New Result for the Pion-Nucleon Sigma Term from an Updated VPIGW Pion-Nucleon Partial-Wave

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气相色谱法测定沃柑中2,4-滴的方法改进

气相色谱法测定沃柑中2,4-滴的方法改进

气相色谱法测定沃柑中2,4-滴的方法改进李琬婧,覃弘毅*(南宁市食品药品检验所,广西南宁 530007)摘 要:采用改进的气相色谱法测定沃柑中的2,4-滴。

试样经过乙腈提取,经过正丁醇加浓硫酸衍生化,弗罗里夕固相萃取柱净化浓缩后测定。

结果表明,2,4-滴在0.010~0.500 mg·L-1线性关系良好,相关系数(r)为0.999,定量限(10倍S/N)为0.001 mg·kg-1,回收率为82.7%~90.7%,RSD为1.6%~4.7%。

关键词:沃柑;气相色谱法;2,4-滴;农药残留Improvement of Gas Chromatography for Determination of2,4-D in Orah MandarinLI Wanjing, QIN Hongyi*(Nanning Institute for Food and Drug Control, Nanning 530007, China) Abstract: The pesticide residue of 2,4-D in Orah Mandarin was determined by improved gas chromatography. The sample was extracted with acetonitrile, derivatized with n-butanol and concentrated sulfuric acid, and cleaned up and concentrated on a Florisil cartrages solid phase extraction column for determination. The results showed that six compounds had the good linear relationship in the range of 0.010~0.500 mg·L-1. The correlation coefficients (r) was 0.999, the limit of quantitation (10 S/N) was 0.001 mg·kg-1, the recovery and relative standard deviations was 82.7%~90.7% and 1.6%~4.7%.Keywords: orah mandarin; gas chromatography; 2,4-dichlorophenoxyacetic acid; pesticide residue2021年柑橘产季全球柑橘产量达到1.585亿t,我国柑橘产量居世界首位,达4 460万t,占全球总产量的28%;出口量91.8万t仅排名第5,表明我国柑橘主要用于国内消费[1]。

Global Study of Nuclear Structure Functions

Global Study of Nuclear Structure Functions
Global Study of Nuclear Structure Functions
S. A. Kulagin∗ Institute for Nuclear Research, 117312 Moscow, Russia R. Petti† CERN, CH-1211 Gen´ eve 23, Switzerland
arXiv:hep-ph/0412425v3 22 Oct 2005
Abstract
We present the results of a phenomenological study of unpolarized nuclear structure functions for a wide kinematical region of x and Q2 . As a basis of our phenomenology we develop a model which takes into account a number of different nuclear effects including nuclear shadowing, Fermi motion and binding, nuclear pion excess and off-shell correction to bound nucleon structure functions. Within this approach we perform a statistical analysis of available data on the ratio of the nuclear structure functions F2 for different nuclei in the range from the deuteron to the lead. We express the off-shell effect and the effective scattering amplitude describing nuclear shadowing in terms of few parameters which are common to all nuclei and have a clear physical interpretation. The parameters are then extracted from statistical analysis of data. As a result, we obtain an excellent overall agreement between our calculations and data in the entire kinematical region of x and Q2 . We discuss a number of applications of our model which include the calculation of the deuteron structure functions, nuclear valence and sea quark distributions and nuclear structure functions for neutrino charged-current scattering.

The decay $rho^{0}to pi^{+}+pi^{-}+gamma$ and the coupling constant g$_{rhosigmagamma}$

The decay $rho^{0}to pi^{+}+pi^{-}+gamma$ and the coupling constant g$_{rhosigmagamma}$

a rXiv:n ucl-t h /441v28Ma y2The decay ρ0→π++π−+γand the coupling constant g ρσγA.Gokalp ∗and O.Yilmaz †Physics Department,Middle East Technical University,06531Ankara,Turkey(February 8,2008)Abstract The experimental branching ratio for the radiative decay ρ0→π++π−+γis used to estimate the coupling constant g ρσγfor a set of values of σ-meson parameters M σand Γσ.Our results are quite different than the values of this constant used in the literature.PACS numbers:12.20.Ds,13.40.HqTypeset using REVT E XThe radiative decay processρ0→π++π−+γhas been studied employing different approaches[1,5].There are two mechanisms that can contribute to this radiative decay: thefirst one is the internal bremsstrahlung where one of the charged pions from the decay ρ0→π++π−emits a photon,and the second one is the structural radiation which is caused by the internal transformation of theρ-meson quark structure.Since the bremsstrahlung is well described by quantum electrodynamics,different methods have been used to estimate the contribution of the structural radiation.Singer[1]calculated the amplitude for this decay by considering only the bremsstrahlung mechanism since the decayρ0→π++π−is the main decay mode ofρ0-meson.He also used the universality of the coupling of theρ-meson to pions and nucleons to determine the coupling constant gρππfrom the knowledge of the coupling constant gρter,Renard [3]studied this decay among other vector meson decays into2π+γfinal states in a gauge invariant way with current algebra,hard-pion and Ward-identities techniques.He,moreover, established the correspondence between these current algebra results and the structure of the amplitude calculated in the single particle approximation for the intermediate states.In corresponding Feynman diagrams the structural radiation proceeds through the intermediate states asρ0→S+γwhere the meson S subsequently decays into aπ+π−pair.He concluded that the leading term is the pion bremsstrahlung and that the largest contribution to the structural radiation amplitude results from the scalarσ-meson intermediate state.He used the rough estimate gρσγ≃1for the coupling constant gρσγwhich was obtained with the spin independence assumption in the quark model.The coupling constant gρππwas determined using the then available experimental decay rate ofρ-meson and also current algebra results as3.2≤gρππ≤4.9.On the other hand,the coupling constant gσππwas deduced from the assumed decay rateΓ≃100MeV for theσ-meson as gσππ=3.4with Mσ=400MeV. Furthermore,he observed that theσ-contribution modifies the shape of the photon spectrum for high momenta differently depending on the mass of theσ-meson.We like to note, however,that the nature of theσ-meson as a¯q q state in the naive quark model and therefore the estimation of the coupling constant gρσγin the quark model have been a subject ofcontroversy.Indeed,Jaffe[6,7]lately argued within the framework of lattice QCD calculation of pseudoscalar meson scattering amplitudes that the light scalar mesons are¯q2q2states rather than¯q q states.Recently,on the other hand,the coupling constant gρσγhas become an important input for the studies ofρ0-meson photoproduction on nucleons.The presently available data[8] on the photoproduction ofρ0-meson on proton targets near threshold can be described at low momentum transfers by a simple one-meson exchange model[9].Friman and Soyeur [9]showed that in this picture theρ0-meson photoproduction cross section on protons is given mainly byσ-exchange.They calculated theγσρ-vertex assuming Vector Dominance of the electromagnetic current,and their result when derived using an effective Lagrangian for theγσρ-vertex gives the value gρσγ≃2.71for this coupling ter,Titov et al.[10]in their study of the structure of theφ-meson photoproduction amplitude based on one-meson exchange and Pomeron-exchange mechanisms used the coupling constant gφσγwhich they calculated from the above value of gρσγinvoking unitary symmetry arguments as gφσγ≃0.047.They concluded that the data at low energies near threshold can accommodate either the second Pomeron or the scalar mesons exchange,and the differences between these competing mechanisms have profound effects on the cross sections and the polarization observables.It,therefore,appears of much interest to study the coupling constant gρσγthat plays an important role in scalar meson exchange mechanism from a different perspective other than Vector Meson Dominance as well.For this purpose we calculate the branching ratio for the radiative decayρ0→π++π−+γ,and using the experimental value0.0099±0.0016for this branching ratio[11],we estimate the coupling constant gρσγ.Our calculation is based on the Feynman diagrams shown in Fig.1.Thefirst two terms in thisfigure are not gauge invariant and they are supplemented by the direct term shown in Fig.1(c)to establish gauge invariance.Guided by Renard’s[3]current algebra results,we assume that the structural radiation amplitude is dominated byσ-meson intermediate state which is depicted in Fig. 1(d).We describe theρσγ-vertex by the effective LagrangianL int.ρσγ=e4πMρMρ)2 3/2.(3)The experimental value of the widthΓ=151MeV[11]then yields the value g2ρππ2gσππMσ π· πσ.(4) The decay width of theσ-meson that follows from this effective Lagrangian is given asΓσ≡Γ(σ→ππ)=g2σππ8 1−(2Mπ2iΓσ,whereΓσisgiven by Eq.(5).Since the experimental candidate forσ-meson f0(400-1200)has a width (600-1000)MeV[11],we obtain a set of values for the coupling constant gρσγby considering the ranges Mσ=400-1200MeV,Γσ=600-1000MeV for the parameters of theσ-meson.In terms of the invariant amplitude M(Eγ,E1),the differential decay probability for an unpolarizedρ0-meson at rest is given bydΓ(2π)31Γ= Eγ,max.Eγ,min.dEγ E1,max.E1,min.dE1dΓ[−2E2γMρ+3EγM2ρ−M3ρ2(2EγMρ−M2ρ)±Eγfunction ofβin Fig.5.This ratio is defined byΓβRβ=,Γtot.= Eγ,max.50dEγdΓdEγ≃constant.(10)ΓσM3σFurthermore,the values of the coupling constant gρσγresulting from our estimation are in general quite different than the values of this constant usually adopted for the one-meson exchange mechanism calculations existing in the literature.For example,Titov et al.[10] uses the value gρσγ=2.71which they obtain from Friman and Soyeur’s[9]analysis ofρ-meson photoproduction using Vector Meson Dominance.It is interesting to note that in their study of pion dynamics in Quantum Hadrodynamics II,which is a renormalizable model constructed using local gauge invariance based on SU(2)group,that has the sameLagrangian densities for the vertices we use,Serot and Walecka[14]come to the conclusion that in order to be consistent with the experimental result that s-waveπN-scattering length is anomalously small,in their tree-level calculation they have to choose gσππ=12.Since they use Mσ=520MeV this impliesΓσ≃1700MeV.If we use these values in our analysis,we then obtain gρσγ=11.91.Soyeur[12],on the other hand,uses quite arbitrarly the values Mσ=500 MeV,Γσ=250MeV,which in our calculation results in the coupling constant gρσγ=6.08.We like to note,however,that these values forσ-meson parameters are not consistent with the experimental data onσ-meson[11].Our analysis and estimation of the coupling constant gρσγusing the experimental value of the branching ratio of the radiative decayρ0→π++π−+γgive quite different values for this coupling constant than used in the literature.Furthermore,since we obtain this coupling constant as a function ofσ-meson parameters,it will be of interest to study the dependence of the observables of the reactions,such as for example the photoproduction of vector mesons on nucleonsγ+N→N+V where V is the neutral vector meson, analyzed using one-meson exchange mechanism on these parameters.AcknowledgmentsWe thank Prof.Dr.M.P.Rekalo for suggesting this problem to us and for his guidance during the course of our work.We also wish to thank Prof.Dr.T.M.Aliev for helpful discussions.REFERENCES[1]P.Singer,Phys.Rev.130(1963)2441;161(1967)1694.[2]V.N.Baier and V.A.Khoze,Sov.Phys.JETP21(1965)1145.[3]S.M.Renard,Nuovo Cim.62A(1969)475.[4]K.Huber and H.Neufeld,Phys.Lett.B357(1995)221.[5]E.Marko,S.Hirenzaki,E.Oset and H.Toki,Phys.Lett.B470(1999)20.[6]R.L.Jaffe,hep-ph/0001123.[7]M.Alford and R.L.Jaffe,hep-lat/0001023.[8]Aachen-Berlin-Bonn-Hamburg-Heidelberg-Munchen Collaboration,Phys.Rev.175(1968)1669.[9]B.Friman and M.Soyeur,Nucl.Phys.A600(1996)477.[10]A.I.Titov,T.-S.H.Lee,H.Toki and O.Streltrova,Phys.Rev.C60(1999)035205.[11]Review of Particle Physics,Eur.Phys.J.C3(1998)1.[12]M.Soyeur,nucl-th/0003047.[13]S.I.Dolinsky,et al,Phys.Rep.202(1991)99.[14]B.D.Serot and J.D.Walecka,in Advances in Nuclear Physics,edited by J.W.Negeleand E.Vogt,Vol.16(1986).TABLESTABLE I.The calculated coupling constant gρσγfor differentσ-meson parametersΓσ(MeV)gρσγ500 6.97-6.00±1.58 8008.45±1.77600 6.16-6.68±1.85 80010.49±2.07800 5.18-9.11±2.64 90015.29±2.84900 4.85-10.65±3.14 90017.78±3.23Figure Captions:Figure1:Diagrams for the decayρ0→π++π−+γFigure2:The photon spectra for the decay width ofρ0→π++π−+γ.The contributions of different terms are indicated.Figure3:The pion energy spectra for the decay width ofρ0→π++π−+γ.The contri-butions of different terms are indicated.Figure4:The decay width ofρ0→π++π−+γas a function of minimum detected photon energy.Figure5:The ratio Rβ=Γβ。

Dropping Sigma-Meson Mass and In-Medium S-Wave pion-pion Correlations

Dropping Sigma-Meson Mass and In-Medium S-Wave pion-pion Correlations

a r X i v :n u c l -t h /990876v 2 31 A u g 1999Dropping σ-Meson Mass and In-Medium S-wave π-πCorrelationsZ.Aouissat 1,G.Chanfray 2,P.Schuck 3,J.Wambach 11IKP,Technische Universit¨a t Darmstadt,Schloßgartenstraße 9,64289Darmstadt,Germany.2IPN Lyon.43Bd.du 11Novembre 1918,F69622Villeurbanne C´e dex,France.3ISN,Universite Joseph Fourier,CNRS-IN2P3,53avenue des Martyrs,F-38026Grenoble C´e dex,France.(February 9,2008)The influence of a dropping σ-meson mass on previously calculated in-medium ππcorrelations in the J =I =0(σ-meson)channel [2,?]is investigated.It is found that the invariant-mass distribution around the vacuum threshold experiences a further strong enhancement with respect to standard many-body effects.The relevance of this result for the explanation of recent A (π,2π)X data is pointed out.In medium s-wave pion-pion correlations have recently attracted much attention both on the theoretical [1–7]and experimental [8]sides.These studies are of rele-vance for the behavior of the in-medium chiral conden-sate and its fluctuations with increasing density [7].In earlier studies we have shown that standard p-wave cou-pling of the pion to ∆-h and p-h configurations induces a strong enhancement of the ππinvariant-mass distri-bution around the 2m πthreshold [2,3],thus signalling increased fluctuations in the σ-channel.This fact was in-dependently confirmed in [5].It has been argued in [2,4]that this effect could possibly explain the A (π,2π)knock-out reaction data from the CHAOS collaboration [8].More recently Vicente Vacas and Oset [6]have claimed that the theory underestimates the experimentally found π−πmass enhancement.This claim may be partly ques-tioned,since the reaction theory calls for a calculation with a finite total three momentum of the in-medium pion pairs ∗.On the other hand Hatsuda et al.[7]ar-gued that the partial restoration of chiral symmetry in nuclear matter,which leads to a dropping of the σ-meson mass [10],induces similar effects as the standard many-body correlation mentioned above.It is therefore natural to study the combination of both effects.This is the ob-jective of the present note.As a model for ππscattering we consider the lin-ear sigma model treated in leading order of the 1/N -expansion [9].The scattering matrix can then be cast in the following formT ab,cd (s )=δab δcdD −1π(s )−D −1σ(s )D π(s ),(1)where s is the Mandelstam variable.In Eq.(1)D π(s )s −m 2π,f π=√1−λ2Σππ(s )−1,(3)where Σππ(s )is the ππself-energy regularized by meansof a form factor which is used as a fit function [2]and allows to reproduce the experimental ππphase shifts.The coupling constant λ2denotes the bare quartic cou-pling of the linear σ-model,related to the mean-field pion mass m π,sigma mass m σ,and the condensate σ via the mean-field saturated Ward identitym 2σ=m 2π+2λ2 σ 2.(4)It is clear from what was said above that the σ-mesonpropagator in this approach is correctly defined,since it satisfies a whole hierarchy of Ward identities.In cold nuclear matter the pion is dominantly coupled to ∆-h,p-h,as well as to the 2p-2h excitations which,on the other hand,are renormalized by means of re-pulsive nuclear short-range correlations,(see [3]for de-tails).Since the pion is a (near)Goldstone mode,its in-medium s-wave renormalization does not induce con-siderable changes.The sigma meson,on the other hand,is not protected against an important s-wave renormal-ization from chiral symmetry.Therefore,following a very economical procedure,we extract an approximate density 1dependence of the mean-field sigma meson mass by tak-ing into account the density dependence of the conden-sate.From eq.(4)it is clear that the density dependence of the sigma-meson is essentially dictated by the density dependence of the condensate.FIG.1.Results for the imaginary part of the in-medium sigma-meson propagator.Except for the vacuum case(full line curve)the remaining in-medium curves are computed at normal nuclear matter density.The dashed-dotted curve is forα=0,dashed forα=0.2and the dotted forα=0.3. For densities below and around nuclear saturation den-sityρ0we take for the in-medium sigma-meson mass the simple ansatz(see also[7])ρmσ(ρ)=mσ(1−α[1]P.Schuck,W.N¨o renberg,G.Chanfray,Z.Phys.A330(1988)119.[2]P.Schuck,Z.Aouissat,F.Bonutti,G.Chanfray,E.Fra-giacomo,N.Grion,J.Wambach,Proceedings of the XXXVI,international Winter Meetingon Nuclear Physics,Ed.I.Iori,Bormio(Italy),January1998;nucl-th/9806069.[3]Z.Aouissat,R.Rapp,G.Chanfray,P.Schuck,J.Wambach,Nucl.Phys.A581(1995)471.[4]R.Rapp,J.W.Durso,Z.Aouissat,G.Chanfray,O.Krehl,P.Schuck,J.Speth,J.Wambach,Phys.Rev.C59(1999)R1237.[5]H.C.Chiang,E.Oset,M.J.Vicente Vacas,Nucl.Phys.A644(1998)77.[6]M.J.Vicente Vacas,E.Oset,nucl-th/9907008[7]T.Hatsuda,T.Kunihiro,H.Shimizu,Phys.Rev.Lett.82(1999)2840.[8]F.Bonutti et al.,Phys.Rev.Lett.77(1996)603.[9]Z.Aouissat,P.Schuck,J.Wambach,Nucl.Phys.A.618(1997)402.[10]G.E.Brown,M.Rho,Phys.Rep.269(1996)333.[11]D.Davesne,Y.J.Zhang,G.Chanfray,to be published.[12]Z.Aouissat,Ph.D.Thesis Report,ISN93-63,Grenoble.3。

N-甲基芸香碱通过调控ERK通路诱导人胶质母细胞瘤细胞凋亡和自噬(英文)

N-甲基芸香碱通过调控ERK通路诱导人胶质母细胞瘤细胞凋亡和自噬(英文)

