2expression within grade II (r =0.680,P \0.001),grade III (r =0.800,P \0.001),and grade IV astrocytomas (r =0.908,Fig.2Representative images of ER b (a ,b ),AIB1(c ,d ),TIF2(e ,f ),and PELP1(g ,h )immunostains in low-grade (grade II,left panel )and high-grade (grade IV,right panel )astrocytomas (original magniﬁcation 9400)P \0.001).Additionally,ER b was inversely correlated to PELP1expression in grade IV astrocytomas (r =-0.314,P =0.038),but not in grade II and grade III astrocytomas.Finally,no correlation between the expression levels of ER b and either AIB1or TIF2was noted in any of three grades of astrocytomas.Survival analysisAmong the clinicopathological characteristics of the patients,only advanced tumor grade was associated with shorter overall survival (P =0.01)on Kaplan-Meier anal-ysis.Univariate analysis revealed that low ER b (P =0.018)expression as well as high AIB1(P =0.049),TIF2(P =0.033),and PELP1(P =0.020)expression were associated with decreased overall survival in the overall study population (Fig.3).However,only low ER b expres-sion was predictive of shorter overall survival in subgroup analysis stratiﬁed by grade (P =0.039).Finally,multivar-iate Cox analysis that included age,gender,grade,and ER b ,AIB1,TIF2,and PELP1expression revealed that high ER b expression [P =0.003,Exp(B )=0.970,95%conﬁdence interval (CI)0.951–0.990]along with lower tumor grade [P =0.023,Exp(B )=0.021,95%CI 0.050–0.798]were independent favorable prognostic factors of overall survival.DiscussionThere is now compelling evidence to show that ER b and its co-activators are important players in complex signaling networks during the development of an increasing number of malignancies [15,27,33].Still,their protein expression proﬁles and possible role in the pathogenesis of astrocytic tumors remain largely unknown.Conﬁrming our initial hypothesis,we showed that ER b expression levels decreased and AIB1,TIF2,and PELP1levels increased in high-grade as compared with low-grade tumors and cor-related with patient survival.We used the validated mor-phological method of immunohistochemistry,which offers the opportunity of investigating protein expression in a semiquantitative manner,as well as their localization at the cellular and subcellular level.Evidence from epidemiological and experimental stud-ies suggests a possible prophylactic role of estrogen in glial tumor growth [1,9].In concordance with our results,ER a expression is reported to be absent to minimal in astrocytic tumors [18,19].Thus,among the estrogen receptors,ER b should probably be considered as the predominant media-tor of estrogen signaling in these tumors.In agreement with previous ﬁndings [16],a substantial decrease of ER b expression in low-relative to high-grade astrocytomaswasFig.3Kaplan-Meier analysis of biomarker expression in the total cohort.Low ER b (a )and high AIB1(b ),TIF2(c ),and PELP1(d )expression were associated with worse overall survivalnoted in our study.Downregulation of ER b expression is a common event during the progression of various tumors, with breast,prostate,and ovarian cancer being the best studied[14].In our study,low ER b expression levels were associated with decreased overall survival.Interestingly,the prog-nostic value of ER b was not tumor grade related,and on multivariate analysis low ER b protein expression was found to be an independent negative prognostic indicator along with the well-established adverse prognostic factor of advanced tumor grade.Our results are in concordance with previousﬁndings[16,17]and suggest that the expression status of ER b,as detected by immunohistochemistry,might distinguish patient subpopulations with different prognosis within the same grade.Importantly,ourﬁndings provide a rationale for therapeutic targeting of ER b downregulation in astrocytic tumors.The loss of ER b in cancer appears to be due to gene silencing by promoter methylation,which,as proposed,is a potentially reversible process[39,40]. Therefore,demethylation of ER b promoter in combination with developing speciﬁc ER b agonists may constitute promising opportunities for targeted approaches in treat-ment of cancer,including gliomas.Undoubtedly,the prognostic value of ER b in astrocytic tumors and its pos-sible utility for clinical decision-making and treatment should be further assessed in large-scale,prospective clin-ical studies,in combination with elucidation of the mech-anistic role of ER b in astrocytic tumorigenesis.AIB1has been the focus of considerable research interest over the past years,and its activity has been linked to a number of biological processes including cell prolif-eration,cell apoptosis,and cell migration,indicating its potent role as an oncogene[22,24,26,27,41].Overex-pression of AIB1has been detected in diverse types of solid tumors,including hormone-sensitive cancers in addition to,traditionally,non-hormone-sensitive tumors [27].Accordingly,our