Effect of task-related extracerebral circulation

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分析三联、四联药物方案治疗胃溃疡的临床效果

系统医学 2023 年 12 月第 8 卷第 24期分析三联、四联药物方案治疗胃溃疡的临床效果王昌盛1，陈兰2，廖小红21.广东药科大学附属第一医院药学部，广东广州510062；2.广东三九脑科医院药剂科，广东广州510510[摘要]目的探讨胃溃疡患者选择四联药物治疗后的临床效果。

方法选取2022年1月—2023年8月广东药科大学附属第一医院收治的76例胃溃疡患者为研究对象，依据投掷硬币法分组，参照组（38例）选择三联药物治疗，研究组（38例）选择四联药物治疗，比较两组治疗总有效率、胃灼痛评分、胃溃疡面积、上腹疼痛评分、临床症状改善时间。

结果研究组治疗总有效率为97.37%，明显高于参照组，差异有统计学意义（χ2= 6.176，P<0.05）。

治疗后，研究组胃灼痛评分、胃溃疡面积、上腹疼痛评分、临床症状改善时间均低于参照组，差异有统计学意义（P均<0.05）。

结论同三联药物比较，胃溃疡患者接受四联药物治疗，可显著提升临床效果，有效改善疾病症状，可促进胃溃疡患者的良好预后。

[关键词]胃溃疡；三联药物；四联药物；疗效[中图分类号]R573 [文献标识码]A [文章编号]2096-1782（2023）12(b)-0175-03 Clinical Effect of Triple and Quadruple Drug Regimens in the Treatment of Gastric UlcerWANG Changsheng1, CHEN Lan2, LIAO Xiaohong21.Department of Pharmacy, the First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, Guang⁃dong Province, 510062 China;2.Department of Pharmacy, Guangdong Sanjiu Brain Hospital, Guangzhou, Guangdong Province, 510510 China[Abstract] Objective To investigate the clinical effect of quadruple drug therapy in patients with gastric ulcer. Methods Seventy-six patients with gastric ulcer admitted to the First Affiliated Hospital of Guangdong Pharmaceuti⁃cal University from January 2022 to August 2023 were selected as the research object and divided into groups ac⁃cording to coin tossing method. The reference group (38 cases) received triple drug therapy, and the study group (38 cases) received quadruple drug therapy. The total effective rate, the score of heartburn pain, the area of gastric ulcer, the score of upper abdominal pain and the improvement time of clinical symptoms were compared between the two groups. Results The total effective rate of the study group was 97.37%, which was significantly higher than that of the reference group, and the difference was statistically significant (χ2=6.176, P<0.05). After treatment, the score of heartburn pain, the area of gastric ulcer, the score of upper abdominal pain and the improvement time of clinical symptoms in the study group were lower than those in the reference group, and the differences were statistically sig⁃nificant (all P<0.05). Conclusion Compared with triple drug, quadruple drug treatment for gastric ulcer patients can significantly improve the clinical effect, effectively improve the disease symptoms, and promote the good prognosis of patients with gastric ulcer.[Key words] Gastric ulcer; Triple drug; Quadruple drugs; Curative effect对于胃溃疡疾病而言，其属于一种胃肠道高发病[1-2]。

氧化应激的适应性反应：细菌真菌植物动物

ReviewAdaptive response to oxidative stress:Bacteria,fungi,plants and animalsVolodymyr I.Lushchak ⁎Department of Biochemistry and Biotechnology,Vassyl Stefanyk Precarpathian National University,57Shevchenko Str.,76025,Ivano-Frankivsk,Ukrainea b s t r a c ta r t i c l e i n f o Article history:