Induction of apoptosis and autophagy in human glioblastoma cells byN -methylflindersine : insights into regulatory role of ERK pathway *LI Siyuan 1, LUO Yuyou 1, LUO Xiongming 3, WANG Zhongyu 1, CHEN Huitong 1,FAN Dong 1, YUAN Xingyi 1, CHEN Le 1, TANG Pei 1, LIU Jing 1△,WANG Zongming 2△, WANG Xin 1△(1Department of Human Anatomy , Histology and Embryology , School of Basic Medical Sciences , Guangdong Pharmaceuti⁃cal University , Guangzhou 510006, China ; 2Pituitary Tumor Center , Department of Neurosurgery , the First Affiliated Hos⁃pital , Sun Yat -sen University , Guangzhou 510062, China ; 3School of Life Sciences and Biopharmaceutics , GuangdongProvincial Key Laboratory of Pharmaceutical Bioactive Substances , Guangdong Pharmaceutical University ,Guangzhou 510006, China )[ABSTRACT ] AIM : Mangrove -associated plants are known for producing natural compounds with antitumor ac⁃tivity. Despite the potential therapeutic value of these compunds , the molecular mechanisms underlying their antitumor ef⁃fects remain unclear. This study aimed to investigate the antitumor properties of N -methylflindersine , an alkaloid derived from the mangrove -associated plant , Micromelum falcatum (Lour.) Tan., and its effects on U87 human glioblastoma cells. METHODS : We identified and isolated 15 compounds from the stem bark of Micromelum falcatum . Among these , we screened N -methylflindersine for its potential inhibitory effects on U87 cell growth. Various assays , including wound healing , Hoechst 33342/PI staining , and protein expression analysis , were conducted to investigate the compound's im⁃pact on cell migration , apoptosis , and autophagy -related proteins. RESULTS : Within 24 h , N -methylflindersine demon⁃strated the ability to reduce U87 cell migration and increase the apoptotic U87 cell population. Furthermore , it downregu⁃lated the anti -apoptosis protein Bcl -2 expression , upregulated the pro -apoptosis protein Bax expression , and elevated the ratio of autophagy -related protein LC3-II/LC3-I in U87 cells. Additionally , the ERK signaling pathway was found to be down -regulated following N -methylflindersine treatment. CONCLUSION : N -methylflindersine appears to induce bothapoptosis and autophagic cell death in U87 cells , resulting in reduced cell growth. This effect seems to be associated with the downregulation of the ERK signaling pathway.[KEY WORDS ] mangrove -associated plants ; N -methylflindersine ; glioblastoma[CLC number ] R739.4; R966; R363.2 [Document code ] A doi : 10.3969/j.issn.1000-4718.2024.04.003Oceans are abundant repositories of natural com⁃pounds. Mangroves are unique marine ecosystems dis⁃tributed along the coasts of tropical and subtropical re⁃gions [1-2]. In addition to widely researched true man⁃groves and mangrove -associated fungi , mangrove -asso⁃ciated plants growing in the intertidal zone have shown great bioactivity and beneficial application [3-4]. For in⁃stance , the aqueous seed extract of Derris trifoliataLour (a common mangrove liana ) has a significantly high 2,2-diphenyl -1-picrylhydrazyl (DPPH ) scaveng⁃ing activity , multiple antibacterial activities , and a moderate inhibitory effect on A549 cells [5]. The leaf wa⁃ter extract of Barringtonia racemosa (another mangrove -associated plant ) modifies the cell cycle and triggers apoptosis in Caco -2 cells [6]. Some mangrove -associatedplants were formerly classified as true mangroves ; how⁃[文章编号] 1000-4718(2024)04-0594-08[Received date ] 2023-11-06 [Accepted date ] 2023-12-06* [Foundation item ] Supported by the National Natural Science Foundation of China for Young Scholars (No. 81802678), the Guangdong Natural Science Foundation Project (No. 2018A030310107), the Guangzhou Basic and Applied Basic Research Founda⁃tion (No. 202102021116; No. 202201010307; No. 202201010299), and the Guangdong Province Administration of Traditional Chi⁃nese Medicine Project (No. 20231210).△Corresponding author WANG Xin E -mail : wangxin@ ; LIU Jing E -mail : liujing@ ; WANG ZongmingE -mail : wangzm23@··594ever,they have received more precise descriptions in recent years, including distinctions in leaf features and salt tolerance[7-8].Some mangrove-associated plant ex⁃tracts from different genera exhibit antitumor activi⁃ty[9-11].This indicates that these plants may be attrac⁃tive sources of antitumor medicines.Glioblastomas are the most aggressive brain tu⁃mors owing to their frequent occurrence and invasive characteristics within the central nervous system (CNS)[12].This rapidly developing malignancy has a World Health Organization (WHO) grade 4 CNS tumor, with a poor prognosis that affects the elderly and chil⁃dren[13].Although surgery,high-dose radiation,and chemotherapy help alleviate patient symptoms,com⁃plete glioblastoma control remains a serious problem that must be conquered globally[12]. Therefore, more in⁃novative antineoplastic medicines are urgently re⁃quired.Xyloketal B is a constituent of the mangrove fungus Xylaria sp.(No.2508)that inhibits U251 cells by attenuating the MEK/ERK and PI3K/Akt path⁃ways[14]. Mangrove-associated compounds may serve as weapons against glioblastomas.In this study,15 compounds were extracted from the stem bark of Micromelum falcatum(a mangrove-as⁃sociated plant), including ten coumarins 1-10, four al⁃kaloids 11-15, and a new compound (compound 13). Compound 13 (N-methylflindersine)was the focus[15]and exhibited an inhibitory effect against U87 cells. This study explored its antitumor properties and poten⁃tial mechanisms.MATERIALS AND METHODS1 Compound extraction and isolation1.1 General All nuclear magnetic resonance (NMR)experiments were performed using a Bruker DRX-500 spectrometer.1H NMR was conducted at 500 MHz,and 13C-NMR was conducted at 125 MHz,equipped with an inverse-detection 5 mm probe operat⁃ing at room temperature. SiMe4 was utilized as the inter⁃nal standard and samples were dissolved in deuterated solvent to test the NMR spectra.Optical rotation was measured using a Polaptronic-HNQW5 high-resolution polarimeter,and the infrared (IR)spectrum was ob⁃tained using a Bruker VECTOR22 infrared spectropho⁃tometer. High-resolution electron ionization mass spec⁃trometry (HREI-MS)and electrospray ionization-mass spectrometry (ESI-MS)were performed using Thermo MAT95XP and API2000 LC/MS/MS mass spectrome⁃ters (Applied Biosystems),respectively.Silica gel served as the medium for the column and thin-layer chromatography (TLC) plates. The TLC plates were vi⁃sualized by applying a mixed solvent consisting of 20% sulfuric acid and 80% ethanol, followed by heating. A Waters 600 high-performance liquid chromatography (HPLC)system equipped with a Waters 996 photodi⁃ode array detector was used for HPLC analysis and semi-preparative reversed-phase (RP)-HPLC (Wa⁃ters)with an octadecylsilane (ODS)column (YMC-Pack ODS-5-A, 250×10 mm i.d., 5 μm, YMC) was performed using the CH3OH/H2O solvent system as elu⁃ents.Column chromatography was performed using a Sephadex LH-20 column (Pharmacia) as the stationary phase.1.2 Plant material In October 2010,M. falcatum (Lour.)Tan.were collected from Hainan Province,China.Prof.Jun Wu from the South China Sea Insti⁃tute of Oceanology, Chinese Academy of Sciences veri⁃fied the identity and deposited a voucher specimen in the herbarium of the South China Sea Institute of Ocean⁃ology.1.3 Extraction and isolation Air-dried M.falca⁃tum(lor)Tan.(11.5 kg)was extracted three times with 95% EtOH (50 L)and 50% EtOH (50 L).The organic solvent was pooled and evaporated under re⁃duced pressure.The residue was dissolved in2.5 L H2O and successively extracted with hexane and ethyl acetate (EtOAc; three times each) to yield 58 g of hex⁃ane extract and 117 g of EtOAc extract. The EtOAc ex⁃tract was fractionated on silica gel (1 755 g, 200~300 mesh)using solvents with ascending polarity:10%~ 70% acetone in n-hexane,followed by 5%~100% MeOH in CHCl3,resulting in 122 fractions.Frs.9-12 (3.70 g,eluted with n-hexane-acetone 90∶10)was subjected to silica gel chromatography with a mixture of chloroform and acetone (40∶1) and further purified on Sephadex LH-20 using methanol (MeOH)to yield 12.4 mg of 12, 21.8 mg of 13 and 7.5 mg of 14. Frs. 33-36 (3.20 g,eluted with n-hexane-acetone 70∶30)was combined and chromatographed on silica gel with chloroform-acetone (15∶1) to afford Frs. 36a-d. Com⁃··595pound 36b was further purified by chromatography on a Sephadex LH-20 column using CHCl3/MeOH to afford compound 9 (151 mg).Frs.40-43 (2.77 g,eluted with a mixture of n-hexane and acetone in a ratio of 70∶30)were chromatographed on a silica gel with chloro⁃form-acetone (7∶1) to afford 426 mg of 5. Frs. 45-47 (4.17 g, eluted with a mixture of n-hexane and acetone in a ratio of 70∶30) were fractionated on silica gel with chloroform-acetone (9∶1 and 6∶1)to afford fraction 47a-d. Compound 47a was purified using chromatogra⁃phy on a Sephadex LH-20 column to afford 20.7 mg of1. Compound 47b was separated by chromatography ona Sephadex LH-20 column and semi-preparative HPLC to afford 10.5 mg of 4 and 11.2 mg of 6.Frs.55-60 (3.35 g,eluted with n-hexane-acetone 60∶40)was fractionated on a silica gel using chloroform-acetone (8∶2) to afford fraction 60a-b. Compound 60a was further purified by chromatography on a Sephadex LH-20 col⁃umn to yield compound 8 (20.9 mg), which was crys⁃tallized from CHCl3. Compound 60b was purified using semi-preparative HPLC with MeOH/H2O as the eluent,yielding compound 10 (12.5 mg).Frs.63-65 (4.36 g, eluted with n-hexane-acetone 50∶50) was separated on silica gel using chloroform-acetone (5∶1)to obtain 15.2 mg of 7) and further purified by semi-preparative HPLC (MeOH/H2O) to yield 11.0 mg 2 and 8.2 mg 3. Frs. 68-71 (3.65 g, eluted with n-hexane-acetone 50:50)was separated on silica gel (200~300 mesh)with chloroform-acetone (3∶1) and purified using Sephadex LH-20 and semi-preparative HPLC to yield 42.5 mg of 11 and 12.6 mg of 15.2 Cell cultureU87, U251 and 4T1 cells were maintained in Dul⁃becco's modified Eagle's medium (DMEM)(Gibco,USA) supplemented with 10% (v/v) fetal bovine serum (FBS)and 1% penicillin/streptomycin.Cells were in⁃cubated in a humidified atmosphere containing 5% CO2 at 37 ℃, and CT-26 was cultured in Roswell Park Me⁃morial Institute (RPMI)1640 medium under similar conditions.3 Cell viability assayCell viability was assessed using the Cell Counting Kit-8 (CCK-8)assay (Biosharp,China).Cells (1×104) were cultured in a 96-well plate with 100 μL medi⁃um. After 24 h, the cells were treated with N-methyl⁃flindersine for another 24 h.Subsequently,10 μL of CCK-8 reagent was added,and incubated for 1 h at 37 ℃,and the absorbance was measured at 450 nm using a microplate reader (Bio-Rad, USA) to calculate the IC50.4 Wound healing assayU87 cells (1×104) were seeded in 6-well plates, al⁃lowed to adhere overnight, and scratched with a pipette tip.The cells were washed with PBS,treated with N-methylflindersine at IC50concentration,and incubated for 24 h. Images were captured at 0 and 24 h after treat⁃ment,and migration was analyzed based on scratch width changes in three different fields per well.5 Hoechst 33342/propidium iodide (PI)fluores⁃cence stainingApoptosis was assessed by Hoechst 33342/PI fluo⁃rescence staining. U87 cells (5×105) were seeded into 24-well culture plates and cultured overnight. The cells were incubated with the IC50of N-methylflindersine in culture medium for 24 h, followed by the addition of a mixture of Hoechst 33342 and PI (Biosharp, China),and stained at 37 ℃ for 30 min. The cellular morpholo⁃gy was examined using a fluorescence microscope (Lei⁃ca,Germany).Three different fields were analyzed within each well.6 Western blot assayU87 cells (2×106)were treated with N-methyl⁃flindersine at IC50for 24 h,lysed,and quantified. Equal amounts of protein were separated by sodium do⁃decyl sulfate-polyacrylamide gel electrophoresis, trans⁃ferred to polyvinylidene difluoride (PVDF)mem⁃branes, probed with primary and secondary antibodies,and visualized using chemiluminescence. The band in⁃tensities were then quantified.7 Statistical analysisStatistical analyses were performed using Graph⁃Pad Prism 5.0 software. The outcomes are presented as mean ± standard deviation (mean±SD).Differences between pairs of datasets were evaluated for statistical significance using the Student's t-test.A P-value less than 0.05 was considered to be statistically significant. RESULTS1 N-methylflindersine inhibited U87 cell viability Initially, the dose-dependent effects of compounds··5961-15 on the viability of human glioblastoma U87 cell line were investigated using a CCK -8 assay. The cells were exposed to increasing concentrations of the com⁃pounds for 24 h , and the viability of the cells was sup⁃pressed in an N -methylflindersine dose -dependent man⁃ner (Figure 1). The IC 50 values of the compounds against U87 cells were determined (Table 1). Nota⁃bly , N -methylflindersine only affected U87 cells with an IC 50 value of 17.372±0.434 μmol/L compared to the much lower IC 50 values of another human glioblastoma cell line , U251, the mouse breast cancer cell line , 4T1, and the mouse colon cancer cell line , CT -26.This indicated that U87 cells exhibited the highest sen⁃sitivity to N -methylflindersine treatment (Table 2).2 N -methylflindersine inhibited migration of U87cellsCancer cells can metastasize to distant parts of thebody , which is a characteristic of tumors. We conducted a wound healing assay to test whether N -methylflinder⁃sine treatment could curb the migration of U87 cells.Inhibition of wound closure was observed after N -methyl⁃flindersine treatment in the micrographs (Figure 2).3 N -methylflindersine induced apoptosis of U87 cellsU87 cells were investigated using Hoechst 33342and PI fluorescence dyes to track nuclei in mid -to -late apoptosis. Activation of apoptosis was observed in U87 cells after N -methylflindersine treatment (Figure 3A ). A western blot assay was used to evaluate the expres⁃sion of the survival protein Bcl -2 and the apoptosis -in⁃ducing protein Bax to investigate whether N -methyl⁃flindersine -induced cytotoxicity in U87 cells involved modulation of pro - and anti -apoptotic genes. There wasa substantial decrease in Bcl -2 protein expression , cou⁃pled with a notable increase in Bax protein levels fol⁃lowing N -methylflindersine treatment (Figure 3B ).4 N -methylflindersine induced autophagy ofU87Figure 1. The structure of N -methylflindersine.Figure 2. N -methylflindersine inhibited the migration of U87cells (detected by wound healing assay ). The area within the white dashed line shows the cells that mi⁃grated in 24 h. Scale bar=400 μm.Table 1. The IC 50 of 15 kinds of compounds for U87 cells Number 123456789101112131415NameMicromeloside A Micromeloside CMicromeloside D Murrangatin Micromelin MinumicrolinHydramicromelin A MurracarpinMicrominutinin Microfalcatin Isovalerate Micrometam B 3-Hydroxy -1-methyl -3-(2-oxopropyl )quinoline -2,4(1H ,3H )-dioneN -MethylflindersineN -Methylswietenidine -BCatunaregin IC 50 (μmol/L )>200>200>200>200>200>200>200>200>200>200>200>20017.372±0.434>200>200Table 2. The IC 50 of N -methylflindersine for 4 cell lines Cell lines U874T1U251CT -26IC 50 of N -methylflindersine (μmol/L )17.372±0.434100~200>200>200··597cellsAutophagic cell death is an important mechanism incancer treatment through drug intervention. N -methyl⁃flindersine significantly increased the ratio of autopha⁃gy -related protein LC3B (LC3-II to LC3-I ratio ) in U87 cells according to Western blot analysis (Figure 4).5 N -methylflindersine suppressed ERK pathway inU87 cellsMitogen -activated protein kinases (MAPKs ) in⁃cluding ERK mediate cell migration and apoptosis. Western blot analysis showed that the phosphorylated ERK (p -ERK )/ERK ratio significantly decreased at theIC 50 of N -methylflindersine relative to that of the corre⁃sponding control (Figure 5).DISCUSSIONMangrove -associated plants are vital sources ofnatural products. In recent years , many compounds ex⁃tracted from mangrove -associated plants exhibit antitu⁃mor activities [4]. Fifteen compounds were successfullyextracted from M. falcatum using advanced natural compound extraction and separation methods. N -methyl⁃flindersine alkaloid exhibits antitumor activity in U87cells and was chosen for further research. N -methyl⁃flindersine might attenuate the migration ability of U87Figure 3. N -methylflindersine induced apoptosis of U87 cells. A : the fluorescence staining results of U87 cells with Hoechst33342 and PI marked after N -methylflindersine treatment (scale bar=100 or 50 μm ); B : the Bax and Bcl -2 protein expres⁃sion levels in U87 cells. Mean±SD. n =3. **P <0.01 vscontrol group.Figure 4. N -methylflindersine induced autophagy of U87cells. The LC3-I and LC3-II protein levels in U87 cells were detected by Western blot. Mean±SD. n =3. *P <0.05 vs control group.··598cells. Combined with the experimental results of CCK -8, N -methylflindersine was hypothesized to inhibit U87 cells , although the underlying mechanisms must be elu⁃cidated.The most extensively investigated mechanism ofcell death is regulated cell death (RCD ), which relies on dedicated molecular machinery that can be modulat⁃ed by pharmacological or genetic interventions. It in⁃volves apoptosis and autophagy -dependent cell death , allowing the body to control the balance between cell growth and death. The effectors of these two pathways are elevated in processes involving medications to pre⁃vent and treat glioblastoma[16].Apoptosis is induced by intracellular and extracel⁃lular stimulation and is mediated by mitochondrial and death receptor -mediated pathways. The B -cell lympho⁃ma 2 (Bcl -2) family of proteins contains pro -apoptotic (such as Bax ) and anti -apoptotic (such as Bcl -2) mem⁃bers and is needed to regulate and execute intrinsic apoptosis. Over the last two decades , targeting Bcl -2 family members has aided in cancer therapeutic phar⁃macological treatments[17]. This study found that N -methylflindersine enhanced the apoptotic rate in U87cells. Simultaneously , Bax protein expression in⁃creased while Bcl -2 protein expression decreased. Thisimplies that N -methylflindersine may trigger U87 cells ’ intrinsic apoptotic mechanism.Subsequently , we focused on type II RCD and au⁃tophagy -dependent cell death in U87 cells. In cancer biology , the intracellular self -degradation pathway known as autophagy is frequently viewed as a double -edged sword. It plays two roles in tumor growth and maintains a balance between cytoprotection and cyto⁃static activity [18]. LC3-I may change to LC3-II during autophagosome formation , mediating membrane closure and the fusion of autophagosomes with lysosomes [19]. A crucial step in autophagy is the accumulation of LC3-II. In this study , N -methylflindersine treatment of U87 cells led to upregulation of the LC3-II protein , even with an increased ratio of LC3-II to LC3-I , demonstrat⁃ing more pronounced autophagy activation. This is in line with recent studies hypothesizing that autophagy functions as a tumor suppressor in gliomas [19-20].The ERK signaling pathway controls a variety ofbiological processes in different tumor types , such as cell proliferation , apoptosis , migration , and inva⁃sion [21-22]. The ERK pathway can facilitate and inhibit apoptosis in diverse contexts , and hyperactive ERK sig⁃naling is frequently linked to anti -apoptotic effects [23]. For example , the activation of ERK , along with its up⁃stream signals c -Raf and MEK in Helicobacter pylori -in⁃fected GES -1 cells can prevent H. pylori -induced apop⁃tosis [24]. Conversely , suppression of the MEK/ERK sig⁃naling pathway activates the intrinsic apoptotic pathwayin A549 cells [25]. Niclosamide inhibits U87-MG cell growth by inducing autophagic responses and blocking the pro -survival signal transduction pathway MAPK/ERK [26]. Although ERK modifications are congruent with changes in autophagy levels , it appears that theERK pathway is also associated with autophagy -depen⁃dent cell death [27-28]. Given that the ratio of LC3-II/LC3-I significantly differed between the two groups , we be⁃lieve that autophagy in U87 cells was hyperactive and caused cell death. Changes in autophagic flow may re⁃quire additional confirmation. In this study , we found that N -methylflindersine led to downregulation of the ERK signaling pathway in U87 cells. Therefore , ERKmay be a target of N -methylrutin in glioblastomas ,al⁃Figure 5. N -methylflindersine suppressed the ERK path⁃way in U87 cells. The p -ERK and ERK protein lev⁃els in U87 cells were detected by Western blot.Mean±SD. n =3. **P <0.01 vs control group.··599though the specific mechanism of action requires fur⁃ther verification.In addition, the CCK-8 assay showed that N-meth⁃ylflindersine only had inhibitory effects on U87 cells,which may be owing to the different inhibitory effects of the same compound on different tumor cells caused by multiple factors,including dose-dependent responses,structural variations, and molecular targets. For exam⁃ple,a study on horsetail ferns found that hexane and chloroform extracts exhibited dose-dependent variations in anticancer activity against human hepatocarcinoma cells,with higher concentrations inducing significant antioxidant and apoptotic effects[29].Chalcones are a class of compounds with diverse biological activities,including anticancer activities by inhibiting various mo⁃lecular targets, indicating that structural variations can affect their potency[30]. Furthermore, the impact of leav⁃ing group variation on the anticancer activity of molyb⁃denocenes demonstrated that different ligands lead to variations in anticancer activity against human cancer cell lines[31].These findings highlight the complex na⁃ture of anticancer activity and the influence of various factors such as dose, structural variations, and molecu⁃lar targets on the inhibitory effects of the compound on different tumor cells.In conclusion,N-methylflindersine extracted and isolated from mangrove-related plants may induce apop⁃tosis and autophagy in U87 cells, which may be related to the ERK signaling pathway.Further research is re⁃quired to elucidate these underlying mechanisms.[References][1]Kulkarni R,Deobagkar D,Zinjarde S.Metals in man⁃grove ecosystems and associated biota:a global perspec⁃tive [J]. 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IUBMB Life, 2006, 58(11):621-631.[24]Zhao S, Wan D, Zhong Y, et al. 1α, 25-Dihydroxyvita⁃min D3 protects gastric mucosa epithelial cells against He⁃licobacter pylori-infected apoptosis through a vitamin D re⁃ceptor-dependent c-Raf/MEK/ERK pathway[J].PharmBiol, 2022, 60(1):801-809.[25]Kim HJ, Choi JW, Ree J, et al. Aloe emodin 3-O-gluco⁃side inhibits cell growth and migration and induces apopto⁃sis of non-small-cell lung cancer cells via suppressingMEK/ERK and Akt signalling pathways[J].Life Sci,2022, 300:120495.[26]Cheng B,Morales LD,Zhang Y,et al.Niclosamide in⁃duces protein ubiquitination and inhibits multiple pro-sur⁃vival signaling pathways in the human glioblastoma U-87MG cell line[J]. PLoS One, 2017, 12(9):e184324.[27]Li W, Ren L, Zheng X, et al. 3-O-Acetyl-11-keto-β-bo⁃swellic acid ameliorated aberrant metabolic landscape andinhibited autophagy in glioblastoma[J].Acta Pharm SinB, 2020, 10(2):301-312.[28]Li X, Cen Y, Cai Y, et al. TLR9-ERK-mTOR signaling is critical for autophagic cell death induced by CpG oligo⁃deoxynucleotide 107 combined with irradiation in gliomacells[J]. Sci Rep, 2016, 6(1):27104.[29]Ruan Y, Kovalchuk A, Jayanthan A, et al. Druggable tar⁃gets in pediatric neurocutaneous melanocytosis: molecularand drug sensitivity studies in xenograft and ex vivo tumorcell culture to identify agents for therapy[J].Neuro On⁃col, 2015, 17(6):822-831.[30]Almasoud HA,Ali D,Yaseen KN,et al.Dose-depen⁃dent variation in anticancer activity of hexane and chloro⁃form extracts of field horsetail plant on human hepatocarci⁃noma cells[J]. Biomed Res Int, 2022, 2022:5778411.[31]Arshad J, Tong KKH, Movassaghi S, et al. Impact of the metal center and leaving group on the anticancer activityof organometallic complexes of pyridine-2-carbothioamide[J]. Molecules, 2021, 26(4):833.(责任编辑:余小慧,罗森)N-甲基芸香碱通过调控ERK通路诱导人胶质母细胞瘤细胞凋亡和自噬*黎思源1,罗宇悠1,罗雄明3,王仲宇1,陈晖彤1,樊东1,袁星怡1,陈乐1,唐佩1,刘靖1△,王宗明2△,王欣1△(1广东药科大学基础医学院人体解剖学与组织胚胎学系,广东广州 510006;2中山大学附属第一医院神经外科垂体瘤中心,广东广州 510062;3广东药科大学生命科学与生物制药学院,药用生物活性物质研究所,广东广州 510006)[摘要]目的:已知红树林相关植物能够产生具有抗肿瘤活性的天然化合物。