immunohistochemical study revealed a signiﬁcant increase of AIB1protein levels in low-grade compared with high-grade astrocytomas.With respect to its prognostic signiﬁcance,high expression of AIB1was correlated with worse overall survival in the overall study group.However,thisﬁnding was not tumor grade independent.A cohort of patients with larger sub-group populations might have the power to reveal such an association.Alternatively,it is plausible that,in astrocytic neoplasms,the negative prognostic value of AIB1is related to its pathogenetic role in the emergence of high tumor grade.TIF2overexpression has been identiﬁed in several neoplasms including colon,prostate,and endometrial cancer[27,35,36,42,43].In the present study,TIF2 exhibited an expression proﬁle very similar to that of AIB1 and was found to be signiﬁcantly enhanced in high-grade compared with low-grade astrocytomas.The observed AIB1and TIF2co-expression supports a functional link between the two proteins and might suggest a combined action in astrocytic tumorigenesis.Studies in colorectal and breast carcinomas as well as in meningiomas have simi-larly provided evidence of strong correlation between AIB1and TIF2expression[35,36,44,45].Survival analysis revealed that,correspondingly to AIB1,high TIF2 expression was associated with worse prognosis,but not in a tumor-grade-independent manner.It is worth noticing that,in the present study,AIB1and TIF2expression was not associated with ER b expression. It could therefore be speculated that ER-independent pathways[46,47]may contribute in AIB1-and TIF2-related pathogenesis in astrocytomas,as previously repor-ted for breast and lung cancers[48,49].Obviously,further work is required to elucidate the precise mechanisms through which AIB1and TIF2are involved in the patho-genesis of astrocytic tumors.PELP1represents a novel ER co-activator and is widely expressed in a number of tissues including brain[30]. PELP1exhibits both nuclear and cytoplasmic localization, in a tissue-and cell-dependent manner,consistent with its role in genomic and nongenomic actions of estrogens, respectively[30,31].Overexpression of PELP1has oncogenic potential[32]and has been noted in various neoplasms including breast,endometrial,prostate,and ovarian cancer[33].Accordingly,the present study dem-onstrated increased PELP1expression in high-grade rela-tive to low-grade astrocytomas.The absence of both PELP1and ER b cytoplasmic expression in our study might suggest that genomic prevails over nongenomic signaling in our cohort.Moreover,PELP1expression was not posi-tively associated with ER b expression;in contrast,an inverse correlation between the two proteins was observed in grade IV astrocytomas.This would not be an unexpected result considering that PELP1has emerged to function as a wide-ranging co-regulator with signiﬁcant participation in multiple cellular signaling pathways,in addition to the ER axis[30,50].The inverse correlation found in grade IV tumors most possibly depicts that ER b and PELP1exert opposing roles in the tumorigenic process.Finally,high PELP1expression was found to predict shorter overall survival.However,after stratiﬁcation by tumor grade or multivariate analysis it was not shown to be an independent prognostic factor.Collectively,theseﬁndings merit further investigation,so that the potential role of PELP1in the pathogenesis and prognosis of astrocytic tumors can be veriﬁed.In summary,we showed that ER b expression decreases with increased astrocytoma grade and is a favorable, independent,prognostic factor of patient prognosis. Expression of steroid receptor co-regulators AIB1,TIF2,and PELP1is not correlated to ER b levels and follows an opposite trend,with increasing levels in high-grade astro-cytomas relative to low-grade tumors.To date,astrocyto-mas,the most common primary brain tumors in adults, have constituted a formidable therapeutic challenge. Unveiling the complex mechanisms that drive tumor ini-tiation and progression is necessary to optimize their diagnostic assessment and to devise novel,individually tailored treatment strategies.Further research at the clinical and molecular level is required to conﬁrm our results and explore whether they could be meaningfully translated into clinical practice.References1.Ohgaki H(2009)Epidemiology of brain tumors.Methods MolBiol472:323–342.doi:10.1007/978-1-60327-492-0_142.Louis DN,Ohgaki H,Wiestler OD,Cavenee WK(2007)WHOclassiﬁcation of tumours of the central nervous system.Interna-tional Agency for Research on Cancer,Lyon3.Wen PY,Kesari S(2008)Malignant gliomas in adults.N Engl JMed359(5):492–507.doi:359/5/492[pii]10.1056/NEJMra0708 1264.Stupp R,Mason WP,van den Bent MJ,Weller M,Fisher B,Taphoorn MJ,Belanger K,Brandes AA,Marosi C,Bogdahn U, Curschmann J,Janzer RC,Ludwin SK,Gorlia T,Allgeier A, Lacombe D,Cairncross JG,Eisenhauer E,Mirimanoff RO(2005) Radiotherapy plus concomitant and 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贸易产品分类bec