白藜芦醇

白藜芦醇

Design,synthesis,and biological evaluation of resveratrol analogues asaromatase and quinone reductase 2inhibitors for chemoprevention of cancerBin Sun a, ,Juma Hoshino a ,Katie Jermihov c ,Laura Marler b ,John M.Pezzuto b ,Andrew D.Mesecar c ,Mark Cushman a,*aDepartment of Medicinal Chemistry and Molecular Pharmacology,School of Pharmacy and Pharmaceutical Sciences,and The Purdue Center for Cancer Research,Purdue University,West Lafayette,IN 47907,United States bCollege of Pharmacy,University of Hawaii at Hilo,Hilo,HI 96720,United States cCenter for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,College of Pharmacy,The University of Illinois at Chicago,Chicago,IL 60612,United Statesa r t i c l e i n f o Article history:Received 5March 2010Revised 11May 2010Accepted 14May 2010Available online 24May 2010Keywords:Resveratrol analogues AromataseQuinone reductase 2a b s t r a c trationalize docking,molec-ular mechanics energy minimization,and computer graphics molecular modeling,and the information was utilized to design several very potent inhibitors,including compounds 82(IC 5070nM)and 84(IC 5036nM).The aromatase inhibitory activities of these compounds are much more potent than that for the lead compound resveratrol,which has an IC 50of 80l M.In addition to aromatase inhibitory activ-ity,compounds 32and 44also displayed potent QR2inhibitory activity (IC 501.7l M and 0.27l M,respec-tively)and the high-resolution X-ray structures of QR2in complex with these two compounds provide insight into their mechanism of QR2inhibition.The aromatase and quinone reductase inhibitors resulting from these studies have potential value in the treatment and prevention of cancer.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionResveratrol (3,5,40-trihydroxystilbene)(Fig.1)was first isolated from the roots of the white hellebore lily Veratrum grandiflorum O.Loes in 1940.1This compound is a naturally occurring phytoalexin produced by a wide range of plants (at least 72plant species)in re-sponse to environmental stress or pathogenic attack.Since the dis-covery of its cardioprotective activity in 1992,resveratrol research has steadily accelerated.This increased attention eventually re-sulted in the discovery of its cancer chemopreventive properties.2Resveratrol was found to be capable of interfering with all three steps of carcinogenesis (initiation,promotion,and progression).2Resveratrol has been reported to exert a variety of biological activities.Some of these include antioxidant,anti-inflammatory,anti-infective,anti-ischemic,cardioprotective,neuroprotective,anti-aging (prolongs lifespan),anti-obesity,anti-viral,and cancer chemopreventive effects.3These effects are mediated through sev-eral biological receptors,including cyclooxygenase (COX),lipooxy-genase (LOX),nuclear factor kappa-light-chain-enhancer ofactivated B cells (NF-kB),quinone reductase 1(QR1),quinone reduc-tase 2(QR2),ornithine decarboxylase (ODC),and aromatase.In the United States,breast cancer is the most commonly diag-nosed type of cancer in women and is the second leading cause of death from cancer in women.Approximately 180,000new cases of breast cancer were detected among women in the United States in 2008.4About two-thirds premenopausal and three-fourths post-menopausal breast cancer patients have estrogen-dependent cancer.5In hormone-dependent breast cancer,estrogens play a critical role in stimulating breast cancer cell proliferation.6High levels of estrogens promote the progression of breast cancer.Two main strat-egies have been devised by medicinal chemists to control or block the pathological activity of estrogens.7The first strategy involves0968-0896/$-see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.bmc.2010.05.042*Corresponding author.Tel.:+17654941465;fax:+17654946790.E-mail addresses:cushman@ ,cushman@ (M.Cushman).On leave from Shandong University,China.the design and synthesis of estrogen receptor antagonists,which has yielded useful anticancer and chemopreventive drugs such as tamoxifen and raloxifene.8,9Aromatase,a pivotal enzyme responsi-ble for the conversion of androgens to estrogens,is‘the other’attrac-tive biological target for the development of new agents for the treatment of breast cancer.10–14As the enzyme responsible for the final step of the estrogen biosynthetic pathway,selective inhibition of aromatase will not interfere with the production of other steroids in the pathway(e.g.,adrenal corticoids).15,16Therefore,aromatase is a useful therapeutic target in the treatment or prevention of estro-gen-dependent breast cancers.17Quinone reductase2(QR2)is a cytosolic FAD-dependentflavo-enzyme that catalyzes the reduction of quinones by reduced N-al-kyl-and N-ribosylnicotinamides.18Recent studies have indicated that QR2may transform certain quinone substrates into more highly reactive species that are capable of causing increased cellu-lar damage.19–21Therefore,it is hypothesized that inhibition of QR2in certain cases may lead to protection of cells against these reactive species.18Several QR2inhibitors have been reported in the literature22and the X-ray crystal structure of QR2in complex with resveratrol has been determined.23,24These observations motivated the present study.Both aromatase and QR2have been targeted for discovery and identification of chemopreventive agents.24An array of resveratrol analogues have been synthesized in our laboratory in an attempt to discover new inhibitors of these two enzymes.The trans-stilbene JH-2-29(Fig.1,Scheme7),with a para-amino group,was found to exhibit versatile biological activities including nitric oxide syn-thase inhibition,aromatase inhibition(IC5022l M),and inhibition of TNF-a-induced NF-j B activity,and was therefore selected as a lead compound for further optimization.In order to improve the potency and selectivity of JH-2-29,a limited number of methoxy-lated compounds were synthesized and observed empirically to enhance aromatase inhibitory activity.In particular,compound 32(Fig.2)was found to possess significant aromatase inhibitory activity,with an IC50value of0.59l M,which is comparable to the clinically useful aromatase inhibitor2-aminoglutethimide (IC500.27l M)(Fig.2).18Molecular modeling was used to investi-gate the mechanism of action,and a variety of structurally related stilbenes were designed and synthesized based on the results.Sev-eral of these synthetic derivatives were found to inhibit aromatase activity with IC50values in the sub-micromolar range.In addition, two compounds,32and44,were discovered to exhibit potent QR2 inhibitory activity,and X-ray crystallographic analysis revealed these two compounds bind to QR2in an orientation similar to that of resveratrol,but both compounds demonstrate greater inhibitory potency than resveratrol.2.Results and discussion2.1.Chemistrytriphenylphosphonium bromide,and the following Heck reaction between appropriately substituted vinylbenzenes11–22and iodo-benzenes23–31as shown in Scheme1and Table1.The coupling constants of the vinylic protons of the trans-stilbenes were about 16Hz.The double bonds of the analogues32,37,41,and49were re-duced by catalytic hydrogenation to provide the corresponding derivatives60,61,62,and63(Scheme2).Dinitro analogues50 and52were reduced with stannous chloride to provide the respec-tive polyamines64and65in satisfactory yields(Scheme3).The ace-tate groups of compounds39and55were hydrolyzed by sodium methoxide in methanol to yield phenol derivatives66and67 (Scheme4).The tert-butyldimethylsilyl(TBDMS)-protected vinyl-benzene69(Scheme5)reacted with4-iodoaniline in a standard Heck reaction,but the yield was very low.The method of Angela et al.was used and the TBDMS group of intermediate compound 69was cleaved by tetrabutylammoniumfluoride(TBAF)to afford the diphenol70,which was acetylated to produce71.Heck reaction between compound71and4-iodoaniline or4-iodonitrobenzene led to the respective trans-stilbenes72and73.Acetate hydrolysis yielded the desired compounds74and75.25The synthetic route to imidazole analogue82began with the preparation of phosphonium salt78from aldehyde5,and ketone80from commercially available compound79(Scheme6).The building blocks78and80were then combined by the Wittig reaction in the presence of potassium car-bonate and18-crown-6,26,27and the resulting E and Z isomers were separated and purified by chromatography to provide the nitro ana-logue82.Reduction by stannous chloride resulted in the amine derivative84(Scheme6).The double bond conformation of com-pound82was confirmed by2D1H–1H NOESY spectroscopy(Fig.3).2.2.MechanismThe aromatase inhibitory activity of the resveratrol analogue32 (IC500.59l M)in comparison with resveratrol itself(IC5080l M) led to an investigation of the mechanism of action of compound 32.Recently,Ghosh et al.solved the crystal structure of human aromatase in complex with androgen.28Compound32was docked into the binding site of Ghosh’s structure(PDB code3eqm)using the GOLD(Genetic Optimization for Ligand Docking)program,29–31 and the energy minimized.In the resulting hypothetical structure (Fig.4),the amine group on ring A hydrogen bonds with the car-bonyl group of Ile305and carboxyl group of Asp309,which plays a critical role in catalysis.28Ring A is involved in a p–p interaction with the indole ring of Trp224,and a CH–p interaction with the methyl group of Thr310.Ring B forms a CH–p interaction with the methyl group of Val370.The methoxyl group on ring B is in-volved in a potential hydrogen bond with the backbone NH of Met374.All of the residues mentioned above are included in the catalytic cleft of aromatase.28The docking and energy minimization results provide a good foundation for design of new analogues.In order to minimize the possible cytotoxicity contributed by the aniline part of compound 32,the amino group on ring A was replaced by hydrogen bond-donating groups,such as hydroxy and aminomethyl groups,which may form hydrogen bonds with Asp309and Ile305(Fig.5).As shown in Figure4,the backbone carbonyl group of residue Leu477,a potential hydrogen bond acceptor located near ring B, suggested the addition of hydrogen bond-donating groups on ring B.Met374,working as a hydrogen bond donor,also suggests incor-poration of hydrogen bond acceptors on ring B.In order to probe the function of the stilbene double bond,compounds with single bonds were also synthesized.Many aromatase inhibitors have imidazole or triazole groups that can coordinate to the iron atom,32–36and these were therefore added to the stilbene frame-work and the resulting structures were modeled in complex with aromatase.B.Sun et al./Bioorg.Med.Chem.18(2010)5352–536653532.3.Aromatase inhibitory activityAn aromatase inhibition assay was performed on synthetic ana-logues of compound JH-2-29,as described in the Section 4.The inhibition percentage at a concentration of 20l g/mL and the IC 50values of the more active compounds are reported in Table 2.Most of the resveratrol analogues with an amino group on the para posi-tion of ring A (32,33,35,36,37,44,45,46,48,51,64,65,72,and 74)consistently exhibited significant activity,with IC 50values in the 0.59–14.51l M range.Substitution of the para -amino group with nitro,halogen,hydroxy,nitrile,acetyl,and aminomethylgroups made the resulting compounds inactive (compounds 38,39,40,41,43,52,53,55,56,57,58,59,66,67,and 75).Changing the position of amino group on ring A also led to reduced activity (compounds 34,42,and 54).Presumably,altering the position of the amino group is expected to influence its ability to simulta-neously hydrogen bond with the Asp309carboxylate and the back-bone carbonyl oxygen of Ile305(Fig.4).Double bond reduction impacted the activity positively (37vs 61,49vs 63,and 41vs 62)or negatively (32vs 60)to some extent.Within the series,the position of amino group on ring A was of greater importance than that of methoxy groups (ring B),and a para -amino group was better than an ortho-or meta -amino group.Compound 84was the most potent aromatase inhibitor,with an IC 50value of 36nM.As expected,molecular modeling indicated that one of the imidazole nitrogens coordinates with the iron atom of heme,which plays a critical role in catalysis (Fig.6).28K M and V max values for those analogues demonstrated to inhibit aromatase can be found in Table 3.V max values for each inhibitor are quite similar to each other and to the V max of the control (34.8pmol/min/mg protein),indicating that these resveratrol ana-logues are competitive inhibitors of CYP19.2.4.Quinone reductase 2inhibitory activity and X-ray structure analysisQR2inhibition assays were performed on analogues of syn-thetic compound JH-2-29,as described in the Section -pounds 32and 44exhibited potent QR2inhibitory activity with IC 50values of 1.7±0.06l M and 0.27±0.01l M,respectively.Both of these substances are more potent than resveratrol,which has an IC 50value of 5.1l M in the same assay.To gain insight into the binding mode of these two inhibitors,the X-ray crystal structures of both compounds 32and 44in com-plex with human QR2were plete X-ray data sets were collected and processed,and the final structural models were refined to 1.74Åand 1.55Åfor compound 32and compound 44complexes,respectively.As revealed by the strong and well-de-fined electron density shown in Figure 7,both compounds 32and 44bind to QR2in an orientation similar to that of resveratrol.However,these two compounds interact directly with the side chain of Thr71,whereas resveratrol binds to this residue indirectly through a water molecule.The direct hydrogen bond interaction between the amino group and the hydroxyl group of residue Thr71is most likely responsible for enhancing the QR2inhibitory activity of compounds 32and 44relative to resveratrol.The3-Table 1Structures of compounds 32–59Compd R 1R 2R 3R 4R 5R 6R 732H OCH 3H OCH 3H NH 2H 33OCH 3H OCH 3H H NH 2H 34OCH 3H OCH 3H NH 2H H 35OCH 3H H OCH 3H NH 2H 36OCH 3OCH 3H H H NH 2H 37H OCH 3OCH 3H H NH 2H 38H OCH 3OCH 3H H CN H 39H OCH 3OCH 3H H OAc H 40H OCH 3OCH 3H H F H 41H OCH 3OCH 3OCH 3H NO 2H 42H OCH 3OCH 3OCH 3NH 2HH 43H OCH 3OCH 3OCH 3H CH 2NH 2H 44H OCH 3OCH 3OCH 3H NH 2H 45OCH 3OCH 3OCH 3H H NH 2H 46OCH 3OCH 3H OCH 3H NH 2H 47H –OCH 2O–H NH 2H H 48H –OCH 2O–H H NH 2H 49H H NO 2H NO 2H H 50H NO 2H H H NH 2H 51H OCH 3H H H NH 2H 52H H NO 2H NO 2H NO 253H OCH 3H OCH 3H NO 2H 54H OCH 3H OCH 3NH 2H H 55H OCH 3H OCH 3H OAc H 56H OCH 3H OCH 3H CH 2NH 2H 57H OCH 3H OCH 3H F H 58H OCH 3H OCH 3H Cl H 59HOCH 3HOCH 3HCNH5354 B.Sun et al./Bioorg.Med.Chem.18(2010)5352–5366methoxy groups on ring B of compounds 32and 44interact with the amide NH 2group of residue Asn161,and there is one more hydrogen bond interaction between this side chain and the 4-methoxy group of compound 44,which may contribute to the higher potency of compound 44relative to 32.The X-ray structures of QR2in complex with compounds 32and 44also indicate that these resveratrol analogues sit parallel to the plane of the isoallox-azine ring of the bound cofactor FAD,occupying a position that is similar to resveratrol in the QR2-resveratrol complex.223.ConclusionsA focused set of 45resveratrol analogues have been designed,synthesized,and tested in an effort to maximize their aromatase and QR2inhibitory pounds 82and 84exhibit excel-lent aromatase inhibitory activity,with IC 50values 70nM and 36nM,respectively.From the preliminary structure–activity rela-tionships and molecular modeling results,it appears that the para -amino group on the trans -stilbene benzene ring is essential for aromatase inhibitory activity,and the introduction of an imidazole moiety improves the activity pounds 32and 44also displayed potent QR2inhibitory activity,and their crystal struc-tures in complex with QR2have been determined.They provide a detailed description of the enzyme active site,and can be utilized in the further modification for discovery of new QR2inhibitors.4.Experimental 4.1.General proceduresMelting points were determined in capillary tubes using a Mel-Temp apparatus and are not corrected.Infrared spectra were ob-tained as films on salt plates using CHCl 3as the solvent except where otherwise specified,using a Perkin–Elmer Spectrum One FT-IR spectrometer,and are baseline-corrected.1H NMR spectra were obtained at 300MHz (1H)and 75MHz (13C)or 500MHz (1H)and 125MHz (13C),using Bruker ARX300and Bruker Avance 500(TXI 5mm probe)spectrometers,respectively.Mass spectral analyses were performed at the Purdue University Campus-Wide Mass Spectrometry Center.Analytical thin-layer chromatography was performed on Baker-flex silica gel IB2-F plastic-backed TLC plates.Flash chromatography was performed with 230–400mesh silica gel.Unless otherwise stated,chemicals and solvents were of reagent grade and used as obtained from commercial sources without further purification.Dichloromethane was distilled from CaH 2and THF from sodium prior to use.4.1.1.General procedure 1(GP 1)for the preparation of aromatic alkenes from aromatic aldehydesMethyl triphenylphosphonium bromide (4.3g,12.05mmol)and sodium amide (0.47g,12.05mmol)in dry diethyletherB.Sun et al./Bioorg.Med.Chem.18(2010)5352–53665355(15mL)were stirred at room temperature under argon for 10h.A solution of benzaldehyde (6mmol)in diethyl ether (10mL)was added to the reaction mixture dropwise under argon in 30min at À10°C.After 10min,the reaction mixture was warmed up to room temperature and stirred for 2h.The reaction mixture was then fil-tered and the solvent was removed in vacuo to yield yellow oil that was purified by silica gel column chromatography,eluting with hexane–dichloromethane mixture,to yield the pure product in good yield.4.1.2.General procedure 2(GP 2)for the Heck reactionsIn a round-bottomed flask,iodobenzene (2.21mmol)was added to a mixture of tetrabutylammonium bromide (1.1g,3.33mmol),potassium acetate (350mg,3.57mmol),palladium acetate (25mg,0.11mmol)and aromatic alkenes (2.44mmol)stirred in DMF (20mL)at room temperature under argon.The reaction mixture was warmed up to 80°C for 3h and then cooled to room tempera-ture.Diethyl ether (25mL)was added and the organic layer was washed with water (3Â20mL)and then dried over anhydrous sodium sulfate.The solution was concentrated and the residuewas purified by silica gel column chromatography,eluting with hex-ane–dichloromethane mixture,to yield the pure product as a solid in moderate yield.4.1.3.General procedure 3(GP 3)for the hydrolysis of acetyl groupsNaOMe (2mg,0.04mmol)was added to a solution of (E )-(sty-ryl)phenyl acetate (0.5mmol)in ethanol (15mL).The reaction mixture was stirred for 10h and the solvent was evaporated in va-cuo.The residue was purified by silica gel column chromatography,eluting with a solution of 1%methanol in dichloromethane,to yield the pure product in good yield.4.1.4.General procedure 4(GP 4)for thecatalytic hydrogenation of nitro groups and stilbenesStilbene compound (0.35mmol)was dissolved in ethyl acetate (15mL)and stirred with palladium on carbon (10%,10mg)under H 2at room temperature for 24h.The mixture was filtered,and the solvent was removed in vacuo to provide a solid that was recrystal-lized from dichloromethane and hexane to yield the pure product in good yield.4.2.Syntheses4.2.1.1,2-Dimethoxy-3-vinylbenzene (11)37This compound was prepared in 97%yield by following GP 1.Colorless oil.1H NMR (300MHz,CDCl 3)d 7.14–6.99(m,3H),6.82(dd,J =10.8Hz,J =1.2Hz,1H),5.77(d,J =17.7Hz,1H),5.30(d,J =11.1Hz,1H),3.84(s,3H),3.81(s,3H).5356 B.Sun et al./Bioorg.Med.Chem.18(2010)5352–53664.2.2.2,4-Dimethoxy-1-vinylbenzene(12)38This compound was prepared in82%yield by following GP1. Colorless oil.1H NMR(300MHz,CDCl3)d7.41(d,J=8.4Hz,1H), 7.04–6.94(m,1H),6.51–6.45(m,2H),5.65(d,J=17.7Hz,1H), 5.17(d,J=11.4Hz,1H),3.83(s,3H),3.81(s,3H).4.2.3.1,4-Dimethoxy-2-vinylbenzene(13)39This compound was prepared in93%yield by following GP1. Colorless oil.1H NMR(300MHz,CDCl3)d7.12–7.01(m,2H),6.80 (s,2H),5.75(d,J=17.7Hz,1H),5.29(d,J=8.4Hz,1H),3.80(s, 3H),3.79(s,3H).4.2.4.1,2-Dimethoxy-4-vinylbenzene(14)39This compound was prepared in87%yield by following GP1. Colorless oil.1H NMR(300MHz,CDCl3)d6.95–6.93(m,2H),6.90 (d,J=1.8Hz,1H),6.69(dd,J=17.4Hz,J=10.6Hz,1H),5.59(d, J=17.4Hz,1H),5.13(d,J=10.6Hz,1H),3.88(s,3H),3.85(s,3H).4.2.5.1,3-Dimethoxy-5-vinylbenzene(15)40This compound was prepared in93%yield by following GP1. Colorless oil.1H NMR(300MHz,CDCl3)d6.59–6.54(m,3H),5.62 (d,J=17.4Hz,1H),5.16(d,J=10.8Hz,1H),3.82–3.81(m,9H). 4.2.6.1,2,3-Trimethoxy-4-vinylbenzene(16)41This compound was prepared in58%yield by following GP1. Colorless oil.1H NMR(300MHz,CDCl3)d7.18(d,J=8.7Hz,1H), 6.95–6.86(m,1H), 6.65(d,J=8.7Hz,1H), 5.63(dd,J=18.0, 1.2Hz,1H),5.17(dd,J=18.0,1.2Hz,1H),3.86(s,6H),3.84(s, 3H);13C NMR(75MHz,CDCl3)d153.2,151.4,142.2,130.9, 124.7,120.5,113.0,107.5,61.1,60.8,55.9.4.2.7.1,2,5-Trimethoxy-3-vinylbenzene(17)42This compound was prepared in42%yield by following GP1. White solid:mp54–55°C.1H NMR(300MHz,CDCl3)d7.02–6.92 (m,2H), 6.48(s,1H), 5.57(dd,J=17.7, 1.2Hz,1H), 5.13(dd, J=14.1,1.2Hz,1H),3.88(s,3H),3.85(s,3H),3.81(s,3H);13C NMR(75MHz,CDCl3)d151.3,149.5,143.2,130.8,118.4,112.0, 109.3,97.6,56.6,56.4,56.0.4.2.8.5-Vinylbenzo[d][1,3]dioxole(18)43This compound was prepared in60%yield by following GP1. Colorless oil.1H NMR(300MHz,CDCl3)d6.99(s,1H),6.85(d, J=8.1Hz,1H), 6.78(d,J=8.1Hz,1H), 6.64(dd,J=16.8Hz, J=10.8Hz,1H), 5.95(s,2H), 5.60(d,J=16.8Hz,1H), 5.15(d, J=10.8Hz,1H);13C NMR(75MHz,CDCl3)d147.9,147.3,136.3, 132.0,120.9,111.8,108.0,105.3,100.9.4.2.9.1,2,3-Trimethoxy-5-vinylbenzene(19)40This compound was prepared in85%yield by following GP1. Colorless oil.1H NMR(300MHz,CDCl3)d6.59–6.54(m,3H),5.62 (d,J=17.8Hz,1H),5.16(d,J=10.8Hz,1H),3.82–3.81(m,9H). 4.2.10.1-Methoxy-3-vinylbenzene(20)44This compound was prepared in73%yield by following GP1. Colorless oil.1H NMR(300MHz,CDCl3)d6.95–6.93(m,2H),6.90 (d,J=1.8Hz,1H),6.69(dd,J=17.4Hz,J=10.6Hz,1H),5.59(d, J=17.4Hz,1H),5.13(d,J=10.6Hz,1H),3.88(s,3H),3.85(s,3H).4.2.11.3,5-Dimethoxy-40-amino-trans-stilbene(32)This compound was prepared in64%yield by following GP2. Yellow solid:mp90–91°C.IR(film)3369,1595,1516,1456, Figure3.Region of a2D1H–1H NOESY spectrum of82.The red circle indicates the NOE signal of H8and H17/21.1203,1151,1066,962cm À1;1H NMR (300MHz,CDCl 3)d 7.31(d,J =8.4Hz,2H),6.99(d,J =16.2Hz,1H),6.83(d,J =16.2Hz,1H),6.65(d,J =8.4Hz,2H),6.62(d,J =2.1Hz,2H),6.34(t,J =2.1Hz,1H),3.81(s,6H),3.75(br,2H);13C NMR (75MHz,CDCl 3)d 160.8,146.2,139.9,129.1,127.7,124.9,115.1,104.0,99.2,55.3;CIMS 256(MH +);HRMS calcd for C 16H 17O 2N m/z 255.1259,found:m/z 255.1261(M +).4.2.12.2,4-Dimethoxy-40-amino-trans -stilbene (33)This compound was prepared in 59%yield by following GP 2.Yellow solid:mp 97–98°C.IR (film)3372,1609,1577,1517,1464,1286,1207,968cm À1;1H NMR (300MHz,CDCl 3)d 7.48(d,J =8.4Hz,1H),7.34(d,J =8.4Hz,2H),7.22(d,J =16.5Hz,1H),6.93(d,J =16.5Hz,1H), 6.66(d,J =8.4Hz,2H), 6.49–6.46(m,2H),3.85(s,3H),3.82(s,3H);13C NMR (75MHz,CDCl 3)d 159.9,157.6,145.3,129.1,127.4,127.0,126.6,120.0,119.6,115.3,104.8,98.4,55.4,55.3;CIMS m/z 256(MH +);HRMS calcd for C 16H 18O 2N m/z 256.1338,found:m/z 256.1344(MH +).4.2.13.2,4-Dimethoxy-30-amino-trans -stilbene (34)This compound was prepared in 42%yield by following GP 2.Yellow solid:mp 69–70°C.IR (film)3369,1598,1577,1504,1456,1287,1158,971cm À1;1H NMR (300MHz,CDCl 3)d 7.49(d,J =8.4Hz,1H),7.35(d,J =16.5Hz,1H),7.11(t,J =7.8Hz,1H),6.94–6.86(m,3H), 6.57(dd,J =2.1Hz,8.1Hz,1H), 6.50(dd,J =2.4Hz,8.7Hz,1H),6.46(d,J =2.4Hz,1H),3.85(s,3H),3.82(s,3H), 3.77(br,2H);13C NMR (75MHz,CDCl 3)d 160.4,157.9,146.1,139.3,129.4,127.1,127.0,123.0,119.5,117.4,114.2,112.8,104.9,98.4,55.4,55.3;ESIMS m/z 256(MH +);HRMS calcd for C 16H 18O 2N m/z 256.1338,found:m/z 256.1340(MH +).4.2.14.2,5-Dimethoxy-40-amino-trans -stilbene (35)45This compound was prepared in 35%yield by following GP 2.Yellow solid:mp 91–92°C.IR (film)3370,1601,1516,1493,1282,1215,1046,969cm À1;1H NMR (300MHz,CDCl 3)d 7.36(d,J =8.4Hz,2H),7.27(d,J =16.2Hz,1H),7.11(d,J =2.7Hz,1H),6.99(d,J =16.2Hz,1H),6.81(d,J =8.7Hz,1H),6.76(d,J =2.7Hz,1H),6.71(d,J =8.4Hz,2H),4.15(br,2H),3.82(s,3H),3.80(s,3H);13C NMR (75MHz,CDCl 3)d 153.7,151.1,146.0,129.3,128.3,127.8,119.4,115.1,112.8,112.2,111.1,56.2,55.7;CIMS m/z 256(MH +).4.2.15.2,3-Dimethoxy-40-amino-trans -stilbene (36)This compound was prepared in 75%yield by following GP 2.Yellow solid:mp 97–98°C.IR (film)3367,2923,1539,1515,1343,961cm À1;1H NMR (300MHz,CDCl 3)d 7.37(d,J =8.1Hz,2H),7.29(d,J =16.8Hz,1H),7.21(dd,J =1.2Hz,7.8Hz,1H),7.07–7.02(m,2H),6.82–6.77(m,3H),3.88(s,3H),3.84(s,3H);13C NMR (75MHz,CDCl 3)d 152.8,146.3,146.1,131.9,129.8,128.0,127.7,123.9,118.8,117.3,115.0,110.4,60.8,55.6;ESIMS m/z 256(MH +);HRMS calcd for C 16H 17O 2N m/z 255.1259,found:m/z 255.1257(M +).4.2.16.(3,4-Dimethoxy-40-amino-trans -stilbene (37)46This compound was prepared in 75%yield by following GP 2.Yellow solid:mp 158–159°C.IR (film)3368,1622,1604,1516,Figure 4.(A)Ribbon diagram illustrating the hypothetical complex structure human aromatase with compound 32bound to the active site,and the hydrogen bonds are labeled as yellow broken lines.(B)Potential interactions between compound 32and Ile305,Asp309,Thr310,Trp224,Val370,Met374in human aromatase active site.The hydrogen bonds are labeled as yellow broken lines.H 3COOH NH 2Hydrogen Bond Donor and AcceptorOAcChem.18(2010)5352–53661465,1137,1023,965cmÀ1;1H NMR(300MHz,CDCl3)d7.32(d,8.4Hz,2H),7.04(d,J=1.8Hz,1H),7.00(dd,J=1.8Hz,8.1Hz, 1H),6.88(s,1H),6.83(d,J=8.1Hz,1H),6.67(d,J=8.4Hz,2H), 3.92(s,3H),3.87(s,3H),3.75(br,2H);13C NMR(75MHz,CDCl3) d149.0,148.3,145.8,131.0,128.1,127.4,126.8,124.8,119.2, 115.2,111.1,108.3,55.8,55.7;CIMS m/z256(MH+).4.2.17.3,4-Dimethoxy-40-cyano-trans-stilbene(38)This compound was prepared in61%yield by following GP2. Yellow solid:mp102–103°C.IR(film)3398,2223,1595,1515, 1463,1267,1139,964cmÀ1;1H NMR(300MHz,CDCl3)d7.60(d, J=8.4Hz,2H),7.53(d,J=8.4Hz,2H),7.14(d,J=16.5Hz,1H), 7.06(d,J=6.3Hz,2H),6.93(d,J=16.5Hz,1H),6.86(d,J=9.0Hz, 1H),3.93(s,3H),3.90(s,3H);13C NMR(75MHz,CDCl3)d149.6, 149.1,142.0,132.4,132.1,129.2,126.5,124.6,120.6,119.0, 111.1,110.0,108.8,55.9;CIMS m/z266(MH+);HRMS calcd for C17H15O2N m/z265.1103,found:m/z265.1100(M+).4.2.18.3,4-Dimethoxy-40-acetoxy-trans-stilbene(39)47This compound was prepared in59%yield by following GP2. Yellow solid:mp125–126°C.IR(film)3368,2927,1760,1515, 1420,1196,964cmÀ1;1H NMR(300MHz,CDCl3)d7.47(d,J=8.4Hz,2H),7.07–6.97(m,5H),6.93(d,J=16.5Hz,1H),6.83 (d,J=7.8Hz,1H),3.93(s,3H),3.89(s,3H),2.29(s,3H);13C NMR (75MHz,CDCl3)d169.5,149.7,149.1,135.3,130.2,128.7,127.1, 125.7,121.7,119.9,111.1,108.6,55.9,55.8,21.1;CIMS m/z299 (MH+).4.2.19.3,4-Dimethoxy-40-fluoro-trans-stilbene(40)This compound was prepared in57%yield by following GP2. Yellow solid:mp117–118°C.IR(film)3368,1600,1515,1461, 1225,1139,1022,964cmÀ1;1H NMR(300MHz,CDCl3)d7.45 (dd,J=5.7Hz,8.4Hz,2H),7.07–7.02(m,4H),6.97(d,J=16.5Hz, 1H),6.91(d,J=16.5Hz,1H),6.86(d,J=7.8Hz,1H),3.95(s,3H), 3.90(s,3H);13C NMR(75MHz,CDCl3)d148.9,133.6,130.2, 128.2,127.7,127.5,125.5,119.7,115.6,115.3,111.1,108.5,55.8, 55.7;CIMS m/z259(MH+);HRMS calcd for C16H15O2F m/z 258.1056,found:m/z258.1051(M+).Table2Figure6.Potential interactions between compound84and Ile305,Asp309,Thr310,Trp224,Val370,Met374and heme in human aromatase active site.The hydrogenbonds are labeled as yellow broken lines.The IC50values are listed in Table2.B.Sun et al./Bioorg.Med.Chem.18(2010)5352–53665359。

sigma

sigma
rizabilities of the nucleon studied in the linear sigma model
Institut fur Kernphysik, Johannes Gutenberg-Universitat Mainz, J. J. Becher-Weg 45, D-55099 Mainz, Germany (June 3, 1996)
2 2 2 2 2 2 2 2 2 2
2
takes account of a number of nucleon resonances and of and correlated 2 exchange in the t channel. Our calculation is based on the pion{nucleon linear sigma model (LSM) 20] in the limit of an in nite sigma mass. Though this model is not a very realistic description of the nucleon, it has built in all the relevant symmetries like Lorentz, gauge and chiral invariance. In particular it ful lls the PCAC relation, which is quite crucial to describe hadron physics at low energies. Evaluating the spin{independent amplitude of VCS in the one{loop approximation we have determined the generalized scalar polarizabilities of the nucleon. In particular, this leads to the rst model prediction of these quantities for the neutron.

对称性与守恒定律

对称性与守恒定律
SU(2)是u,d夸克对称,破坏2--3% SU(3)SU(4)SU(5)SU(6) 同位旋破坏主要来自多重态不同分量质 量差印起的运动学效应
奇异数(Strangeness)和重 子数
1947年宇宙线实验(after pion),1954年
加速器实验发现一批奇异粒子(photos)
特性一:协同产生,独立衰变
ant,自旋相同,所 有内部相加性量子数反号。反粒子就是 自己的称Majorana 粒子
Charge Conjugation
C A C' ( A) A ,C' ( A)为相因子
C变换性质:CC=1
若Q为相加性守恒量,
QC A QC' ( A) A Q' ( A)C' ( A) A
在费米尺度,强作用比EM作用强2-3数量 级,其强作用性质相似。
介子
Particles J Q mass
I_3
pi+
0 1 139.56
1
pi0
0 0 1 34.97
0
pi-
0 –1 139.56 -1
所有强子都有确定的同位旋!
与自旋类似,粒子内部抽象空间角动量
强作用同位旋守恒意味着I, I_3守恒
Rho介子通过强作用衰变到三个pion严格警戒, Rho0 通过EM作用到两 gamma严格警戒 自旋必为奇数。
Pion-Nucleon Scattering
同位旋守恒给出很强的限制和预言
(pi+,pi0,pi-) + (p,n)共10个反应道(电荷 守恒),互相独立!?
时间反演不变--》8个独立
同位旋空间转动不变(I_3变号)--》4个 独立,两个独立振幅(复数)

The_Retraining_Churn_Data_Mining_Model_in_DMAIC_Ph

The_Retraining_Churn_Data_Mining_Model_in_DMAIC_Ph

Journal of Communication and Computer 10 (2013) 1063-1069The Retraining Churn Data Mining Model in DMAIC PhasesAndrej TrnkaDepartment of Mass Media Communication, Faculty of Mass Media Communication, University of SS. Cyril and Methodius, Trnava 917 01, Slovak RepublicReceived: May 30, 2013 / Accepted: July 01, 2013 / Published: August 31, 2013.Abstract: Six Sigma is a rigorous, focused, and highly effective implementation of proven quality principles and techniques. A company’s performance is measured by the sigma level of their business processes. Traditionally companies accepted three or four sigma performance levels as the norm. The Six Sigma standard of 3.4 problems-per-million opportunities is a response to the increasing expectations of customers. DMAIC is an acronym for five phases of Six Sigma methodology: Define, Measure, Analyze, Improve, Control. This paper describes possibility of using Bayesian Network for retraining data mining model. Concrete application of this proposal is in the field of the churn. Churn is a derivation from change and turn. It can be defined as a discontinuation of a contract. Data mining methods and algorithms can predict behavior of customers. We can get better results using Six Sigma methodology. The goal of this paper is proposal of implementation churn (with Bayesian network) to the phases of Six Sigma methodology.Key words: Bayesian network, data mining, DMAIC, Churn, Six Sigma.1. IntroductionSix Sigma methodology and its phases (Fig. 1) have been widely adopted by industries and non-profit organizations throughout the world. Six Sigma methodology was first espoused by Motorola in the mid-1980s. The successful implementation of the Six Sigma program in Motorola led to huge benefits. Motorola recorded a reduction in defects and manufacturing time, and also began to reap financial rewards. The Six Sigma has become the most prominent trend in quality management not only for manufacturing and service industries, but also for non-profit organizations and government institutes [1-5].The main target of Six Sigma is to minimize variation because it is somehow impossible toCorresponding author: Andrej Trnka, Ph.D., research fields: data mining, Six Sigma, statistical process control. eliminate it totally. Sigma (σ) in the statistical field is a metric used to represent the distance in standard deviation units from the mean to a specific limit. Six Sigma is a representation of six standard deviations from the distribution mean. If a process is described as within Six Sigma, the term quantitatively means that the process produces fewer than 3.4 DPMO (defects per million opportunities). Table 1 shows how exponential the sigma scale is between levels 1 and 6 [6].We can talk about Lean Six Sigma, too. Lean Six Sigma for services is a business improvement methodology that maximizes shareholder value by achieving the fastest rate of improvement in customer satisfaction, cost, quality, process speed, and invested capital [7]. But for our research, we can ignore the fast, so using the Six Methodology is proper.In our previous research we implemented selected data mining methods and algorithms to the DMAICphases of Six Sigma Methodology. The main area ofThe Retraining Churn Data Mining Model in DMAIC Phases1064Fig. 1 Representation of Six Sigma methodology by BPMN.Table 1 Six Sigma scale.Sigma DPMO Efficiency (%)1 691,462 30.9 2 308,538 69.13 66,807 93.34 6,210 99.45 233 99.986 3.4 99.9999966Source: Ref. [6].the implementation was manufacturing processes. But Six Sigma methodology can be used in customer services, too.Some authors have used data mining algorithms in manufacturing processes, but without Six Sigma methodology [8-11].Data mining is the process of discovering interesting patterns and knowledge from large amounts of data. The data sources can include databases, data warehouses, the Web, other information repositories, or data that are streamed into the system dynamically [12]. One of the data mining task is to predict the customer’s churn.2. ChurnMobile phone providers fight churn by detecting patterns of behavior that could benefit from new services, and then advertise such services to retain their customer base. Incentives provided specifically to retain existing customers can be expensive, and successful data mining allows them to be precisely targeted to those customers who are likely to yield maximum benefit [13]. Churn is defined as a discontinuation of a contract. Reducing churn is important because acquiring new customers is more expensive than retaining existing customers. In order to manage customer churn to increase profitability, companies need to predict churn behavior, however, this problem not yet well understood[14, 15].Churning customers can be divided into two main groups, voluntary and non-voluntary churners. Non-voluntary churners are the easiest to identify, as these are the customers who have had their service withdrawn by the company. There are several reasons why a company could revoke a customer’s service, including abuse of service and non-payment of service.Voluntary churn is more difficult to determine,because this type of churn occurs when a customer makes a conscious decision to terminate his/her service with the provider. Voluntary churn can beThe Retraining Churn Data Mining Model in DMAIC Phases 1065sub-divided into two main categories, incidental churn and deliberate churn.Incidental churn happens when changes in circumstances prevent the customer from further requiring the provided service. Examples of incidental churn include changes in the customer’s financial circumstances, so that the customer can no longer afford the service, or a move to a different geographical location where the company’s service is unavailable. Incidental churn usually only explains a small percentage of a company’s voluntary churn. Deliberate churn is the problems that most churn management solutions try to battle. This type of churn occurs when a customer decides to move his/her custom to a competing company. Reasons that could lead to a customer’s deliberate churn include technology-based reasons, when a customer discovers that a competitor is offering the latest products, while their existing supplier can not provide them. Economic reasons include finding the product at a better price from a competing company. Examples of other reasons for deliberate churn include quality factors such as poor coverage, or possibly bad experiences with call centers [16-17].3. Data Mining ModelFor our research we used IBM SPSS Modeler 14. Telecommunications provider is concerned about the number of customers it is losing to competitors. Historic customer data can be used to predict which customers are more likely to churn in the future. These customers can be targeted with offers to discourage them from transferring to another service provider.This model focuses on using an existing churn data to predict which customers may be likely to churn in the future and then adding the following data to refine and retrain the model [18].Fig. 2 shows the built model in IBM SPSS Modeler, which contains the historical data.In analysis we used two data sets. These data sets had identical structure of variables. First data set contained 412 rows (records) and the second data set contained 451 rows.Fig. 2 Summary model for churn.The Retraining Churn Data Mining Model in DMAIC Phases 1066The first analysis with Feature Selection showed that several variables were unimportant when predicting churn. These variables were filtered from data set to increase the speed of processing when the model is built.The step in analysis is using Bayesian networks to predict the churn. A Bayesian network provides a succinct way of describing the joint probability distribution for a given set of random variables. In our analysis we used Tree Augmented Naive Bayes. This algorithm is used mainly for classification. It efficiently creates a simple Bayesian network model. The model is an improvement over the naive Bayes model as it allows for each predictor to depend on another predictor in addition to the target variable. Its main advantages are its classification accuracy and favorable performance compared with general Bayesian network models. Its disadvantage is also due to its simplicity; it imposes much restriction on the dependency structure uncovered among its nodes [19]. After learning the model from first data set we attached the second data set and we trained the existing model.4. ResultsTo compare and evaluate the generated models we had to combine the two data sets. The generated Bayesian Network model shows two columns. The first column contains a network graph of nodes that displays the relationship between the target and its most important predictors. The second column indicates the relative importance of each predictor in estimating the model, or the conditional probability value for each node value and each combination of values in its parent nodes.Fig. 3 shows relationship between the target variable. Due to confidentiality of provider data, we changed the names of variables and we used generic names of variables.Fig. 4 shows predictors (variables) importance.To display the conditional probabilities for any node, it is necessary to click on the concrete node and the conditional probability is generated. Fig. 5 shows conditional probability for most important variable—variable 2.To check how well each model predicts churn, we used an analysis node. This node shows the accuracyin terms of percentage for both correct and incorrect predictions. The analysis shows that both models havea similar degree of accuracy when predicting churn. Tables 2-5 show results for output variable churn.For the other view to data analysis we usedFig. 3 Created Bayesian network.The Retraining Churn Data Mining Model in DMAIC Phases1067Fig. 4 Predictors importance.Fig. 5 Conditional probability of variable 2.Table 2 Comparing churn_1 with churn.Total 863 Correct 654 75.78% Wrong 209 24.22%Table 3 Comparing churn_2 with churn.Total 863Correct 655 75.9% Wrong 208 24.1%Table 4 Agreement between churn_1 and churn_2.Total 863Correct 682 79.03% Wrong 181 20.97%Table 5 Comparing agreement with churn.Total 682Correct 565 82.84% Wrong 117 17.16%evaluation graph to compare the model’s predictedaccuracy by building a gains chart. Fig. 6 shows evaluating model accuracy.The graph shows that each model type produces similar results. However, the retrained model (churn_2) using both data sets is slightly better because it has a higher level of confidence in its predictions. Therefore, we used another algorithm ofBayesian network—Markov Blanket.The Markov Blanket [19] for the target variable node in a Bayesian network is the set of nodescontaining target’s parents, its children, and its children’s parents. Markov blanket identifies all theFig. 6 Evaluation graph of analysis (TAN Bayes Network).The Retraining Churn Data Mining Model in DMAIC Phases1068Fig. 7 Evaluation graph of analysis (Markov Blanket Bayes Network).Fig. 8 Churn in proposed control phase. variables in the network that are needed to predict the target variable. This can produce more complex networks, but also takes longer to produce. Using feature selection preprocessing can significantly improve performance of this algorithm.Fig. 7 shows the same analysis, but with using Markov Blanket algorithm. The evaluation graph shows that churn_2 has higher level of confidence than churn_2 with TAN Bayes Network.5. ConclusionsThe churn can be implemented to the DMAIC phases of Six Sigma methodology. We suggest implementing churn in to the control phase with message event to the step process control. Fig. 8 shows proposed place of churn in control phase.The red tasks and gateways represent our origin proposal. The green task churn is the new proposed task in Control phase of DMAIC.AcknowledgmentsThis paper supports the project VEGA 1/0214/11. Grateful acknowledgment for translating the English edition goes to Juraj Mistina. The results of this article were published in World Congress on Engineering and Computer Science 2012, WCECS 2012, San Francisco, USA, October 24-26, 2012.References[1] C.C. Yang, Six Sigma and Total Quality Management, in:Quality Management and Six Sigma, ed. A Coskun, Croatia, Sciyo, 2010.[2] J. Antony, R. Banuelas, Key ingredients for the effectiveimplementation of Six Sigma Program, Measuring Business Excellence 6 (2002) 20-27.[3] H. Wiklund, P.S. Wiklund, Widening the Six Sigmaconcept: An approach to improve organizational learning, Total Quality Management 13 (2002) 233-239.[4] L. Sandholm, L. Sorqvist, 12 requirements for Six Sigmasuccess, Six Sigma Forum Magazine 2 (2002) 17-22. [5] Ch.CH. Yang, An integrated model of TQM and GE SixSigma, International Journal of Six Sigma and Competitive Advantage 1 (2004) 97-111.[6] B. El-Haik, A. Shaout, Software Design for Six Sigma,John Wiley & Son, Hoboken, New Jersey, 2010.The Retraining Churn Data Mining Model in DMAIC Phases 1069[7]W. Bentley, P.T. Davis, Lean Six Sigma Secrets for theCIO, CRC Press, Boca Raton, Florida, 2010.[8]M. Kebisek, P. Schreiber, I. Halenar, Knowledgediscovery in databases and its application in manufacturing, in: Proceedings of the InternationalWorkshop “Innovation Information Technologies: Theory and Practice”, Dresden, Sep. 6-20, 2010, pp.204-207.[9]R. Halenar, Matlab possibilities for real time ETL method,Acta Technica Corviniensis: Bulletin of Engineering 5(2012) 51-53.[10]P. Vazan, P. Tanuska, M. Kebisek, The data miningusage in production system management, World Academy of Science, Engineering and Technology 7(2011) 1304-1308.[11]M. Kudla, M. Stremy, Alternatívne metódy ukladaniapološtruktúrovaných dát (Alternative methods for storingsemi-structured data,), in: Applied Natural Sciences 2007,International Conference on Applied Natural Sciences,Trnava, Slovak Republic, Nov. 7-9, 2007, pp. 404-409. [12]J. Han, M. Kamber, J. Pei, Data Mining Concepts andTechniques, Elsevier, Waltham, Massachusets, 2012. [13]I. Witten, E. Frank, M. Hall, Data Mining PracticalMachine Learning Tools and Techniques, Elsevier, Burlington, Massachusets, 2012.[14]J. Ahn, S. Han, Y. Lee, Customer churn analysis: Churndeterminants and mediation effects of partial defection inthe Korean mobile telecommunications service industry,Telecommunications Policy 30 (2006) 552-568.[15]K.Ch. Lee, N.Y. Jo, Bayesian network approach topredict mobile churn motivations: Emphasis on generalBayesian network, Markov blanket, and what-if simulation, in: Second International Conference, FGIT2010, Jeju Island, Korea, Dec. 13-15, 2010.[16]H. Kim, C. Yoon, Determinants of subscriber churn andcustomer loyalty in the Korean mobile telephony market,Telecommunications Policy 28 (2004) 751-765.[17]J. Hadden, A. Tiwari, R. Rajkumar, D. Ruta, Churnprediction: Does technology matter?, International Journal of Electrical and Computer Engineering 1 (2006)397-403.[18]IBM SPSS Modeler 14.2 Applications Guide, IBM,2011.[19]IBM SPSS Modeler 14.2 Algorithms Guide, IBM, 2011.。