ESA/STAT/AC.124/89 April 2007 UNITED NATIONSDEPARTMENT OF ECONOMIC AND SOCIAL AFFAIRSSTATISTICS DIVISIONMeeting of the Expert Group onInternational Economic andSocial ClassificationsNew York, 16-18 April 2007Future revision of the Classification by Broad Economic Categories (BEC)UNSDFuture revision of the Classification by Broad Economic Categories (BEC)Proposal by UNSDThis is a proposal to put a new work programme regarding BEC on the agenda of the Expert Group on International Economic and Social Classifications. The proposal is (1) that BEC should be reviewed to ensure that its current structure relating to transportable goods is still appropriate, (2) that the draft correlation table1 between the BEC categories and subheadings of Harmonized Commodity Description and Coding System, 2007 edition, should be reviewed, and (3) that a possible extension of the BEC to services products should be considered as well as the development of a link between the BEC and the Central Product Classification (CPC).Background informationThe BEC was initially developed by UNSD for internal purposes to reclassify merchandise imports (reported in terms of SITC) into the product categories relevant to the SNA. With time, many countries started to use BEC for a variety of purposes including economic analysis and setting tariffs. UNSD has created correspondence tables between BEC and each new revision of SITC and the Harmonized System. Since the CPC is based on HS, it also can be correlated to BEC. However, over the years the BEC structure remained unchanged and the rules for assigning particular SITC or HS headings to BEC clearly need revisiting if BEC is to continue as a useful tool. The issue is quite urgent as HS07 and SITC, Rev.4 are already in force. Question is also raised about extending BEC to trade in services using CPC services categories as a starting point. UNSD prepared a draft correspondence table between HS07 and BEC, which was submitted to the EG for a review. The EG is requested to comment on the status and modalities of the possible future revision of BEC.The introductory pages of the most recent BEC publication (2000) are given in the annex and they include a full overview of its headings.1 See /unsd/trade/methodology%20IMTS.htmAnnex 1Introductory pages of BEC publicationHistorical background1. The Statistical Commission, recognizing the growing need for international trade statistics analyzed by broad economic categories, recommended, at its thirteenth session, that data on large economic classes of commodities, distinguishing food, industrial supplies, capital equipment, consumer durables and consumer non-durables, should be compiled for each reporting country and for the world and principal regions to supplement the summary data already compiled on the basis of the sections of the Standard International Trade Classification (SITC). 22. Following this recommendation, the Statistical Office of the United Nations Secretariat prepared a draft classification of international trade by Broad Economic Categories (BEC), which would provide for such a compilation. Successive drafts of the classification were considered by the Statistical Commission at its fourteenth and fifteenth sessions and were referred back to the Statistical Office by the Commission with specific guidelines for further study and consultation. At its sixteenth session, the Commission reviewed a further draft (E/CN.3/408) which had been prepared by the Statistical Office based upon the guidelines of the Commission and the comments of selected countries and international organizations. The Commission was unanimous in considering that the draft classification met the requirements which it had specified at previous sessions and by its resolution 9 (XVI) requested the Secretary-General to publish the classification for international use and as a guideline for national use. 33. The original BEC was defined in terms of divisions, groups, subgroups and basic headings of the Standard International Trade Classification, Revised4 and issued in 1971. 5 The BEC has been revised twice since 1971. The first revision defined BEC in terms of the Standard International Trade Classification, Revision 26 and was issued in 1976. 7 The second revision defined BEC in terms of the Standard International Trade Classification, Revision 38 and was issued in 1986. 