引用次数最多的100篇SCI文章

引用次数最多的100篇SCI文章

Rank Authors1Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.2Laemmli, U. K.3Bradford, M. M.4Sanger. F., Nicklen, S. & Couslon, A. R.5Chomczynski, P. & Sacchi, N.6Towbin, H., Staehelin, T. & Gordon, J.7Lee. C., Yang, W. & Parr, R. G.8Becke, A. D.9Folch, J., Lees, M. & Stanley, G. H. S.10Thompson, J. D., Higgins, D. G. & Gibson, T. J11Kaplan, E. L. & Meier, P.12Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D.13Sheldrick, G. M.14Altschul, S. F. et al.15Murashige, T. & Skoog, F.16Perdew, J. P., Burke, K. & Ernzerhof, M.17Folstein, M. F., Folstein, S. E. & McHugh, P. R.18Bligh, E. G. & Dyer, W. J.19Southern, E. M.20Saitou, N. & Nei, M.21Livak, K. J. & Schmittgen, T. D.22Shannon, R. D.23Otwinowski, Z. & Minor, W.24Cox, D. R.25Becke, A. D.26DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith27Reynolds, E. S.28Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & H 29Bland, J. M. & Altman, D. G.30Weber, K. & Osborn, M.31Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W.32Scatchard, G.33Baron, R. M. & Kenny, D. A.34Kohn, W. & Sham, L. J.35Mosmann, T.36Iijima, S.37Fiske, C. H. & Subbarow, Y38Davis, B. J.39Hohenberg, P. & Kohn, W.40Feinberg, A. P. & Vogelstein, B.41Felsenstein, J.42Grynkiewicz, G., Poenie, M. & Tsien, R. Y.43Kresse, G. & Furthmüller, J.44O’Farrell, P. H.45Tamura, K., Dudley, J., Nei, M. & Kumar, S.46Zadeh, L. A.47Sheldrick. G. M.48McKhann, G. et al.49Monkhorst, H. J. & Pack, J. D50Burton, K.51Radloff, L. S.52Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. 53Hamilton, M.54Beck, A. T., Ward, C. H., Mendelsohn, M., Mock, J. & Erbaugh, 55Kyte, J. & Doolittle, R. F.56Gornall, A. G., Bardawill, C. J. & David, M. M.57Dempster, A. P., Laird, N. M. & Rubin, D. B.58Metropolis, N., Rosenbluth, A. W., Rosenbluth, M. N., Teller, A. 59Benjamini, Y. & Hochberg, Y.60Smith, P. K. et al.61Oldfield, R. C.62Friedewald, W. T., Levy, R. I. & Fredrickson, D. S.63Saiki, R. K. et al.64Duncan, D. 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H., Wilk, L. & Nusair, M.97Köhler, G. & Milstein, C.98Matthews, D. R. et al.99Brunauer, S., Emmett, P. H. & Teller, E.100Ronquist, F. & Huelsenbeck, J. P.101Ross, R.Numbers from Thomson Reuters; extracted 7 October 2014TitleProtein measurement with the folin phenol reagent. Cleavage of structural proteins during the assembly of A rapid and sensitive method for the quantitation of mi DNA sequencing with chain-terminating inhibitors.Single-step method of RNA isolation by acid guanidini Electrophoretic transfer of proteins from polyacrylamid Development of the Colle-Salvetti correlation-energy f Density-functional thermochemistry. III. 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10.1137/0111030/10.1137/011103010.1016/0927-0256(96)00008-0/10.1016/0927-0256(96)00008-010.1038/353737a0/10.1038/353737a010.1016/S0022-5320(69)90033-/10.1016/S0022-5320(69)90033-110.1093/nar/28.1.235/10.1093/nar/28.1.23510.1103/PhysRevB.45.13244/10.1103/PhysRevB.45.1324410.1093/nar/7.6.1513/10.1093/nar/7.6.151310.1107/S0108767390010224/10.1107/S010876739001022410.1139/p80-159/10.1139/p80-15910.1038/256495a0/10.1038/256495a0/article/10.1007%2FBF00280883#page-1 10.1021/ja01269a023/10.1021/ja01269a02310.1093/bioinformatics/btg1/10.1093/bioinformatics/btg180/10.1056/NEJM19990114340020710.1056/NEJM19990114340020SubjectBiology lab technique Biology lab technique Biology lab technique Biology lab technique Biology lab technique Biology lab technique Physical chemistry Physical chemistry Biology lab technique Bioinformatics Medical statistics Bioinformatics Crystallography Bioinformatics Biology lab technique Physical chemistry Psychology/psychiatry Biology lab technique Biology lab technique Phylogenetics Biology lab technique Physical chemistry Crystallography Medical statistics Physical chemistry Biology lab technique Biology lab technique Bioinformatics Medical statistics Biology lab technique Biology lab technique Biology lab technique Psychology/psychiatry Physical chemistry Biology lab technique PhysicsBiology lab technique Biology lab technique Physical chemistry Biology lab technique Phylogenetics Biology lab technique Physical chemistry Biology lab technique Phylogenetics Mathematics/statisticsCrystallography MedicinePhysicsBiology lab technique Psychology/psychiatry Biology lab technique Psychology/psychiatry Psychology/psychiatry Biology lab technique Biology lab technique Mathematics/statistics Mathematics/statistics Mathematics/statistics Biology lab technique Psychology/psychiatry MedicineBiology lab technique Mathematics/statistics PhysicsBiology lab technique Biology lab technique Mathematics/statistics Crystallography Mathematics/statistics Crystallography Medicine Mathematics/statistics Biology lab technique Bioinformatics Bioinformatics Physical chemistry Biology lab technique Biology lab technique Physical chemistry Biology lab technique Crystallography Crystallography Biology lab technique Physical chemistry Physical chemistry Biology lab technique Mathematics/statistics Physical chemistry Physical chemistry Biology lab technique Crystallography Physical chemistryBiology lab technique Crystallography Physical chemistry Biology lab technique MedicinePhysics Phylogenetics Medicine。

美国和加拿大的生物科学业务:美勒普希格马克说明书

美国和加拿大的生物科学业务:美勒普希格马克说明书

Inorganicson tapWithdrawal systems and safety accessories for acids & basesThe life science business of Merck operates as The life science business of Merck operates as MilliporeSigma in thehealth acids and bases is through the use hazard class. Always use appropriateParameter Canister 25 L Fassett ® 25 L Height 48.8 cm 50 cm Width 24.2 cm 28.5 cm Depth 29.5 cm 32.9 cm Volume 27 L 30 L Filling quantity 25 L 25 L Weight (empty) 1.25 kg 1.5 kg Number per pallet 118Openings KS 60 × 6CCS 60x6MaterialPEPEManual withdrawal system for acids and bases (PE)•Made of specially tested high purity polyethylene (PE)•Suitable for use with all acids and bases (except HNO 3 and H 2SO 4)Manual withdrawal systems for acids & basesManual withdrawal system specially for Nitric acid and Sulfuric acid (PVDF)•Made of specially tested high purity polyvinylidene fluoride (PVDF)•Developed specifically for use with aggressive acids, e.g. HNO 3 and H 2SO 4Features & Benefits:•Unique concept allows safe and easy withdrawal of chemicals, preventing accidental contact with contents and vapors •Flexible, lightweight withdrawal systems with integrated outlet valve and individual pressurizing options •Integrated check valve protects the pump ball from chemical vapors •Integrated venting system avoids vacuum development•No operating supplies required: manual pressure buildup by hand or foot pump ball •Lower costs through use of larger volumes of 10 l or moreTechnical DataPump ballSqueeze to pressurizeOutlet valveRotate to dispense Outlet valve Open ClosedCheck proper operationOpen the container*Insert dip tube and tighten*Check outlet valve is closed.Screw in dispensing head and tightenPlace receptacle under the outletand open the outlet valvePressurize by squeezing the red pump ball and fill the receptacleClose outlet valveSafe withdrawal in 8 simple steps*use drum key 1.67503.0001(Always follow local safety regulations and the detailedinstructions provided in the manual of the withdrawal system in use.)Parameter PE drum 200 L Height 93.5 cm Diameter 58.5 cm Volume 220 L Filling quantity 200 L Weight (empty)8.4 kg Number per pallet 2Openings S70 × 6 and S38 × 6MaterialPlug: PP white Gasket: PE blueParameter Combi drum (metal/PE) 25 L*Combi drum(metal/PE) 180 L*Heigh 52 cm 88.5 cm Diameter 29 cm 58.8 cm Volume 28 L 203 L Filling quantity 25 L 180 L Weight (empty) 3.4 kg 22 kg Number per pallet S56 x 4 2 x S56 x 4Openings S56 × 62 × S56 × 6Material Steel with PE inlinerSteel with PE inliner*With PE inlinerNEWIntegrated check valveProtects pump ball from chemical vaporsa c i d s a n db a s e s , m a n u a l p r e s s u r e b u i l d -u pD i s p e n s e h e a d (P V D F ) f o r N i t r i c a c i d a n d S u l f u r i c a c i d , m a n u a l F o o t p u m p b a l l f o r d i s p e n s e h e a d s 167500 a n d 167501D i p t u b e (P V D F ) f o r a c i d s a n d b a s e s i n 25 l F a s s e t t s ®m k e y (P E ) f o r n i n g /c l o s i n g t a i n e r s w i t h 2” o r ” s c r e w c a p s m a d e o f s t i c m a t e r i a l sn t a i n e r k e y f o r D i p t u b e (P V D F ) f o r S u l p h u r i c a c i d i n 25 L c a n i s t e r sMerck KGaAFrankfurter Strasse 250 64293 Darmstadt, GermanyLit. No. PB8651EN00 Ver. 1.02017 - 06034© 2017 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Merck,the Vibrant M, Sigma-Aldrich, EMSURE, EMPARTA and EMPLURA are trademarks of Merck KGaA,Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.To place an order or receive technical assistanceOrder/Customer Service: /order Technical Service: /techserviceSafety-related Information: /safetycenter /safety-accessories。

Milligard PES 过滤器 Steam-in-Place 熔化疗灭菌推荐指南说明书

Milligard PES 过滤器 Steam-in-Place 熔化疗灭菌推荐指南说明书

Tech NoteRecommendations for Steam-in-Place Sterilization of Filtration Trains containing Milligard® PES PrefiltersIntroductionSteam-in-place (SIP) sterilization is commonly usedfor process equipment such as piping, housings, bioreactors, and filtration trains used in the manufacture of biotechnology products.Milligard® PES cartridge and capsule filters are designed to be used as stand-alone filters for reducing levels of bioburden and particulates or as prefilters, protecting downstream sterilizing filters from premature plugging. These filters can be sterilized by SIP, autoclave, and gamma irradiation. However, if prefilter integrity willbe assessed, it is recommended that Milligard® PES filters are wet before use. By contrast, our sterilizing-grade Durapore® and Millipore Express® filters are recommended to be dry before SIP sterilization. The different recommended wetting requirements for these filters prompted additional studies to clarifybest practices for SIP sterilization of a filtration train containing a Milligard® PES cartridge prefilter upstream of Millipore Express® or Durapore® sterilizing-grade cartridge filters.Materials and MethodsFilter conditions for SIP TestsAll SIP testing was performed using 10 in. cartridge filters containing Milligard® PES 1.2/0.2 µm nominal membrane upstream of a 10 in. cartridge filter containing either Millipore Express® SHR with prefilter (Millipore Express® SHRp) or Durapore® 0.22 µm membranes.Table 1 summarizes four filter conditions and methods that were evaluated based on whether Milligard® PES prefilter and sterilizing-grade filter were wet or dry before SIP, whether the filters were SIP treated in series (Milligard® PES prefilter followed by sterilizing-grade filter), or whether a plumbing bypass wasset up around the Milligard® PES prefilter. After SIP sterilization, the integrity of all prefilters and sterilizing filters was confirmed using an automated Integritest®instrument.Table 1: Summary of filter conditions and SIP treatments1*Dry Dry Series2Wet Dry Series3Wet Dry Steam bypassfrom inletaround Milligard®PES filter todownstream filter 4Wet Wet Series* T his test was also performed with a cartridge filter containing Durapore®0.22 µm membrane in place of the Millipore Express® SHRp membrane.The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.Figure 1 shows the generalized setup of the prefilter and downstream filter in the filtration train. For all four SIP tests, steam at 135 °C was continuously introduced into the system through valve V1 at ‘heat up’ and during the 30-minute ‘temperature hold’ steps; steam exited through valve V7. At the end of the ‘temperature hold’, steam was stopped and sterile-filtered compressed air at ambient temperature was introduced into the system through valve V2. The compressed air was designed to cool the system and maintain sterility simulating potential customer applications. Compressed air pressures are listed within the discussion of each method. A drain and valve (V3) positioned upstream of the Milligard® PES prefilter removed system condensation. The downstream sterilizing-grade filter had a similar drain (mid-point drain) and valve configuration (V5) between prefilter and downstream filter. The drains upstream of each filter were critical elements of system setup to reduce the risk of filter damage due to water condensation.V4 and V6 are vent valves that relieved any differential pressure that built up during the sterilization operation, also protecting the filters from damage.Temperature and pressure were measured throughout the system with multiple thermocouples (TC) and pressure transmitters (PT). During these studies, all valves were operated manually and were adjusted to minimize temperature and pressure differentials across the system.Confirmation of Flow PerformanceFlow rates of cartridge filters containing the three pore sizes of Milligard® PES membrane or sterilizing-grade Millipore Express® SHRp membrane were measured at a constant pressure of 10 psi; all membranes were dry before water flow rate measurement. Following testing, filters were dried, SIP sterilized (dry prefilter, dry sterilizing-grade filter) and then water flow rates were re-measured at 10 psi.After SIP sterilization, the integrity of all filters was confirmed using an automated Integritest® instrument.Results and DiscussionFilter Condition and Considerations for SIP TreatmentsIt is recommended that Milligard® PES prefilters are wet before SIP treatment, and we recognize this presentsa challenge when performing SIP sterilization with a dry sterilizing-grade filter. To clarify best practices for SIP sterilization of a filtration train containing Milligard®PES filters, four methods were compared for performing SIP treatment with filtration trains containing Milligard® PES prefilters and Millipore Express® SHRp filters. These methods comprised different wet or dry conditions for the prefilters and sterilizing-grade filters, both serial SIP of the filters in the filtration train, as well as SIP where a bypass was connected aroundthe Milligard® PES prefilter. The bypass enabled bi-directional steaming of the Milligard® PES filter and forward steaming of the sterilizing-grade filter at the same time, reducing the pressure differential across the wet Milligard® PES membrane.SIP sterilization includes three phases: ‘heat up’,‘temperature hold’, and ‘cool down’. The minimum temperature for sterilization is 121 °C (250 °F) witha hold of at least 30 minutes. The impact of the SIP test method on both temperature and pressure across the filtration train are summarized below. After SIP treatment and flow rate tests, both the Milligard® PES prefilters and sterilizing-grade filters passed filter integrity tests.Steam inFigure 1: SIP Sterilization Test System Schematic. The prefilter in this schematic was Milligard® PES 1.2/0.2 µm nominal and the sterilizing-grade filter was either a Millipore Express® SHRp or a Durapore® 0.22 µm filter.23Figure 2A and B show the temperature and pressure profiles, where the Milligard ® PES 1.2/0.2 µm nominal prefilter and Millipore Express ® SHRp sterilizing filter were both dry before SIP treatment. This method was also performed with a Durapore ® 0.22 µm sterilizing-grade filter downstream of the Milligard ® PES prefilter. Test results with the two different sterilizing-grade filters were similar and, as Millipore Express ® SHRp membrane's bubble point is higher than that ofDurapore ® 0.22 µm membrane, it represents ‘worst-case’ for the SIP study, so results with this membrane are shown in this report.Temperature Profiles & Differential Pressure Across System:• System heated up quickly (<20 minutes), and the temperature was consistent throughout the system. The temperature stabilized at 135 ºC, above the 121 ºC threshold. The cooling compressed airpressure was 10-15 psi, although it could be set to any pressure, and the whole system cooled down to ambient temperature in ~45 minutes.• Differential pressure was less than 1 psi across both filters during the whole SIP method, minimizing risk of filter damage.Implementation:• The drain valve (V5) between the filters was open to minimize the push of condensation to the downstream filter. Minimal valve adjustments needed.Recommendation:• We recommend wetting Milligard ® PES filters before SIP or integrity testing. However, if the Milligard ® PES filter does not need to be integrity tested, the filters in the filtration train do not need to be wet before SIP sterilization.0.020.040.060.080.0100.0120.0140.006051015202530354045505565707580859095T e m p e r a t u r e (ºC )Time (elapsed, 5-minute intervals)TC 01 prefilter inlet lowTC 02 prefilter inlet high TC 04 prefilter core top TC 05 sterilizing filter inlet low TC 06 sterilizing filter inlet high TC 07 sterilizing filter core outletTC 08 sterilizing filter core topTC 10 test system midpointTC 11 test system outletTC 12 prefilter core outTemperature holdHeat upCool down0.05.010.015.020.025.030.035.006051015202530354045505565707580859095P r e s s u r e (p s i )Time (elapsed, 5-minute intervals)PT 01 prefilter upstream (high)PT 03 Test system midpointPT 05 sterilizing filter upstream (high)PT 06 test system outletTemperature holdHeat upCool downFigure 2A: Method 1 SIP Temperature ProfileFigure 2B: Method 1 SIP Pressure Profile4Figure 3A and B show the temperature and pressure profiles measured when the Milligard ® PES 1.2/0.2 µm nominal prefilter was wet and Millipore Express ® SHRp sterilizing filter was dry before SIP sterilization in series. Temperature Profiles & Differential Pressure Across System:• During heat up, there were temperature delays in the system and although the temperature stabilized at 133-135 ºC above the 121 ºC threshold, there was ~1 ºC temperature difference across theprefilter during the temperature hold. There was no temperature difference across the Millipore Express ® SHRp filter. • Differential pressure across the Milligard ® PES prefilter reached ~7 psi during heat up and reduced to ~2-3 psi during the temperature hold. There was no pressure difference across the Millipore Express ® SHRp filter. • Cool down air pressure was initially set at ~15 psi, however, as the Milligard ® PES filter was wet, air could not blow through the prefilter to the downstream filter. To cool the system, air pressure was increased to 50 psi, above the Milligard ® PES filter's air water bubble point, to cool down the system and maintain system sterility.Implementation:• To cool down the system, inlet air pressure should be set above the prefilter membrane bubble point: recommended cool down air pressure for Milligard ® PES 1.2/0.2 µm nominal filters is 50-60 psi; forMilligard ® PES 1.2/0.45 µm filters is 40-50 psi, and for Milligard ® PES 1.2/0.8 µm filters is 25-35 psi. • Valve adjustments, especially at the heat up step, were necessary to minimize differential pressure across the filters.• The drain valve (V5) between the filters must be open to minimize the push of condensation to the downstream filter. Recommendation:• This method is feasible but not recommended for SIP sterilization of filtration trains containing Milligard ® PES prefilters.6051015202530354045505565707580850.020.040.060.080.0100.0120.0140.0T e m p e r a t u r e (ºC )Time (clock 30-second intervals)Temperature holdHeat upCool downTC 01 prefilter wet upstream high TC 02 prefilter wet upstream low TC 04 prefilter wet core top TC 05 sterilizing filter upstream low TC 06 sterilizing filter upstream high TC 07 sterilizing filter core top TC 08 sterilizing filter core outletTC 10 Test system mid pointTC 11 Test system outletTC 12 prefilter wet core outlet-5.05.015.025.035.045.055.00605101520253035404550556570758085P r e s s u r e (p s i )Time (clock 30-second intervals)Temperature holdHeat upCool downPT 01 prefilter wet upstreamPT 02 Test system mid pointPT 03 sterilizing filter dry upstreamPT 04 Test system outletFigure 3A: Method 2 SIP Temperature ProfileFigure 3B: Method 2 SIP Pressure Profile. Some traces are not visible as the trend lines are superimposed. The circled area indicates no air flow through the wet Milligard ® PES filter.5The Milligard ® PES prefilter was prewet and the Millipore Express ® SHRp filter was dry as with Method 2. However, rather than the filters being SIP treated in series, a bypass was connected from the steam inlet, around the wet Milligard ® PES prefilter, and reconnected to the system before the mid-point drain. This configuration enabled both the prefilter and sterilizing filter to besteamed simultaneously and minimized pressure buildup across the wet prefilter. Figure 4A and B show the measured profiles.Temperature Profiles & Differential Pressure Across System:• Heat up was rapid and temperature was consistent in the whole system during the temperature hold. The cooling compressed air pressure does not need to be above the prefilter's membrane bubble point. Onedisadvantage was the slow cool down of the Milligard ® PES prefilter, a result of bi-directional air flow without convection. • No pressure differences were measured, resulting in minimal risk to both the prefilter and sterilizing filter.Implementation:• Steam went through the filters simultaneously resulting in rapid heat up and consistent system temperature. Milligard ® PES prefilter received bi-directional steam and air.• The drain valve (V5) before the mid-point drain was partially open to minimize the likelihood thatcondensation reached the downstream sterilizing-grade filter.• Minimum valve adjustments were needed. Recommendation:• If Milligard ® PES prefilter is to be integrity tested after use, it is recommended that this method is used for SIP sterilization of filtration trains containing Milliard ® PES prefilters.12060510152025303540455055657075808590951001051101150.020.040.060.080.0100.0120.0140.0T e m p e r a t u r e (ºC )Time (5 min intervals)Temperature holdHeat upCool downTC 01 prefilter wet upstream lowTC 02 prefilter wet upstream high TC 04 prefilter wet core top TC 06 sterilizing filter dry upstream upper TC 06 sterilizing filter dry upstream lower TC 07 sterilizing filter dry core outlet TC 08 sterilizing filter dry core topTC 10 Test system midpointTC 11 test system outletTC 12 prefilter wet core outlet0.05.010.015.020.025.030.035.040.045.050.001206051015202530354045505565707580859095100105110115P r e s s u r e (p s i )Time (5 min intervals)Temperature holdHeat upCool downPT 01 prefilter wet upstreamPT 03 Test system midpointPT 05 sterilizing filter dry upstreamPT 06 Test system outletFigure 4A: Method 3 SIP Temperature ProfileFigure 4B: Method 3 SIP Pressure Profile. Some traces are not visible as the trend lines are superimposed.6Both the Milligard ® PES prefilter and Millipore Express ® SHRp filters were wet before SIP treatment. This method is only possible when two sterile compressed air inlets are available. Figure 5A and B show the measured profiles.Temperature Profiles & Differential Pressure Across System:• Temperature differences were measured throughout the system during heat up and temperature hold steps. Although the entire system exceeded the requirement for sterilization (121 ºC for 30 minutes), temperature stabilization was slow (~45 minutes).• Pressure differences were measured across the filters, increasing the risk of filter damage: during heat up the pressure differential across the prefilter reached 10 psi, which reduced to 5 psi during the temperature hold.Implementation:• Multiple manual adjustments of vent valves werenecessary to minimize differential pressure across the filters and possible filter damage. • During cool down, the cooling inlet air pressure was set to >50 psi, above the Milligard ® PES prefilter's bubble point. Because the Millipore Express ® SHRp filtermembrane's air water bubble point (>90 psi) is higher than the normal housing air pressure, a second sterile compressed air supply was required downstream of the sterilizing filter to maintain system sterility.• The mid-drain valves (V5) must be open to minimize the likelihood that condensation reaches the downstream sterilizing-grade filter.Recommendation:• This method requires more manual control, an additional sterile compressed air source for cooldown, and risks filter damage due to high differential pressures. Based on these considerations, we do not recommend this method for SIP sterilization of filtration trains containing Milliard ® PES prefilters.60510152025303540455055657075808590951001051100.020.040.060.080.0100.0120.0140.0T e m p e r a t u r e (ºC )Time (5 min intervals)Temperature holdHeat upCool downTC 01 prefilter wet upstream low TC 02 prefilter wet upstream high TC 04 prefilter wet core top TC 05 sterilizing filter wet upstream high TC 06 sterilizing filter wet upstream low TC 07 sterilizing filter wet core out TC 08 sterilizing filter wet core topTC 10 test system midpointTC 11 test system outletTC 12 prefilter wet core outlet-5.015.035.055.075.095.0115.006051015202530354045505565707580859095100105110P r e s s u r e (p s i )Time (5 min intervals)Temperature holdHeat upCool downPT 01 prefilter wet upstreamPT 02 Test system midpointPT 03 sterilizing filter wet upstreamPT 04 Test system outletFigure 5A: Method 4 SIP Temperature ProfileFigure 5B: Method 4 SIP Pressure ProfileConfirmation of Post SIP Flow Rate PerformanceWater flow rates of cartridge filters containingMilligard ® PES membranes of different pore sizes were tested before and after SIP treatment, to confirm the sterilization procedure did not impact filtration performance, Figure 6.Flow rates for all filters containing Milligard ® PES membranes were the same before and after SIPoperation, confirming the sterilization operation, even with dry membranes, did not impact flow rate.Measurements of filter integrity following SIPtreatment indicated that all filters met the bubble point specifications for integral filters, even though they were sterilized dry.However, in general, when integrity testing is to be performed on Milligard ® PES filters, it is highlyrecommended to thoroughly wet the filters to minimize potential risks to filter integrity measurements that could result from incomplete filter wetting. In thetests described here, integrity measurements were not impacted, but that may not always be the case.SummaryMilligard ® PES cartridge filters can be sterilized by SIP as part of a filtration train with Durapore ® or Millipore Express ® sterilizing-grade cartridge filters. Four different approaches to SIP were tested and the conclusions are outlined below.• If the Milligard ® PES prefilter is to be integrity tested after SIP or use, it is highly recommended that the prefilter is wet before SIP or autoclavesterilization treatments 1. SIP treatment should ideally be performed using a bypass connection around the prefilter, and the system setup should includedrainage between the wet Milligard ® PES prefilter and dry sterilizing filter, as outlined in Method 3. • If the Milligard ® PES prefilter does not need to beintegrity tested after use, the prefilter can be dry and the sterilizing filter must be dry before SIP. In these instances, the dry prefilter and sterilizing filter can be SIP sterilized in series using the procedures outlined in Method 1.Whichever approach is adopted, each customer should perform studies under their own process conditions to confirm their sterilization operations are compatible with measurements of filter integrity.References1. Wetting Instructions, Integrity Testing, Sterilizing and DryingGuidelines. Filters with Milligard ® PES Membrane. MS_UG1375EN, Rev 2, 7/2019.50100150200250Millipore Express ®SHRpMilligard ® PES 1.2/0.2 μm nominalMilligard ® PES 1.2/0.45 μm Milligard ® PES 1.2/0.8 μmF l o w R a t e (L /M i n )Flow Rates before and after SIP SterilizationFigure 6: Flow rates of filters before and after SIP. In all cases, dry filters were subjected to SIP.© 2020 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Merck, the vibrant M, Millipore, Milligard, Millipore Express, Durapore, and Integritest are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.Lit. No. MK_TN5022EN Ver. 1.02019-2588401/2020Merck KGaAFrankfurter Strasse 250 64293 Darmstadt, GermanyTo place an order or receive technical assistancePlease visit /contactPSFor additional information, please visit 。