9 Each of these revisions was prepared and issued in response to requests by the Statistical Commission10, 11 and resolutions adopted by the Economic and Social Council (resolution 1948 (LVIII) of 7 May l975 and resolution 1985/7 of 28 May l985). The present revision of the BEC is the third and it is published for technical reasons only, to replace the second revision, in order to provide full details of the SITC, Rev. 3 headings corresponding to BEC categories 41* and 62*. These categories were incomplete in the second revision due to the omission of one page. The present revision also incorporates the corrigendum that had been issued to the second revision, and a revised introduction.2Official Records of the Economic and Social Council, 1965, Thirty-ninth Session, Supplement No. 13 (E/4045-E/CN.3/339, para. 30).3Ibid., 1970, Fiftieth Session, Supplement No. 2 (E/4938-E/CN.3/417), chap. XIII.4 United Nations publication, Sales No. 61.XVII.6.5Classification by Broad Economic Categories (United Nations publication, Sales No. E.71.XVII.12).6 United Nations publication, Sales No. E.75.XVII.6.7Classification by Broad Economic Categories Defined in Terms of SITC, Rev. 2 (United Nations publication, Sales No. E.76.XVII.7).8 United Nations publication, Sales No. E.86.XVII.12.9Classification by Broad Economic Categories Defined in Terms of SITC, Rev. 3 (United Nations publication, Sales No. E.86.XVII.24).10Official Records of the Economic and Social Council, 1974, Fifty-eighth Session, Supplement No. 2 (E/5603, para. 118 (b)).11Ibid., 1985, Supplement No. 6 (E/1985/26, para. 57 (f) and (i)).The classification4. The BEC is set out below:THE CLASSIFICATION BY BROAD ECONOMIC CATEGORIES (BEC) 121* Food and beverages11* Primary111* Mainly for industry112* Mainly for household consumption12* Processed121* Mainly for industry122* Mainly for household consumption2* Industrial supplies not elsewhere specified21* Primary22* Processed3* Fuels and lubricants31* Primary32* Processed321* Motor spirit322* Other4* Capital goods (except transport equipment), and parts and accessories thereof41* Capital goods (except transport equipment)42* Parts and accessories5* Transport equipment, and parts and accessories thereof51* Passenger motor cars12 The asterisk following each reference to a BEC classification code number is a device to distinguish the BEC classification code number from the numbered sections, divisions and groups of the SITC, Rev. 3, which is used to define the composition of each BEC item.52* Other521* Industrial522* Non-industrial53* Parts and accessories6* Consumer goods not elsewhere specified61* Durable62* Semi-durable63* Non-durable7* Goods not elsewhere specifiedDescription of the classification5. The BEC includes nineteen basic categories. The basic categories are those that are not further subdivided in the above listing of the classification, namely, 111*, 112*, 121*, 122*, 21*, 22*, 31*, 321*, 322*, 41*, 42*, 51*, 521*, 522*, 53*, 61*, 62*, 63* and 7*. Each category of the BEC is defined in terms of divisions, groups, subgroups and basic headings of the SITC, Rev.3. The categories of the BEC are a rearrangement and aggregation of the basic headings of the SITC, Rev. 3 reflecting the end-uses of the commodities covered by each basic heading. Each SITC, Rev. 3 basic heading is allocated entirely to one BEC category. The allocation is done on the basis of the main end-use of the commodities in each SITC, Rev. 3 basic heading, although it is recognized that many commodities that are traded internationally may be put to a variety of uses.6. TheBEC:(i) Distinguishes the five categories specified by the Commission at its thirteenth session; namely food, industrial supplies, capital equipment, consumer durables and consumer non-durables;(ii) Provides separate categories for "fuels and lubricants" and "transport equipment" in pursuance of the Commission's discussions at its fourteenth session, 13 and within these categories includes special subcategories for "motor spirit" and "passenger motor cars";(iii) Provides within the categories relating to capital goods, a distinction between equipment proper and parts and accessories;13Official Records of the Economic and Social Council, 1966, Forty-second Session, Supplement No. 3(E/4283-E/CN.3/354, para. 34).(iv) Provides within the categories for "Food and beverages", "Industrial supplies not elsewhere specified" and "Fuels and lubricants", a distinction between "primary" commodities and "processed" commodities;(v) Provides within the categories for "Primary food and beverages", "Processed food and beverages" and "Transport equipment" (other than parts and accessories and passenger motor cars), a distinction between commodities for industry and those for household consumption. Purposes of the classification7. The BEC was originally devised mainly for use by the Statistical Office of the United Nations Secretariat for the summarization of data on international trade by large economic classes of commodities (see para. 1 above). In addition, it was designed to serve as a means for converting external trade data compiled on the SITC into end-use categories that are meaningful within the framework of the System of National Accounts (SNA), 14 namely categories approximating the three basic classes of goods in the SNA: capital goods, intermediate goods and consumption goods. 