浙江纯洁精密科技有限公司 MAT 人協力测试方法说明书

浙江纯洁精密科技有限公司 MAT 人協力测试方法说明书

Application NoteThe MAT is also mentioned by the FDA "Guidance For Industry – Pyrogen and Endotoxins testing: Questions and Answers" as an alternative to the rabbit pyrogen test which should be validated according to USP <1225>.Additionally, the USP <151> Pyrogen Test mentions that, "A validated, equivalent in vitro pyrogen or bacterialendotoxin test may be used in place of the in vivo rabbit pyrogen test, where appropriate.”Principle of the MATThe monocyte activation test (MAT) is the human in vitro alternative to the rabbit pyrogen test, and allows the detection of the full range of pyrogens, including endotoxins and non-endotoxin pyrogens (NEPs). By putting the product to be tested in contact withhuman monocytic cells, it will mimic what happens in the human body: in presence of pyrogens, the monocytes are activated and produce cytokines such as interleukin-6.The cytokines are then detected using an immunological assay (ELISA) involving specific antibodies and an enzymatic color reaction.Detection of pyrogens in fetal bovine serum (FBS) with the PyroMAT ® SystemIntroductionWhat is a pyrogen?A pyrogen is, by definition, a substance that produces a rise in temperature in a human or animal. Pyrogens constitute a heterogeneous group of contaminants comprising microbial and non-microbial substances. The most widely known pyrogen is the endotoxin (LPS = Lipo-Polysaccharide), which is produced by gram-negative bacteria. Other microbial substances include those derived from gram-positive bacteria like Lipoteichoic Acid (LTA), particles from viruses and pyrogens originating from yeasts and fungi. Non-microbial pyrogenic substances can be rubber particles, microscopic plastic particles or metal compounds in elastomers.Why to conduct a pyrogen test?Pyrogenic substances in pharmaceutical products can induce life-threatening fever reactions after injection into the human body. Therefore, it is a regulatory requirement to test such products for pyrogens to ensure product quality and patient safety.Purpose of the test is to prove that the amount of pyro-gens contained in the product will not exceed a certain threshold, known as the contaminant limit concentration (CLC), that will guarantee the patient safety.The monocyte activation test (MAT) method has been qualified and validated for the detection of pyrogens by the European Center for the Validation of Alternative Methods (ECVAM) in 2005 and by the InteragencyCoordinating Committee on the Validation of Alternative Methods (ICCVAM) in 2008.It has been among the compendial methods for pyrogen detection in the European Pharmacopeia since 2010 (Chapter 2.6.30) [1].The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.2Principle of the PyroMAT ® SystemThe PyroMAT ® System uses cryo-preserved Mono-Mac-6 (MM6) human monocytic cells as a source of monocytes. The response to pyrogenic substances is determined by measurement of Interleukin-6 (IL-6) produced by the Mono-Mac-6 cells. For this purpose, the ELISA-microplate supplied in the kit is coated with an antibody specific to IL-6.IL-6 molecules released by MM6 cells supernatant during incubation phase are transferred in the ELISA plate, and bound by the immobilized primary antibody.A secondary antibody, linked to an enzyme, is added to form an IL-6 bound complex. After washing any unbound molecules, the IL-6 bound complex is detected in a color reaction started by the addition of an appropriate substrate.The color development is proportional to the amount of initial IL-6 production in the supernatant and measured with an absorbance reader.Quantification of pyrogens with the MATFor the quantification of the pyrogenic load of a sample, method A in accordance with the European Pharmacopeia [1] can be conducted.Method A involves a comparison of the preparation being examined with a standard endotoxin dose-response curve. The contaminant concentration of the preparation being examined is to be less than the CLC (Contaminant Limit Concentration) to pass the test.To ensure both the precision and validity of the test, preparatory tests need to be conducted to assure that:• The criteria for the endotoxin standard curve are satisfied • The solution does not interfere with the test • The test detects endotoxin and non-endotoxin contaminants • The solution does not interfere with the detection systemMaterial and EquipmentTo perform the MAT and a product specific validation, we recommend using:• PyroMAT ® Cells (Ref: Pyr0MATCELLS) • PyroMAT ® Kit (Ref: Pyr0MATKIT)• Reference Standard Endotoxin (Ref: 1.44161.0001). • NEP Control HKSA (Ref: MATHKSA)• NEP Control Flagellin (Ref: MATFLAGELLIN) • IL-6 control (Ref: Pyr0MATIL6)Additional equipment and consumables required:• Incubator, 37 °C, humidified • Water bath (37 °C)• Microplate reader to measure absorbance at 450 nm and 630 nm (reference wavelength)• Cryo-freezer (< -80 °C)• Freezer (-20 °C)• Refrigerator (2-8 °C)• Vortexer• 50 mL centrifuge• Multichannel pipettes with suitable containers• Adjustable pipettes: (10 μL – 100 μL; 100 μL – 1000 μL)with suitable sterile, pyrogen-free pipette tips • Pyrogen-free glass tubes• 2 mL endotoxin-free reaction tubes with caps • 50 mL endotoxin-free tubes with capsThe PyroMAT ® KitSpeak to a Specialist3Quantification of pyrogens with the PyroMAT ® systemPreparatory tests – Product Specific Validation (PSV)European pharmacopeia, chapter 2.6.30Before routine testing of a pharmaceutical product with MAT, a product specific validation (PSV) must be performed according to the method chosen for routine testing to ensure the validity of the criteria for theendotoxin standard curve, the detectability of endotoxin and non-endotoxin contaminants in the sample and that the sample does not interfere with the test or detection system.Interferences with the test can be removed by diluting the product up to a certain limit, referred to as the maximum valid dilution (MVD).The MVD is the maximum dilution factor at which it is still possible to detect the pyrogen limit (i.e., the CLC). It is directly linked to the limit of detection (LOD) of the system.The more sensitive the system is, the more the product can be diluted to remove interferences.The MVD of a test solution can be calculated using the following formula:MVD = CLC x CLODCLC = Contaminant Limit Concentration (EU/mg or EU/mL) C = Concentration of the test solution (mg/mL or mL/mL) LOD = Limit of Detection (EU/mL).The CLC is the acceptance criterion for the pass/fail decision, expressed in endotoxin equivalents per milligram or milliliter (EEU/mg or EEU/mL) or per unit of the biological activity of the product.It is calculated by the following expression:CLC = KMK = threshold pyrogenic dose per kilogram of body mass (EU/kg)M = maximum recommended bolus dose of product per kilogram of body mass (mg/kg or mL/kg).When the product is to be injected at frequent intervals or infused continuously, M is the maximum total dose administered in a single hour period.When testing for interfering factors, dilutions of the preparation being examined with geometric steps not exceeding the MVD should be performed. The same dilutions spiked with endotoxin at a justified concentration (in case of method A, a concentration near the estimated middle of the endotoxin standard curve) should then be performed.These dilutions must be tested in parallel in the same experiment and together with an endotoxin standard curve, which shall be used to calculate the concentration of endotoxin-equivalents in each solution.The mean recovery of the added endotoxin spike isthen calculated for each dilution. The test is considered free of interference when recovery of the addedendotoxin is within the range of between 50 and 200%.Where practicable, interference testing should beperformed on at least 3 different lots of the preparation being examined to investigate on possible batch-to-batch variation. If the interference cannot be removed by dilution or specific sample preparation of the product within the MVD range, Method C is preferred over Method A and B.For validation of the detection of non-endotoxincontaminants, historical batches that have been found to be contaminated with non-endotoxin contaminants causing positive responses in the rabbit pyrogens test or adverse drug reaction in man can be used. Where those batches are not available, validation should be done including at least 2 non-endotoxin ligands fortoll-like receptors, one of which is to be spiked into the preparation being examined.Once the optimum dilution of the preparation being examined has been identified, this dilution needs to be tested for interference in the detection system. The agreement between a dilution series in presence and absence of the preparation being examined is to be within ± 20% of optical density.Sample specifications: FBS (fetal bovine serum)FBS derived from clotted blood is the most widely used undefined supplement in eucaryotic, especially mammalian, cell culture. Though there are efforts to establish human or synthetic alternatives, fetalbovine serum still is a product that often is used in the production of vaccines and therapeutics.Being a raw material used within the production process of injectables administered to man, FBS has to be tested for harmful substances like endotoxin or other pyrogenic substances like (1 3)-β-d-glucan [2,3]. Quality of FBS is also important for researchers using cell cultures as pyrogenic contaminants may create problems by affecting the bioactivity of the cultured cells and therefore influence the experimental results obtained [4].For the examined FBS the criteria of quality was an endotoxin level of equal or smaller than 10 EU/mL. Therefore, the contaminant limit concentration of the product is:CLC = 10 EU/mLFor the PyroMAT ® system, the LOD is 0.05 EU/mL, so MVD = 10 EU/mL x 1= 2000.05 EU/mLRequestInformation4Product specific validation for testing FBS (fetal bovine serum) with the PyroMAT ® systemAssurance of the criteria for the endotoxin standard curve:A standard curve using Reference Standard Endotoxin was performed to verify that the criteria for endotoxin standard curve were valid.• The regression of response on log dose was statistically significant (p < 0.01)• The regression of response on log dose did not deviate significantly from linearity (p > 0.05)Test for interfering factors and method validation for detection of non-endotoxin contaminants:A dilution series from undiluted product up to the MVD was prepared and a test for interfering factors andmethod validation for non-endotoxin monocyte-activating contaminants (NEPs) according to EP was performed, comparing endotoxin-spiked sample dilutions as well as NEPs-spiked sample dilutions with the same unspiked sample dilutions.Tests results showed non-endotoxin pyrogen (NEPs) control was detectable in all dilutions of the product from undiluted sample to MVD.The undiluted product was found to show interference with the detection of the endotoxin spike, the reaction was strongly inhibited. From an 1:10 dilution to the MVD, the product reproducibly showed both detection of the NEP and a spike recovery within the range 50-200%, allowing to rule out interference with the test. Test for interference in the detection system:The so found optimum dilution was forwarded to a test for interference in the detection system. A dilution series of IL-6 control was tested in absence and presence of the sample dilution. All IL-6 control dilutions showed less than 20% difference between dilution in absence and presence of the product, therefore no interference with the detection system could be found.ConclusionAll criteria of the product specific validation were fulfilled and the dilution 1:10 was chosen as the firstvalid dilution for the Method A.Assurance of criteria for the standard curve Valid Test for interfering factors Valid Detection of NEP contaminants Valid Interference in the detection systemValidSpeak to a Specialist5Quantitative method A according to EPPlanning Test ExecutionOnce a valid dilution has been identified through the Product Specific Validation, Method A (quantitative test) can be performed to assess the pyrogenicity of samples from this product.The lowest dilution / highest concentration of thesample, that was found free of interference in the PSV is to be chosen as initial dilution and 2-fold serial dilutions are tested.For quantification, the middle section of the dose-response curve of the endotoxin standard is to prefer, giving the most exact results, while quantification within the upper plateau of the standard curve can lead to inaccuracy due to reaching the endpoint of the reaction and therefore is not recommended.For most exact results, we therefore recommend to analyze sample dilutions which do not exceed the measuring range of 0.05 to 0.4 EU/mL.Testing of FBS (fetal bovine serum) with method A The test setup was performed according to the user guide of the PyroMAT ® system.An endotoxin standard curve was performed for the test.Three dilutions of the product were tested according to method A described in EP: the dilutions 1:10, 1:20 and 1:40 of the sample were all tested with and without endotoxin spike. A quantification of the found endotoxin equivalents for all dilutions of the unspiked and spiked sample was done using the endotoxin standard curve. The endotoxin spike recovery for all three sample dilutions was calculated.Heat killed Staphylococcus aureus (HKSA) was used as additional control for detection of non-endotoxin pyrogens within the sample, tested with the highest concentration of the product to be examined.Data interpretationThe data analysis was performed with Gen5 software and the PyroMAT ® Method A available on our website. Information related to the sample were completed directly on the software: sample name, CLC.For interpretation, the layout for method A wasmodified with the appropriate dilution factors for this sample matrix.Request Information6After reading the plate, the data interpretation was performed with the software.The standard curve was valid for all the criteria.Speak to a Specialist7The chosen sample dilutions were appropriate for the sample with all tested dilutions being within the MVD and spike recovery within 50-200%.The data analysis showed validity of the test being fulfilled for all sample dilutions.The sample itself showed a pyrogenic load of <0.5 EEU*/mL which is below the CLC (10 EU/mL) and therefore being considered “not pyrogenic”.* EEU: Endotoxin Equivalent UnitThe NEP-control confirmed detection of non-endotoxin pyrogens in the system and in the sample.ResultsThe examined FBS showed inhibition of the reactionof the Monocytes in the undiluted sample that could be overcome by dilution within the authorized dilution range (not exceeding the MVD).The data show that PyroMAT ® system is suitable for detecting both endotoxin and non-endotoxin pyrogenic contaminations in preparations of FBS.Request InformationReferences[1] European Pharmacopoeia, chapter Monocyte activation test (2.6.30)[2] Kirikae T, Tamura H, Hashizume M, Kirikae F ,Uemura Y, Tanaka S, Yokochi T, Nakano M. “Endotoxin contamination in fetal bovine serum and its influence on tumor necrosis factor production by macrophage-like cells J774.1 cultured in the presence of the serum” International Journal of Immunopharmacology Volume 19, Issue 5, May 1997, Pages 255-262[3] Fishel S, Jackson P , Webster J, Faratian B. “Endotoxins in culture medium for human in vitrofertilization” Fertility and Sterility Volume 49, Issue 1, January 1988, Pages 108-111[4] Ryan J. “Endotoxin and cell culture” Technical bulletin, /lifesciencesMK_AN1804EN Ver. 3.004/2022© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Merck, the vibrant M, PyroMAT and Millipore are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.Merck KGaAFrankfurter Str. 250 64293 DarmstadtTo place an order or receive technical assistanceOrder/Customer Service: /order Technical Service:/techservice/ info-pyromat/ library-pyromatLearn more at /pyrogen-testing。

TRIZMA 预设晶体盐产品说明

TRIZMA 预设晶体盐产品说明

TRIZMA® Pre-set crystals Cell Culture Tested Product Number T 0444 Store at Room TemperatureProduct DescriptionThis product is cell culture tested (1.82 mg/ml) and is designated as Biotechnology Performance Certified. It has been tested for endotoxin levels and analyzed for the absence of nucleases and proteases.This product is a pre-mixed combination of TRIZMA base and Trizma HCl in convenient foil pouches. When dissolved in 1 L of deionized water, this product gives a 0.1 M TRIZMA solution with a pH of 7.5 at25 °C. TRIZMA is the registered trademark fortris(hydroxymethyl) aminomethane, commonly called Tris.Sigma Technical Bulletin 106B contains additional information on temperature and concentration effects, and on the use of pH electrodes with TRIZMA buffers. Tris is an established basimetric standard and buffer used in biochemistry and molecular biology.1 It may be used by itself as a buffer or as a component of mixed buffer formulations.2 These different buffer formulations include:• Tris-EDTA (TE) buffer• Tris magnesium buffer• Tris-acetate-EDTA (TAE) buffer• Tris-borate-EDTA (TBE) buffer• Tris-buffered saline (TBS)• Tris-buffered saline with dextrose (TBS-D)• Tris-glycine buffer• Tris-phosphate EDTA buffer• Tris-SDS buffer• Tris-sucrose• Tris-Tricine-SDS bufferTris salts are used in protein crystallization at various pH values (Product Nos. 82009, 70437, 75403, 86684, 73513).3,4,5,6 The use of low-ionic strength Tris buffers in the formation of intermediate filaments of lamin from Caenorhabditis elegans has been described.7Tris has been utilized in studies of double stranded complexes of peptide nucleic acids (PNA) and their complementary DNA sequences, by use of anion exchange HPLC.8 The use of Tris in capillary electrochromatography and UV analysis of tocopherols and tocotrienols has been reported.9 Precautions and DisclaimerFor Laboratory Use Only. Not for drug, household or other uses.Preparation InstructionsThis product is soluble in water (666 mg/ml), yielding a clear, colorless solution.Storage/StabilityTRIZMA solutions can be autoclaved. Tris has a significant temperature coefficient:• From 5 °C to 25 °C, the pH decreases an average of 0.03 pH units per °C.• From 25 °C to 37 °C, the pH decreases an average of 0.025 pH units per °C.Thus it is necessary to choose the proper mixture to give the desired final pH at the desired temperature. The pH of 0.05 M solutions of this product at various temperatures is as follows:5 °C = pH 8.0725 °C = pH 7.5037 °C = pH 7.22References1. Gomori, G., Preparation of Buffers for Use inEnzyme Studies. Methods Enzymol., 1, 138-146(1955).2. Molecular Cloning: A Laboratory Manual, 3rd ed.,Sambrook, J. and Russell, D.W., CSHL Press(Cold Spring Harbor, NY: 2001), pp. 5.8, 5.30,5.43, 5.60, 5.76, 10.25-10.26, 12.75, 12.84, 12.87,13.52, 16.29-16.31, A1.2-1.3, A1.7-A1.8, A1.17-A1.18, A1.22, A8.42-A8.43.3. Brzozowski, A. M., et al., Structural analysis of achimeric bacterial α-amylase. High-resolutionanalysis of native and ligand complexes.Biochemistry, 39(31), 9099-9107 (2000).4. Knapp, S., et al., Crystallization and preliminarycrystallographic analysis of an amylopullulanasefrom the hyperthermophilic archaeon Pyrococcus woesei. Proteins, 23(4), 595-597 (1995).5. Andrykovitch, M., et al., Crystallization andpreliminary X-ray diffraction studies of NusG, aprotein shared by the transcription and translation machines. Acta Crystallogr. D Biol. Crystallogr.,58(Pt 12), 2157-2158 (2002).6. Campos, A., et al., Crystallization and preliminaryX-ray analysis of FlhD from Escherichia coli.J. Struct. Biol., 123(3), 269-271 (1998).7. Karabinos, A., et al., The single nuclear lamin ofCaenorhabditis elegans forms in vitro stableintermediate filaments and paracrystals with areduced axial periodicity. J. Mol. Biol., 325(2),241-247 (2003). 8. Lesignoli, E., et al., Recognition and stranddisplacement of DNA oligonucleotides by peptide nucleic acids (PNAs). High-performance ion-exchange chromatographic analysis. J.Chromatogr. A., 922(1-2), 177-185 (2001).9. Abidi, S. L., and Rennick, K. A., Capillaryelectrochromatographic evaluation of vitamin E-active oil constituents: tocopherols andtocotrienols. J. Chromatogr. A, 913(1-2), 379-386 (2001).TRIZMA is a registered trademark of Sigma-Aldrich Biotechnology.GCY/RXR 5/03Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side ofthe invoice or packing slip.。

Scepter 2.0 单细胞计数仪说明书

Scepter 2.0 单细胞计数仪说明书

Scepter™ 2.0 Cell Counter Precise, handheld cell countingThe life science business of Merck KGaA,Darmstadt, Germany operates as/scepterupgrade Precise, handheld cell countingScepter™ 2.0 cell counters mark the next generation in Scepter™ technology, highlighted by:Compatibility with More Cell TypesIncreased Cell Concentration RangePowerful Software for Complex, Effortless Cell AnalysisScepter™ 2.0 Cell Counter• Recommended based on size • Customer Validated• MilliporeSigma Validated“At last, an alternative to lining up for the Coulter Counter ®, and far easier than sweating over fragile hemocytometers.”Amy A. Caudy is a Lewis-Sigler Fellow at Princeton University’s Lewis-Sigler Institute for Integrative GenomicsThe Scientist , Dec. 2010. Top Ten Innovations of 2010.The power of precisionTrust Scepter™ devices with your most valuable samples to get reproducible and reliable counts. The reliability of Scepter™ cell counters is particularly apparent with smallercell types. Because the Scepter™ cell counter measures volume using the Coulter Principle, it can quantify cells based on size and will discriminate larger cells from smaller debris, unlike vision-based techniques, which rely on object recognition software and cannot reliably detect small cells.Scepter™ sensor technologyCompatible with 60 μm and 40 μm sensors, theScepter™ 2.0 cell counter can meet even more of your cell- and particle-counting needs. Use the 60 μm sensor for particles between 6 and 36 μm. Use the 40 μm sensor for particles between 3 and 17 μm.• Precise volumes are drawn into the Scepter™ sensor.• As cells flow through the aperture in the sensor, resistance increases. This increase in resistance causes a subsequent increase in voltage.• Voltage changes are recorded as spikes with each passing cell.• Spikes of the same size are bucketed into a histogram and counted. This histogram gives you quantitative data on cell morphology that can be used to examine the quality and health of your cell culture.Particles are detected by Ohm’s Law V=IR (V=voltage, I=current, and R=resistance).Table 1.Cell types validated with the Scepter™ cell counter and the recommended Scepter™ sensor.Cell Type Measuredsize (μm)40 μm sensor 60 μm sensorMeg-0116-17•MG-6315-17•Mouse ES Cell5-13••Mesenchymal Stem Cell 15-16•MRC-5•NCI-H14610-13•NIH 3T315•NTERA2, clone D113••OK 17-18•PBMCs 7-12•PC129-13•Primary Astrocytes 7•Primary Neuronal Cell •Raji 12-15••Ramos11-12••Rat Dorsal Root Ganglion Cells 7•Rat Whole Blood 4.6•Red Blood Cells 5-7•Rat Neural Stem Cell 11-13•RAW 264.712-15•RBL 11-13••RIN-mF513-14•SF913•SH-SY5Y 12•Sk-Br-315-20•SK-MEL-2817-19•SK-N-MC 14-15•SK-N-SH 14-15•Splenocytes 7-9•SW-48015•SW-62013-14••T8414-18•T98G 17•TF-113-14••U25116-20•U2OS 16-19•U26612•U87-HumanGlioblastoma cell line 12-14•U93711-13••WI-3812-15•Y7913-14•Yeast- Pichia Pastoris 5•Yeast- S.cerevisiae 6•Cell Type Measuredsize (μm)40 μm sensor 60 μm sensor2102 Ep 15-19•454 beads •A17215•A25314-18•A37516•A43115-17•A549•Algae (various)7-9•B3513-16••B Cells6-11•C2C1212••C30512-14••C612-13•CA4610-12••Caco-217•CHO 14-17•COS-112••Cos-715•D28312•Daudi 10-12••DU-14515-17•Epithelia 14-15•HCT-11610••HEK29311-15•HeLa 12-14•HepG212•HFF 18-20•Hs2714••HT-108014-16•HT-2911•HUH7- Hepatoma line •Human ES Cells 9-12•HUVEC 14-15•IMR-3212-14••IMR-9015••Jurkat 13•K56222•KB 14•KG-110-13••L614-16•LNCaP15-16•Luminex ® beads 5-6•MCF715-17•MDCK13-15•Scepter™ cell counterHemocytometer= Average % CV= Coulter counter % CVVision-based counter1234567Figure 2.The average percent coefficient of variation (CV) for each counting method shown was calculated using cell concentration measurementsat 50,000 cells/mL samples of 19 different cell lines. The Scepter™ cell counter is more precise than vision-based counting and hemocytometry, and approaches the precision of the Coulter Counter ® standard (yellow bars). Error bars represent standard deviation.of cell diameter or cell volume FormatCounting methods Samplevolume needed Samplevolume counted Cells counted in a 100,000 cell/mL sample Average % CV Hemocytometer Slide and microscope Manual, vision-based 10 μL 0.1 μL /square 10/square 41.8Brand L Benchtop Automated vision-based system10 μL 0.4 μL 4032.1Scepter™ Cell Counter HandheldImpedance-based cell detection100 μL50 μL50009.1Intuitive analysis softwareFrom simple counts to complex volume measurements used to assess cell health parameters, Scepter™ Software Pro provides an intuitive, intelligent platform to perform high-level cell analysis based on the size measurements captured with the Scepter™ cell counter.Using the Scepter™ Software Pro on your computer, you can:• Compare several samples and data sets side by side using histogram overlay and multiparametric tables • Create and save gates to be used from one experiment to the next • Create attractive graphical presentations and reports with your dataA View of Scepter™ Software ProData:Data files from your Scepter™ cell counter Current Plot:Working plot and data file Group Stats:Customizable statistics from your selected data filesMultifunctional Plot:Multiple data sets/histogram overlays Reports:Export, print selected graphs/files, cut and pasteAnalysis Templates:Saved gating parametersPrepare the sample:Start with a single-cell suspension, diluted to a total volume of 100 μL (recommended) in phosphate buffered saline (such as EmbryoMax ® 1x DPBS) to 10,000-500,000 cells/mL (operating range for 60 μm sensor) in a 1.5 mL microcentrifuge tube.Perform cell count:• Turn on the Scepter™ cytometer by pressing the toggle on the back of the instrument and wait for on-screen instructions to appear.• When prompted, attach a sensor to the end of the Scepter™ unit with the electrode sensing panel facing toward the front of the instrument, and you’ll see detailed instructions for each step of the counting process.• Pipette once to draw sample into the sensor. 50 μL of your cell suspension is drawn into the microfabricated, precision-engineered channel embedded in the sensor. The cell sensing zone detects each cell drawn into the sensor and thus cell concentration is calculated.• The sensing zone also measures cell sizes and cell volumes with sub-micron and sub-picoliter resolution, enabling the Scepter™ cytometer to display a As easy as pipetting100MDMA231 cellsFlow cytometryScepter™Trypan blue + hemocytometerNIH 3T3 cells% V i a b i l i t y1009080706050403020100Flow cytometryScepter™Trypan blue + hemocytometer% V i a b i l i t yA.B.ApplicationsMilliporeSigma continues to expand the capabilities of Scepter™ technology, and the latest generation Scepter™ 2.0 device features enhanced analytical powers, enabling you to count even more cell types and sizes.Figure 3.Rapid cell analysis using the Scepter™ 2.0 device provides reliable assessments of cell viability compared to flow cytometry (ViaCount ® assay) and hemocytometry (using Trypan blue staining). MDMA231 cells (A) and NIH 3T3 cells (B) were treated with camptothecin 24 hours prior to analysis.“Cell counting is normally a very tedious process and usually only provides minimal information on the cell population. This instrument, which is only slightly larger than an automatic pipette, allows you to count cells in your tissue-culture hood, simplifies the procedure, and provides much useful data, such as the fraction of intact cells.”H. Steven Wiley is a lead biologist at the Environmental Molecular Sciences Laboratory at the Pacific Northwest National LaboratoryThe Scientist , Dec. 2010. Top Ten Innovations of 2010.Scepter™ 2.0 cell counter for counting heterogeneous cell populationsCount blood cells and other cells with small diameters with the highest precision. Biological samples such as primary isolates or cultured cells are often heterogeneous mixtures of cells that differ by type and/or function. Such differences in cellularattributes are most commonly determined by multicolor fluorescent antibody detection of cell type specific surface marker(s) using flow cytometry. Notably,in addition to variations in protein expression, many cell types and physiological states are also uniquely distinguishable on the basis of size alone. The ability to identify population subsets on the basis ofphenotypic differences and further determine their relative frequencies (and concentrations) is critical to many aspects of research.Scepter™ 2.0 cell counter for cell healthInstantly gauge the health of your cell cultures without even leaving the culturehood. Because the Scepter™ cell counter displays high-resolution histograms of entire cell populations, you can differentiate live cells from dead cells and debris by simply gating on the histogram peak corresponding to larger-diameter cells. No staining is required! The resulting calculation for % viable cells agrees with viability calculations obtained using flow cytometry (ViaCount ® reagent) and Trypan blue staining/hemocytometry (shown here with MDMA231 and NIH 3T3 cells).% C o e ffic i e n t o f V a r i a t i o n (%C V )(40 µm sensor)Vision-based Counter Counting MethodPBMCsAverage % CV Z2 Coulter Counter C o u n tC o u n tForward ScatterFlow CytometryPlot PO3, ungatedScepter™ (40 µm)Current AcquisitionDiameter (µm)100200300400500600700800900100001000200030004000500060005.6 µm Polystyrene MILLIPLEX ® Antibody-Conjugated MicrospheresC o u n tDiameter (µm)1,500,000500,000250,000125,00050,000750,0000.0E+00M e a s u r e d C e l l C o n c e n t r a t i o n (c e l l s /m L )Theoretical Cell Concentration (cells/mL)4.0E+058.0E+051.2E+061.6E+06Theoretical Cell ConcentrationZ2 Coulter Counter Scepter™ - 40 µm SensorsScepter™ 2.0 │ Precise, handheld cell countingScepter™ 2.0 cell counter for countingheterogeneous cell populations (continued)Distinguishing lymphocytes from monocytes in freshly isolated PBMCs . The assessment of immune profiles of the various immune cell subsets can help identify molecular signatures that may facilitate research. The Scepter™ cell counter, when used in combination withScepter™ Software Pro, provides a tool for rapid determination of lymphocyte and monocyte concentrations as well as the relative frequency of these cell types in PBMC isolates.Figure 4.The Scepter™ 2.0 cell counter counts PBMCs with greater precision than other counting methods, as reflected by low coefficients of variation. % CVs were calculated using average cell counts of four replicate samples.Figure 6.Scepter™ Software Pro displays imported size distribution histograms as either a single sample histogram or as overlaid histograms for multiple samples. Shown is an overlaid histogram for serially diluted 5.6 μm MILLIPLEX ® map microspheres.Figure 7.The Scepter™ cell counter counts yeast cells with good accuracy and linearity. Measured yeast cell concentrations were compared totheoretical concentrations. The solid blue line represents the theoretical values. Dotted lines represent best linear fit to data. Both the Scepter™ and Coulter Counter ® platforms show a loss of linearity and accuracy upon an increase in cell concentration.Figure 5.Representative comparison of histogram plots for human PBMCsamples acquired on the Scepter™ cell counter (diameter histogram on right) and guava ® easyCyte™ flow cytometry (forward scatterhistogram on left) platforms. Analysis plots derived from both platforms demonstrate three distinct peaks corresponding to 1) dead cell/debris, 2) lymphocyte and 3) monocyte fractions. The difference in counts displayed (Y-axis) is due to differences in sample dilution between the guava ® easyCyte™ flow cytometer and the Scepter™ cell counter.Cell Fraction Scepter™ 1Forward Scatter 2Staining 31Lymphocyte 586563Monocyte 4235372Lymphocyte 687271Monocyte 3228293Lymphocyte 666971Monocyte343129Precise and accurate bead counting with the Scepter™ 2.0 cell counterMicron-sized beads are used in a variety of biological applications, ranging from daily validation of flowcytometer performance to purification of fusion protein constructs from cell lysates. Accurate determination of bead counts at the onset of each assay allows for standardization of bead concentrations acrossmultiple samples and minimizes errors and variation in downstream results. The Scepter™ cell counter is well suited for precise counting for beads of numerous types and can improve reproducibility of bead-based assays, such as immunoprecipitation and multiplexed detection.Scepter™ devices can facilitate yeastcell counting for brewing and wine industriesIntroduction of a consistent yeast cell concentration is required for successful beer and wine fermentation as well as to maintain batch-to-batch reproducibility and ensures consistent fermentation over many cycles. Scepter™ cell counter can be used to monitor yeast size and concentration by yielding interpretable histograms that could be gated to provide this depth of information.Ordering InformationDownloadable Scepter™ Software 1O-Rings2Scepter™ Test Beads 1PHCCBEADS Scepter™ USB Cable 1PHCCCABLE Scepter™ Sensors, 60 μm50PHCC60050500PHCC60500Scepter™ Sensors, 40 μm 50PHCC40050500PHCC40500Universal Power Adapter1PHCCP0WER Scepter™ O-Ring Kit (includes 2 O-rings and 1 filter cover)1PHCC0CLIP Table 2.Lymphocyte and monocyte subset frequencies from three individual PBMC samples. Aliquots from each sample were analyzed using the guava ® easyCyte™ flow cytometry and Scepter™ platforms. 1 Values were derived from the diameter histogram plot. 2 Values were derived from the forward scatter histogram plot based on total events measured on guava ® easyCyte™ flow cytometry platform. 3 Staining frequencies derived as follows: % Lymphocytes = (% CD3+ T cells) + (%CD16/56+ NK cells) + (%CD19+ B cells); % Monocytes = % CD14+ cells.vMilliporeSigma400 Summit Drive Burlington, MA 01803。

南开大学——牛津大学生命科学学术研讨

南开大学——牛津大学生命科学学术研讨

南开大学——牛津大学生命科学学术研讨吴卫辉(南开大学生命科学学院,天津300071)为推动“双一流”建设和国际化进程,南开大学 对标世界一流,精准施策发力,促进交叉融合,打造 学科高地,积极提升教学科研水平,不断拓展国际学 术交流合作。

自2018年8月以来,南开大学与英国 牛津大学达成合作意向,积极磋商探讨合作领域并 达成共识。

2019年6月1丨~13日,双方在南开大学生命科 学学院举办南开大学——牛津大学生命科学学术研 讨会。

会议开幕仪式上,南开大学校长曹雪涛与牛津 大学纳菲尔德医学院院长理查德•约翰•科纳尔共同 签署合作备忘录,双方将在学生联合培养、学术科研 交流合作、共建联合研究机构等领域,深入拓展、夯 实战略合作伙伴关系,开启国际学术交流合作的新 模式。

曹雪涛校长在开幕式致辞中指出,南开大学建 校之初心就是要面向世界,融汇中西,“知中国,服务 中国”。

张伯苓老校长曾提出“务使我南开学校,能与 英国之牛津、剑桥,美国之哈佛、耶鲁并驾齐驱,东西 称盛”的宏愿。

发展需要结伴而行,在迎接百年校庆、开启新百年征程之际,与牛津大学及其纳菲尔德医 学院这样世界级的顶尖伙伴达成合作,将加快推动 南开大学“双一流”建设和国际化进程,具有战略性 意义。

我们期待通过双方共同努力,在生命科学、医学、药学及统计与数据科学等领域进行学科交叉、创 新探索,并希望在更多学科领域开展全面深入合作,解决科研难点,培养优秀人才,产出更多成果,造福 社会。

理查德•约翰•科纳尔院长表示,牛津大学非常 重视与南开大学的合作,此次合作也是牛津大学纳 菲尔德医学院推动全球化战略的重要举措。

伙伴在于分享“难题”,我们如今生活在一个超乎传统认知的 时代,医学及生命科学面临着生存环境快速变化、生 物技术掀起变革、人口老龄化问题日益凸显等众多挑 战。

我们希望与南开大学一道,通过合作凝聚合力,共 同致力于教学科研,为师生搭建创新平台,破解人类 健康的众多奥秘和亟待解决的科学问题,对世界范 围内的医学及生命科学等领域产生深远影响。