15 Specifically, the subcategories of the BEC can be aggregated to approximate these three classes of goods. This aggregation permits external trade statistics to be considered jointly with other sets of general economic statistics, such as national accounts and industrial statistics, for national, regional or world level economic analysis.8. The classification was also expected by the Statistical Commission to serve as a guideline for national classifications of imports according to broad economic categories. 16 However, at its sixteenth session, the Statistical Commission "recognized that countries might wish to adapt the classification for national purposes in different ways to meet national requirements," 17 and concluded that "consequently, the classification was not to be regarded as a standard classification in the same sense as was, for example, the Standard International Trade Classification (SITC)." 18Correspondence of BEC with the basic classes of goods in the SNA9. The correspondence of the 19 BEC basic categories with the basic classes of goods in the SNA (capital goods, intermediate goods and consumption goods) is set out below.THE CLASSIFICATION BY BROAD BASIC CLASSES OF GOODS INECONOMIC CATEGORIES (BEC) THE SNA1* Food and beverages11* Primary14Ibid., 1968, Forty-fourth Session, Supplement No. 10 (E/4471-E/CN.3/390, paras. 116 and 118).15A System of National Accounts (United Nations publication, Sales No. E.69.XVII.3, para. 1.50).16Official Records of the Economic and Social Council, 1968, Forty-fourth Session, Supplement No. 10(E/4471-E/CN.3/390, para. 123).17Ibid., 1970, Fiftieth Session, Supplement No. 2 (E/4938-E/CN.3/417, para. 95).18Ibid.111* Mainly for industry Intermediate goods112* Mainly for household consumption Consumption goods12* Processed121* Mainly for industry Intermediate goods122* Mainly for household consumption Consumption goods2* Industrial supplies not elsewhere specified21* Primary Intermediate goods22* Processed Intermediate goods3* Fuels and lubricants31* Primary Intermediate goods32* Processed321* Motor spirit (see para. 11 below)322* Other Intermediate goods4* Capital goods (except transport equipment), and parts and accessories thereof 41* Capital goods (except transport equipment) Capital goods42* Parts and accessories Intermediate goods5* Transport equipment, and parts and accessories thereof51* Passenger motor cars (see para. 11 below)52* Other521* Industrial Capital goods522* Non-industrial Consumption goods53* Parts and accessories Intermediate goods6* Consumer goods not elsewhere specified61* Durable Consumption goods62* Semi-durable Consumption goods63* Non-durable Consumption goods7* Goods not elsewhere specified (see para. 11 below)The basic classes of goods in the SNA in terms of BEC10. The composition of the three basic classes of goods in the SNA in terms of the basic categories of the BEC is shown below:1. Capital goodsSum of categories:41* Capital goods (except transport equipment)521* Transport equipment, industrial2. Intermediate goodsSum of categories:111* Food and beverages, primary, mainly for industry121* Food and beverages, processed, mainly for industry21* Industrial supplies not elsewhere specified, primary22* Industrial supplies not elsewhere specified, processed31* Fuels and lubricants, primary322* Fuels and lubricants, processed (other than motor spirit)42* Parts and accessories of capital goods (except transport equipment)53* Parts and accessories of transport equipment3. Consumption goodsSum of categories:112* Food and beverages, primary, mainly for household consumption122* Food and beverages, processed, mainly for household consumption522* Transport equipment, non-industrial61* Consumer goods not elsewhere specified, durable62* Consumer goods not elsewhere specified, semi-durable63* Consumer goods not elsewhere specified, non-durable11. The above groupings include only 16 of the 19 BEC basic categories as categories 321* Motor spirit, 51* Passenger motor cars and 7* Goods not elsewhere specified, are omitted. Category 321* Motor spirit and Category 51* Passenger motor cars are used extensively both for industry and for household consumption. Category 7* Goods not elsewhere specified, includes among other commodities, a range of military equipment, postal packages and special transactions and commodities not classified according to kind and can be a mix of the SNA classes of goods. These three BEC categories are of particular importance in international trade and of great interest to economists and others studying international flows of commodities. As indicated in paragraphs 16 and 18 below, these commodities were considered to be sufficiently important to warrant establishment in the BEC of separate categories. Users may choose to make their own assignments of these three categories of goods among the basic SNA classes of goods. Description of the categories12. The BEC makes provision for the main categories originally requested by the Commission (see paras. 1 and 6 above). In addition, in order to provide elements which enable the construction of aggregates approximately comparable to those for the three basic classes of goods in the SNA, a number of subcategories were established to supplement these main categories. The subcategories reflect the various end-uses of commodities. Some features of the commodity composition of each of the BEC categories are outlined in the following paragraphs. 13. In each of the categories 1* Food and beverages, 2* Industrial supplies not elsewhere specified and 3* Fuels and lubricants, subcategories of primary and processed commodities were established. In general, commodities have been classified as "primary" if they are characteristically products of primary sectors of the economy - i.e., farming, forestry, fishing, hunting, and the extractive industries. In addition, commodities which characteristically are products of other sectors, such as manufacturing are also classified as primary if nearly all the value of the product is contributed by one of the primary sectors of the economy. For example, cotton undergoes physical transformation when ginned, but as almost all the value of ginned cotton derives from the agricultural sector, it is classified in the BEC as a primary commodity, not as a product of the textile industry in which cotton ginning is included in the International Standard Industrial Classification of All Economic Activities (ISIC). 19 Canned and prepared foods, on the other hand, owe much of their value to the food-processing industry and are therefore generally excluded from the primary category in the BEC and are consequently classified as processed. Thus, a commodity is classified as primary if it is a product of farm, forest, fishing and hunting, or of an extractive industry, to whose value transformation has made only a minor contribution. Waste and scrap materials are also classified as primary commodities in the BEC. If a commodity is not defined as primary, it is classified as processed in the BEC. 14. In category 1* Food and beverages, in order to facilitate analysis in terms of the SNA classes, categories 11* Primary food and beverages and 12* Processed food and beverages, were subdivided to provide for commodities "mainly for industry" and "mainly for household consumption". The main or usual end-use of many of the food items in BEC category 1* Food and beverages, is household consumption, while other food items in that category are used as intermediate goods in industry. For instance, tea is mainly used for household consumption, as are processed canned foods. Wheat, however, is commonly supplied to the milling industry to 19 United Nations publication, Sales No. E.68.XVII.8.produce flour, and flour, in turn, is commonly supplied to bakeries to produce bread: therefore, both wheat and flour may be considered as intermediate goods for the food industry. In addition, many food items may in some instances be used for industry, and in other instances for household consumption. In the case of category 11* Primary food and beverages, the great majority of items are capable of dual use, and the only practicable way of making an assignment between the SNA classes of goods in this category is by adopting conventions. Thus, for example, the conventions are adopted that food grains, when traded internationally, are normally for use by industry, and that fresh fruit and vegetables, when traded internationally, are normally for household consumption. In the case of category 12* Processed food and beverages, the majority of items can be allocated to an SNA class with greater certainty, but a considerable application of conventions is nevertheless involved in the case of several commodities.15. In category 2* Industrial supplies not elsewhere specified, no breakdown beyond primary and processed was called for as the assignment to the SNA classes of goods is entirely to intermediate goods.16. In category 3* Fuels and lubricants, the processed component was further subdivided to identify 321* Motor spirit and 322* Other processed fuels and lubricants. Motor spirit was separately identified because of its importance as a commodity in trade. It is a commodity commonly used by both industry and consumers, that is, it can be both an intermediate and consumption good. It is separately identified in the BEC but not allocated to a specific SNA class (see para. 11 above). Commodities in categories 31* Primary fuels and lubricants and 322* Other processed fuels and lubricants are entirely intermediate goods.17. Category 4* Capital goods (except transport equipment), and parts and accessories thereof, is divided into two subcategories in which commodities are classified according to whether their main end-use is as capital goods (41* Capital goods, excluding transport