CellASIC ONIX M04T-01微流体梯度板用户指南说明书

CellASIC ONIX M04T-01微流体梯度板用户指南说明书

User GuideCellASIC ® ONIX M04T-01 Microfluidic Gradient PlateFor research use only. Not for use in diagnostic procedures.IntroductionThe CellASIC ® ONIX M04T Microfluidic Plate is a 4-chamber cell culture plate designed for use with the CellASIC ® ONIX2 Microfluidic System and CellASIC ® ONIX2 Manifolds,enabling real-time imaging of 12 µm-sized suspension cells 1. This bio-inspired plate provides a controlled and dynamic microenvironment for cells, which when used in conjunction with the ONIX2 system, permits perfusion-based, long-term, live-cell analysis with automated solution switching. Theeasy-to-use format and novel microfluidics-based technology redefine the standard for live cell imaging experimentation.Applications• Trapping and monitoring of mammalian suspension of 5–12 µm-sized cells 1• Time-lapse analysis of suspension cells• Temperature and gas atmospheric control (temperature shift, anoxic conditions, etc.)• Long-term continuous perfusion experiments• Solution exchange experiments (induction, inhibition, drug dosing, etc.)• Comparison of up to 4 different cell types or exposureconditions (media type and reagent concentration) in parallelPlate DescriptionA B C D123456Solution inlets78Cell inletOutletFigure 1. Plate configurationThe M04T plate has 4 independent units (A–D), each with 6 inlet wells (1–6), a cell inlet (8), and a large outlet well (7). Each row of wells (A–D) addresses the corresponding culture chamber . The plate is shipped pre-primed with a PBS (phosphate-buffered saline) solution, which can be replaced with a buffer of choice prior to experiment. Each chamber has an array of 104 barrier trap pads 12.0 μm in height to hold cells in a single focal plane during long-term analysis. The plate is for single use only.1. The trap has height of 12 µm with vertical barrier gap spacing of 3 µm (See “Cell Trapping Mechanism” for more detail), suitable to use with cells that are 5–12 µm insize. Depending on cell morphology, it is also possible to capture cells of larger sizes.Culture chamber (A–D)Flow inletsFigure 2. Chamber viewing windowAll four culture chambers are located under a single viewing window to minimize travel distance for high-magnificationphase objectives.iii. Wide-opening cell trap123456Figure 3. Culture chamberSchematic indicating the dimensional features of tissue culture chamber with cell trap array. Notice that the colors represent different heights. White: 12 µm; light blue: 25 µm; dark blue: 40 µm. Zoomed-in area (red rectangle) shows the barrier surrounding each trap marked by royal blue with vertical gap height of 3 µm. Additional description on trap design can be found in Figure 4. The culture chamber hexagon marquee is 3.0×6.0 mm with a ceiling height of 25 µm (area with light blue color). Within each chamber , the culture array area is 3.0×3.0 mm with 104 interdigitating individual traps with two different openings (wide and narrow) and heights of 12 µm. Wide and narrow openings enable a balance between initial trapping efficiency and retention of cells over time, allowing more experimental flexibility. For example, narrow opening traps will have lower number of suspension cells initiallytrapped, while retaining these cells within the trap for a longer period, compared to the wide-opening traps. Nine position markers indicate unit number and relative position.The inlet/outlet functions and minimum/maximum recommended volumes for each culture unit are listed below.Inlet 1Inlet for solution switching50300 Inlet 2Inlet for solution switching50300 Inlet 3Inlet for solution switching50300 Inlet 4Inlet for solution switching50300 Inlet 5Inlet for solution switching50300 Inlet 6Inlet for solution switching50300 Outlet 7Outlet from culture chamber50795 Inlet 8Inlet for cell loading into culturechamber / Additional outlet fromculture chamber50265 Cell Trapping MechanismPDMS Trap PDMS TrapFigure 4. Cell trapping mechanismA. C ross-sectional view of an individual trap. Themicrofabricated polydimethylsiloxane (PDMS) trap has x-y dimension of 100 μm and height of 12 μm. Due to its height, the traps can hold suspension cells gently against the glass viewing surface and maintain these cells within single focal plane during live cell imaging, facilitating more detailedimaging of suspension cells.B. I mage of traps with retained cells. Shown are narrow (top)and wide (bottom) opening traps with captured Jurkat cells.Notice the barriers bracketing each trap pad’s perimeteron three sides, which act to retain motile cells within FOV (field of view) during imaging. Narrow and wide traps have openings of 26 µm, and 88 µm, respectively, with vertical gap height of 3 µm. Each trap is “open” on the side facing the direction of flow during cell loading.Manifold DescriptionThe CellASIC® ONIX2 heated (CAX2-MXT20) or basic(CAX2-MBC20) manifolds connect the microfluidic plate to the CellASIC®ONIX2 Microfluidic System.Well group 8GasenvironmentVacuum12345678Gas inletWell groups Well groupsFigure 5. Lines to CellASIC® ONIX2 Microfluidic SystemFlow control is achieved using air pressure above the liquid in each well. Multiple wells on a plate are grouped together and addressed by a single pneumatic line via the manifold. Each set of wells is called a “well group.” A vacuum line is used to seal the plate to the manifold, and a gas line enables atmospheric control.Flow PropertiesFlow properties of wells 1–6 are shown in Figure 6. The figure shows the flow rate out of the well as a function of pressure.If more than one channel is pressurized, multiply the well flow rate by the number of pressurized channels to derive the overall flow rate.FlowRate(mL/hr)15304560Pressure (kPa)015304560Figure 6. Flow rate for wells 1–6Plate StorageStore at room temperature. Do not store in direct sunlight. LimitationsThe plate is incompatible with acetic acid and organic solvents such as acetone, ethanol, and methanol. Plates should be tested for compatibility with other acids or organic solvents prior to use.Plate OperationIf temperature control is needed, use the CellASIC® ONIX2 Manifold XT (CAX2-MXT20). Refer to the CellASIC® ONIX2 Microfluidic System User Guide for setup instructions.Plate Priming1. For units to be used (A–D, see Figure 1), replace the PBSin the solution inlet (wells 1–6) and cell inlet (well 8) wells with 100–300 µL of your desired media, e.g., RPMI with10% FBS. Aspirate and empty well 7 (waste well). Makesure to leave all the wells of any unused units filled PBSsolution while emptying well 7.NOTE: It is best to perform the priming and cell loading(see next section) steps at room temperature as certain cell types can become sticky at 37 °C.2. Seal the microfluidic plate to the ONIX2 manifold, accordingto the CellASIC® ONIX2 Microfluidic System User Guide.3. Open the CellASIC® ONIX2 Software, select one of the NewExperiment options, and find the M04T plate on the drop down list. On the Manual Mode tab (Figure 7), click on the Run liquid priming sequence button.NOTE: The preloaded priming sequence should contain the following steps for well group 1–6 and 8:i) 7 kPa (1 psi) for 3 minutes.ii) 50 kPa (7.3 psi) for 20 seconds.iii) 7 kPa (1 psi) for 20 seconds.iv) 50 kPa (7.3 psi) for 20 seconds.For more information on creating custom protocols, refer to the CellASIC® ONIX2 Microfluidic System user Guide.4. Unseal the plate by pressing the Seal button on theinstrument or in the Tools drop-down menu, click UnsealPlate. Remove the manifold from the plate. Proceed to cell loading step.Cell Loading1. Prepare cell suspension of 3~10 x 106 cells/mL for loading.This concentration can be optimized depending on thedesired trapping efficiency.NOTE: It is critical that cells are in good health. Only usecells in their logarithmic growth phase.NOTE: Since trap height is 12 µm, some large suspension cells, such as RAW264.7 may be difficult to trap. However, in most cases, cells with size larger than 12 µm can becaptured with the trap. To date, we have done live cellimaging using the following commonly used cell lines:Jurkat, Raji, THP-1, U937, K562, and HL-60.2. Aspirate media from cell inlet well 8.3. Pipette 80 µL of prepared cell suspension into cell inletwell 8. Make sure cell solution covers the hole at thebottom of the well.4. Seal the microfluidic plate to the ONIX2 manifold accordingto the CellASIC® ONIX2 Microfluidic System User Guide.5. Open the CellASIC® ONIX2 Software, select one of the NewExperiment options, and find the M04T plate on the drop-down list. On the Manual Mode tab (Figure 7), click on the Run cell loading sequence button.NOTE: for initial experiment, we recommend pressurizingwell groups 1–6 and 8 at 3 kPa (0.4 psi) for 20 seconds.This loading protocol must be repeated minimum of 4 times.6. Assess the overall trapping level by microscope. To increasetrapping, repeat the loading protocol.NOTE: Increasing the number of loading will also increase trapping of cellular debris and dead cells. Do not increasethe number of loading beyond the minimum required.NOTE: Alternatively, it is also possible to capture more cells by increasing the loading pressure to 4~5 kPa. However,as before, it is important to strike the balance between the trapping of healthy cells vs. cellular debris and dead cells.Do not increase the pressure beyond the minimum required.7. Once desired level of cell trapping has been achieved,leave the plate at room temperature for 30 minutes before proceeding to setting up imaging experiment.8. Unseal the plate by pressing the Seal button on theinstrument or in the Tools drop-down menu, click onUnseal Plate. Remove the manifold from the plate.Proceed to setting up the experiment.Setting up the dynamic live cell imaging experiment: Cell Culture and Solution Switching1. Aspirate and replace media from desired wells withappropriate solution (any well s 1–6). Add up to 300 µL ofdesired solution to the wells. Optional: cells can be removed from well 8 at this point.2. Seal the microfluidic plate to the ONIX2 manifold accordingto the CellASIC® ONIX2 Microfluidic System user Guide.3. Open the CellASIC® ONIX2 Software, select one of the NewExperiment optio ns, and find the M04T plate on the drop-down list. Click on the Protocol Editor tab and enter thedesired steps and conditions. For an example, see Figure 8.NOTE: It is important to keep perfusing cells duringculturing to provide adequate nourishment and removalof potential waste products. We recommend minimumpressure of 14 KPa (2 psi) from at least one inlet duringculturing to keep cells healthy.NOTE: When designing the duration of experiment, flowrate data from Figure 6 should be used as reference. Forexample, when perfusion setting of 14 kPa (2 psi) is used, 300 µL of solution from a given well will easily be able toperfuse cells for an overnight 16-hour experiment without emptying. However, if 50 kPa (7.3 psi) is used, then 300 µL of solution will run out within 10 hours.For information on creating a protocol, refer to theCellASIC® ONIX2 Microfluidic System User Guide.4. To start imaging, place the sealed plate/manifold assemblyon an inverted microscope. Survey the trap array andidentify the traps to be used for live cell imaging.5. During extended perfusion experiments, empty well 7periodically to avoid outlet overflow into the manifold tubing and perfusion system, since the capacity of the waste well is about 800 µL. On the Run tab in the CellASIC® ONIX2Software, click the Pause button. Press the Seal buttonon the instrument or in the Tools drop-down menu, clickUnseal Plate. Remove the manifold from the plate, andaspirate well 7.6. Reseal the manifold to the plate, then on the Run tab, clickResume to restart the perfusion protocol.Software OperationThe figures below show two modes for running experiments using the CellASIC ® ONIX2 software. Refer to the CellASIC ® ONIX2Microfluidic System User Guide for details on software features.Manual pressure activation (click colored bar to activate well group)(set point)= 0.0 kPaGas flow control (On/Off, source,and flow)Culturechamber layoutpressure is on)Status bar (shows current system conditions and operations)Figure 7. Manual Mode allows interactive operation of the ONIX2 System. Operating parameters can be set manually and this mode also provides the option to run short automated setup sequences that are prepopulated with plate-specific defaults. These setup sequences can be edited if desired.Perfusion: Setspressure and well specified duration Temperature Set:Gas Set: Sets conditionsPause: Closes while maintaining temperature and gas environmentsProtocol steps are listed in this pane Additional comments or details for each step may be captured in this paneFigure 11. Protocol Editor mode allows the creation and editing of an experimental protocol. A protocol is comprised of a sequence of environmental control and/or perfusion steps. Steps can be added and altered as desired. When the protocol is ready, it can be executed using the Run tab.SpecificationsCulture Plate Dimensions Length × widthHeight without lid 127.3 mm (5.0 in.) × 85.2 mm (3.4 in.)14.3 mm (0.6 in.)Number of Traps Wide opening Narrow opening 104 64 40Culture Chamber Dimensions LengthWidthTrap heightChamber heightCulture array lengthGlass bottom thickness (#1.5 slide)6.0 mm (0.24 in.)3.0 mm (0.12 in.)12.0 µm25.0 µm3.0 mm170 µmPlate materials of construction Polycarbonate, silicone, acrylic, glass Product Ordering InformationThis section lists catalogue numbers for the CellASIC® ONIX products. You can purchase these products and find the most up-to-date software, plate maps, and user guides at /cellasic.Microfluidic PlatesCellASIC® ONIX Plate for Bacteria Cells(4-chamber, trap heights of 0.7, 0.9, 1.1,1.3,2.3, and 4.5 µm)5B04A-03-5PKCellASIC® ONIX Gradient Plate forMammalian Cells (4-chamber)5M04G-02-5PK CellASIC® ONIX Open-top Plate forMammalian Cells (4-chamber)5M04L-03-5PK CellASIC® ONIX Switching Plate forMammalian Cells (4-chamber)5M04S-03-5PK CellASIC® ONIX Pad Trap Plate(4-chamber, trap heights 12.0 µm)5M04T-01-5PK CellASIC® ONIX Plate for Haploid YeastCells (4-chamber, trap heights of 3.5, 4.0,and 4.5 µm)5Y04C-02-5PKCellASIC® ONIX Plate for Diploid YeastCells (4-chamber, trap heights of 5.0, 6.0,and 7.0 µm)5Y04E-01-5PKCellASIC® ONIX Pad Trap Plate(4-chamber, trap height of 4.0 µm)5Y04T-04-5PK CellASIC® ONIX2 Microfluidic System and ManifoldsCellASIC® ONIX2 Microfluidic System1CAX2-S0000 CellASIC® ONIX2 Manifold XT(temperature controlled)1CAX2-MXT20 CellASIC® ONIX2 Manifold Basic(no temperature control)1CAX2-MBC20Replacement Parts/AccessoriesCellASIC® ONIX2 Filter Multiconnector(includes filters)1CAX2-AMC00 CellASIC® ONIX2 Software USB Drive1CAX2-SSW01 CellASIC® ONIX2 Gasket1CAX2-AGK20 CellASIC® ONIX2 Self Check Plate1CAX2-ASP20 CellASIC® ONIX2 Cleaning Plate1CAX2-ACP20 CellASIC® ONIX2 Replacement Filter Pack(9 × 4 mm and 1 × 13 mm Millex® 0.45 µmPTFE filters)1CAX2-AFP00CellASIC® ONIX2 Accessory Fittings(quick-connect gas fitting, 2/pk)1CAX2-ABF00 CellASIC® ONIX2 Temperature CalibrationPlate1CAX2-ACT20 CellASIC® ONIX2 Premixed Gas Regulator(for use with 103 L or 112 L gas cylinderswith a C10 connection)1CAX2-ABR00 CellASIC® ONIX2 Microfluidic ServicesCellASIC® ONIX2 Essential Service Plan1CAX2-ESVC CellASIC® ONIX2 Total Service Plan1CAX2-TSVC CellASIC® ONIX2 Installation1CAX2-INST NoticeWe provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. 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3M Novec 7000和7100工程液体产品说明书

3M Novec 7000和7100工程液体产品说明书

Cat. No.Product DescriptionSHH0001-500ML Novec™ 7000 Engineered Fluid SHH0001-1L Novec™ 7000 Engineered Fluid SHH0002-500ML Novec™ 7100 Engineered Fluid SHH0002-1L Novec™ 7100 Engineered Fluid A5597-1GAL Xylene SubstituteA5472-1GAL Formalin Free Tissue Fixative H2779-1L HistoChoice® Clearing AgentH2904-100ML HistoChoice® Tissue FixativeH2904-1L HistoChoice® Tissue Fixative Xylene SubstituteRené J Buesa and Maxim V PeshkovAnnals of Diagnostic Pathology, 13(4), 246-256 (2009) After the hazardous effects of xylene became indisputable in the 1970s, many potential substitutes became available, some with as many if not more hazards. This article discusses the inadequacy of 5 vegetable oils as substitutes, as well as the characteristics of 22 D-limonene-based substitutes, all less effective in their chemical role, some capable of inducing health problems, and costing more than twice as much as xylene. Some of the 35 alkane-based substitutes discussed are effective for tissue processing, less toxic, with a cost about the same as xylene, but are not very effective for dewaxing and other staining tasks. Isopropanol (2-propanol) alone or mixed with molten paraffin is a technically acceptable and cost-effective substitute for xylene for tissue processing, but in this study, we demonstrate that the best clearing agents from the sectioning quality and diagnostic value point of view, with automated or manual protocols, are mixturesof 5:1 and 2:1 isopropanol and mineral oil, followed by undiluted mineral oil, all at 50 degrees C, making thema safer and cheaper substitute than xylene. Using a1.7% dishwasher soap aqueous solution at 90 degreesC to dewax before staining and oven drying the stained sections before coverslipping will eliminate xylene from the staining tasks. Tissue processors retorts and conduits can be dewaxed with a 2% solution of a strong glassware laboratory detergent. These 4 methodologies will make the histology laboratory xylene-free but, due to the natural resistance to change, many histotechs will be reluctantto adopt them if they think that their technical expertise could be jeopardized, and the only way these changes will succeed is if the pathologists, as stewards of the histologylaboratory, commit to their implementation.Go Greener Histology & HematologySigma-Aldrich® is Committed to Bringing Safer Greener Alternatives to Our Customers.• Novec fluids are non-flammable, low toxicity fluidsfor freezing histology samples.• Xylene Substitute is non-toxic version of xylenewhich is compatible with all tissue• Formalin Free Tissue Fixatives is a less toxicalternative to formalin. It is also compatible witha number of applications like PCR, IHC and in-situhybridization• HistoChoice® Clearing Agent is suitable as analternative to toluene and xylene for dewaxing paraffintissue sections• HistoChoice Tissue Fixative is designed for molecularbiology and used for in-situ hybridizationThe life science business of Merck operates asMilliporeSigma in the U.S. and Canada.Formalin Free Tissue FixativesM E Boon and L P KokBiotechnic and Histochemistry, 83(6), 261-277 (2008)The German, F. Blum, introduced formalin as a fixative in 1893. Formalin rapidly became popular for hardening and preserving gross human and animal specimens. As a result, microscopy for diagnostic pathology by combining paraffin embedding and formalin fixation was developed. Alcohol-based fixatives have coagulation of proteins as their main preservative effect. Because there is no cross-linking, immunostaining is not compromised, and DNA and RNA is not damaged. Ethyl alcohol was used by Dutch scientists of the 18th century, but was replaced by the cheaper formalin. Addition of low molecular weight polyethylene glycol (PEG) optimized the coagulant fixative, Kryofix. The polyethylene glycol prevents excessive hardening and enhances the speed of coagulation of proteins. Kryofix was used on a large scale for skin biopsies in Leiden between 1987 and 2001. DNA preservation by the formulated coagulant fixative, BoonFix, is related to the concentration of ethyl alcohol, PEG and acetic acid. BoonFix has been used since 2004 in Leiden for over 40,000 diagnostic skin biopsies and more than 100,000 cervical samples.A literature review and three decades of experience with coagulant, formalin-free fixatives in pathology suggest that when health authorities realizethat formalin invalidates expensive tests, it might eventually be eliminated legislatively from diagnostic pathology. Finally, coagulant fixation is optimal for microwave histoprocessing where ethyl alcohol is followed by isopropanol. HistoChoice® Clearing Agent1. I mmunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula).Janine A Duckworth et. alReproduction, 133 (1), 177-186 (2007)In a previous study, three infertility-relevant epitopes of possum ZP2 (Pep12 (amino acids 111-125), Pep31 (amino acids 301-315), and Pep44 (amino acids 431-445)) were identified using sera from possums (Trichosurus vulpecula) immunized with recombinant possum zona pellucida 2 (ZP2) constructs, and a synthetic peptide library of possum ZP2 protein. In this study, the three peptides were conjugated to keyhole limpet hemocyanin and 300 mug of each conjugated peptide were administered subcutaneously to female possums (n = 20 per peptide) in complete Freund's adjuvant. Immunogen doses were repeated 3 and 6 weeks later using incomplete Freund's adjuvant. Control animals were immunized with either phosphate-buffered saline only (n = 10) or 300 mug keyhole limpet hemocyanin (n= 10), administered with the same adjuvants. Serum antibodies from animals immunized against these three epitopes bound to the corresponding possum ZP2 peptides, recombinant possum ZP2 protein constructs, and native zona. Possum fertility was assessed following superovulation and artificial insemination. Peptides Pep12 and Pep31 had no significant effects on fertility parameters (P > 0.05). However, animals immunized with Pep44 had lower egg fertilization rates (immunized 19.5% versus control 60.5%,P < 0.05) and produced significantly fewer embryos than control animals (immunized 0.5 embryos versus control 2.4 embryos, P < 0.05). The number of Pep44-immunized females that produced embryos was reduced by 64%. Identification and characterization of possum infertility-relevant epitopes on possum ZP2 protein will assist development of safe, humane, and possum-specific immunocontraceptive vaccines for controlling the introduced possums in New Zealand.2. A simplified method for combined immunohistochemistry andin-situ hybridization in fresh-frozen, cryocut mouse brain sections. Sathyanesan Samuel Newton et. alBrain Research Protocols, 9 (3), 214-219 (2002)A method is described to perform combined immunohistochemistry and in situ hybridization in mouse brain sections. The protocol is specific to sections mounted on glass slides. In contrast to earlier methods that require either paraffin embedding or perfusion ofthe brain with paraformaldehyde, this protocol can be carriedout on fresh-frozen, cryostat cut post-fixed sections. This simple and concise protocol increases the applicability of the techniqueas the RNAse-free immunodetection of antigen is useful by itself for immunologically identifying specific cells of interest and then examining gene expression in those cells using techniques such as real-time PCR and microarray analysis. The use of fresh-frozen, cryocut sections enables reliable detection of easily perturbable post-translational modifications such as phosphorylation and improves the quality of results obtained in subsequent in situ hybridization by reducing the background signal and interference from lower cell layers. Inducible transgenic mice that express either a dominant negative mutant form of the cAMP response element binding protein (mCREB) or CREB, in discrete brain regions, were used in this study. The combined immunohistochemistry and in situ hybridization protocol was used to examine colocalization of enkephalin or dynorphin mRNA, both downstream targets of CREB-mediated gene expression, in cells expressing transgenic mCREB or CREB. HistoChoice Tissue FixativeJ Melrose et. alBiotechnic and Histochemistry, 83(1), 47-53 (2008) Histochoice is a proprietary nontoxic, non-cross-linking fixative designedby the manufacturer to replace formaldehyde based fixation protocols. We compared Histochoice and formalin fixation for several cartilaginous tissues including, articular and growth plate cartilage, meniscus and intervertebral disc. The tissues were stained with general histology stains including toluidine blue for tissue proteoglycans, picrosirius red to evaluate collagenous organization, and hematoxylin and eosin to assess cell morphology. The chondroitin sulfate and heparin sulfate substituted proteoglycans aggrecan and perlecan were also immunolocalized in some of the tissues to provide a comparison. Histochoice did not fix deep into the tissue blocks resulting in focal loss of aggrecan and other matrix components from the more central regions of the blocks. This was evident in toluidine blue stained sectionsof immature tibial articular cartilage where loss of glycosaminoglycan was significant in Histochoice fixed tissues. Histochoice fixation worked well, however, in the aggrecan and perlecan immunohistology applications where its non-cross-linking traits were conducive to epitope retrieval and identification by primary antibodies to extracellular matrix components.To place an order or receive technical assistance Order/Customer Service: /orderTechnical Service: /techserviceSafety-related Information: /safetycenter Copyright © 2017 Merck KGaA. All Rights Reserved. Merck and the Vibrant M are trademarks of Merck. HistoChoice is a registered trademark and Sigma-Aldrich is a trademark of Sigma-Aldrich Co. LLC. or its affiliates.Lit. No. sh8707 2017 - 0672309/2017。

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R.A. Arndt † Dept. of Physics, Virginia Polytechnic and State University, Blacksburg, VA 24061
I.I. Strakovsky and R.L. Workman Dept. of Physics, The George Washington University, Washington, D.C. 20052
fact that D¯ +(t) can be expressed as a power series in t [5], the coefficients determined
from dispersion relation are determined from the
subtraction forward C¯+
constants. The coefficients and “derivative” E¯+ DRs,
up to O(t), respectively.
d¯+00 and d¯+01, The smaller
∗Present address: TRIUMF, Vancouver, B.C. V6T-2A3 ; EMAIL: marcello.pavan@triumf.ca †Present affiliation: Dept. of Physics, The George Washington University, Washington, D.C. 20052
relations [5]. The canonical result Σ = 64 ± 8 MeV was based on hyperbolic dispersion
relation [4] calculations using the groups’ πN [8] and ππ [5] phase shifts.
arXiv:nucl-th/9912034v1 15 Dec 1999
Abstract
A new result for the πN sigma term from an updated πN partial–wave and dispersion relation analysis of the Virginia Polytechnic Institute (now George Washington University) group is discussed. Using a method similar to that of Gasser, Leutwyler, Locher, and Sainio, we obtain Σ =90±8 MeV (preliminary), in disagreement with the canonical result 64±8 MeV, but consistent with expectations based on new information on the πNN coupling constant, pionic atoms, and the ∆ resonance width.
Σ = Fπ2D¯ +(ν = 0, t = 2m2π)
(1)
where Fπ=92.4 MeV is the pion decay constant, ν is the crossing energy variable, and t is the four-momentum transfer. Since the Cheng-Dashen point lies outside the physical πN
New Result for the Pion–Nucleon Sigma Term from an Updated VPI/GW πN Partial–Wave and Dispersion Relation Analysis
M.M. Pavan ∗ Lab for Nuclear Science, M.I.T., Cambridge, MA 02139
(2)
In the GLLS approach, the Karlsruhe KH80[8] or KA84[9] πN phases shifts are used as fixed input above about Tπ=70 MeV, and the D and higher phases are used below the cutoff as well in six forward dispersion relations (B±, C±, E±). By fitting the low energy data, d¯+00 and d¯+01 can be determined. Their result [2,6] was Σd ≃50 MeV, and ∆D=12 MeV, leading to Σ ∼ 62 MeV, in agreement with the Karlsruhe results [5,4]. However, since the dispersion relations were constrained to be satisfied, the subtraction constants, which are energy independent, must be the same at low energies where the data were fit as at high energies where they were fixed input. Therefore, Σd could not have come out significantly different than the Karlsruhe result. Nonetheless, this analysis provided a very useful validation of the method. The technique has been criticized [10] since the E DR is more sensitive to the higher partial waves than the other DRs, so it could be rather uncertain due to uncertainty in the higher phases. What the GLLS analyses showed was that this is in fact not the case, and the method can be used reliably to extract Σd.
The only recent dispersion theoretic determinations have been by Sainio [6], based on
the method of Gasser, Leutwyler, Locher, and Sainio (GLLS)[2]. The method exploits the
Experimental Σ Term
The “experimental” sigma term Σ is related to the πN isoscalar amplitude D¯ + (bar signifies the pseudovector Born term is subtracted) at the “Cheng-Dashen point” [7]:
scattering region, the experimental πN amplitudes must be extrapolated in order to obtain
Σ. The most theoretically well-founded extrapolation approach is based on dispersion
Introduction
The pion nucleon sigma term (Σ) continues to be a puzzle some thirty years after initial attempts to determine it. The keen interest in Σ comes from the fact that it vanishes in the massless quark (chiral) limit of QCD, and becomes non-zero only for a non-zero light (up or down) quark mass, so it is a crucial parameter in the understanding of chiral symmetry breaking (see e.g. Refs.[1,2]). The nucleon’s strange quark content can be inferred from Σ (see e.g. Ref.[2]), so Σ is also relevant to quark confinement, not yet fully understood, since one must understand the mechanism for accommodating strange quarks in an ostensibly light quark object [3]. Thus Σ is a parameter of fundamental significance to low energy QCD, making it crucial to obtain its value as precisely as possible. The canonical result for Σ ≃ 64 MeV [4,5] implies a large nucleon strangeness content [2], and much effort has been spent trying to understand that. This article outlines recent work of the (former) Virginia Polytechnic Institute (VPI), (now George Washington University (GWU)) group to extract the “experimental” value of the sigma term (Σ) from the πN scattering data as part of ongoing πN partial-wave (PWA) and dispersion relation (DR) analyses.
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