equipment) or as intermediate goods (42* Parts and accessories). This category includes machinery, such as electrical generators and computers, and other manufactured goods, such as medical furniture, which are used by industry, government and non-profit private institutions. They are, in fact, producers' goods that are defined in the SNA as part of fixed capital formation and are thus capital goods. Parts and accessories essential to the maintenance of machinery, and unassembled components of machinery, etc. used as supplies to assembling plants, are inputs to industry and are, for this reason, considered as intermediate goods. However, see paragraph 18 below under category 5* Transport equipment, and parts and accessories thereof, for the treatment of unassembled vehicles.18. Category 5* Transport equipment, and parts and accessories thereof covers finished ships, road vehicles, aircraft, railway and tramway rolling stock and their parts and other accessories. In the SNA, the finished commodities are classified as capital goods or as (durable) consumer goods, while parts and accessories are treated as intermediate goods. The category was broken down into 51* Passenger motor cars, 52* Other transport equipment (i.e., other than passenger motor cars) and 53* Parts and accessories. Category 51* Passenger motor cars was identified as a separate category because of the importance of passenger motor cars in trade. They are commodities commonly used in industry as capital goods or as consumer durables; that is, they can be a capital good or a consumption good. The subcategory is separately identified in the BEC but not allocated to a specific SNA class (see para. 11 above). Most of subcategory 52* Other transport equipment traded internationally is classified as capital goods, but several items, such as motorcycles and bicycles, are normally used by consumers and consequently are classified as consumption goods. Subcategories 521* Industrial and 522* Non-industrial were introduced to cover these. Subcategory 53* Parts and accessories, is considered as intermediategoods. However, unassembled vehicles (part of SITC headings 781, 782 and 783), which are in fact, input into assembling plants and should be allocated to BEC category 53*, are classified together with the corresponding assembled vehicles in categories 51* and 52* because assembled and unassembled vehicles are classified within the same SITC heading.19. Category 6* Consumer goods not elsewhere specified, while fully assignable as consumption goods in the SNA, has three subcategories in the BEC reflecting the durability of consumption goods. Subcategory 61* Durable consumer goods, includes commodities which have an expected lifetime of more than one year and of a relatively high value, such as refrigerators and washing machines, together with other commodities with a useful life of three years or more; subcategory 62* Semi-durable consumer goods, includes those which have an expected lifetime of use of more than one year but less than three years and are not relatively of high value; and subcategory 63* Non-durable consumer goods includes those with an expected lifetime of a year or less.20. Category 7* Goods not elsewhere specified includes, among other commodities, a range of military equipment, postal packages and special transactions and commodities not classified according to kind. No subcategories are made for this category in the BEC and it is not assigned to any of the basic classes of the SNA (see para. 11 above).General remarks21. It is proposed that in order to facilitate international comparison by end-use, the 19 categories and the three derived classes of the BEC - capital goods, intermediate goods and consumption goods - be recognized internationally as a useful classification for all commodities traded internationally. They are not intended to interfere with or supersede a classification that is more compatible with the internal statistical system of any country. Countries may wish to utilize the BEC for national purposes as a supplement to existing classifications.。

International Journal of System Control and Information Processing.

2. Modeling of Boiler Superheated Steam Temperature Plant
In this section, a coal-fired boiler (#2 furnace 600MW unit) in a certain thermal power plant is taken as an example to introduce the model structure chart of reconstructive boiler steam temperature system (Fig.1). The warm water reduction system of super heater uses two-level four-point cooling structure. First level desuperheater is arranged at two sides between low temperature superheater and dividing panel superheater; Second level desuperheater is arranged at two sides between rear platen superheater and steam lead pipe connected. Except the influence of swinging burner, two-level spray desuperheating adjustment is mainly applied in the main steam temperature control system.
An improved single neuron self-adaptive PID control scheme of superheated steam temperature control system

TI_How_to_check_Off-headingOff-course-alarm-1stE-Rev4

This document describes how to check Off-heading alarm and Off-course alarm of Autopilot (PT500) at quay.1. Off-heading alarm1.1 Preparations1) Rudder limit: Set [10 degrees] as the rudder limit value to does not reach the rudder 10 deg or more for safety.2) Off-heading alarm limit: Set [10 degrees] and [10sec.] as Off-heading alarm limit and detection time.3) Low speed alarm limit: check low speed alarm limit value with “Adjust mode 10”4) Set ships speed: Set ships speed higher than low speed alarm limit value with Manual, not to occur the low speed alarm.5) Steering mode: Select [AUTO] mode.6) Verify that gyrocompass is settling to "actual heading"1.2. Procedure1) Press “Heading set key [H/S]” on Auto steering unit for 2sec. to read the present heading as Set course.2) Confirm “Control mode” changes from “ALTER” to “STEADY”.To indicate the “control mode display”, press the “UP” or “DOWN” key on Auto steering unit.At that time, confirm the rudder angle is around midship.<NOTE*1>This indication is only displayed on Auto Unit (from the following Software version).Before the following software version, Auto Unit displays “CRS DEV’N ALARM” once the alarm is detected instead of the indication.Model PT500A PT500D PT500D (SUB-AUTO)Parts number PB343 PB344 PB345Software Ver. S R PROM-ID CR155S CR156R CR157POff-heading alarm is effective once Control mode changes to “STEADY”.It usually takes several minutes to change “STEADY” mode after set course keep on Gyro Compass heading.In case the Software version does not have display function of Control mode, set the present TechnicalInformations YDK-J01-BZ-10-001EPT500 AUTOPILOTCMZ700/900 GYROCOMPASSHow to check Off-heading alarm and Off-course alarm of Autopilot (PT500) at quay. *13) Deviate Gyro Compass heading more than 10 degree with C. Operation Unit “Command Code 24 (Manual alignment of Master Compass) “(Page 3 Operation procedure flow for “Manual alignment of Master Compass” is for your reference)4) Off-heading alarm occur beyond Off-heading detection time [10sec.]EX; 1) When the actual heading is 090 deg., make the set course [090.0]2) Set the Gyro Compass heading more than 100°.At this time the Rudder moves about PORT 10 degrees.《Caution 》1) After confirmation of OFF-heading alarm, carry out “the automatic alignment of mastercompass” to align the gyrocompass heading to actual heading.(Please refer to the operation flow of next page.)2) The Gyro Compass heading might have heading error about 3 degrees for 30 minutes after executing master compass alignment.3) Restore ships speed setting to “AUTO” after above checks.Actual heading & Set course=090° °Procesure for alignment Gyro Compass heading. (with C. Operation unit)2．Off-course alarmIn case PASS is not set Off-course alarm is not activated.2.1 Preparations1) Rudder limit: Set [10 degrees] as the rudder limit value to does not reach the rudder 10 deg or more for safety.2) Off-course alarm limit: Set [10 degrees] and [10sec.] as Off-course alarm limit and detectiontime.3) Low speed alarm limit: check low speed alarm limit value with “Adjust mode 10”4) Set ships speed: Set ships speed higher than low speed alarm limit value with Manual, not tooccur the low speed alarm.5) Steering mode: Select [AUTO] mode.2.2. Procedure1) Press “Heading set key [H/S]” on Auto steering unit for 2sec. to read the present heading as Set course.2) Confirm “Control mode” changes from “ALTER” to “STEADY”.To indicate the “control mode display”, press the “UP” or “DOWN” key on Auto steering unit.At that time, confirm the rudder angle is around midship.3) In case Magnetic Compass is selected as PASS, deviate Magnetic Compass heading more than Off-course alarmlimit value [10 degrees] with Magnet (Magnetized Screwdriver) from current Magnetic Compass heading.In case Gyro Compass is selected as AUX COMPASS, deviate Gyro Compass heading more than Off-course alarm limit value [10 degrees] with C. Operation Unit “Command Code 24 (Manual alignment of Master Compass) “ and Off-course alarm occur beyond Off-course detection time[10sec.]EX; 1) When the actual heading is 090 degrees, make the set course [090.0]2) Set the Gyro Compass heading more than 100 degrees《Caution 》1) Restore ships speed setting to “AUTO” after above checks.Actual heading & Set course =090° ° *1。

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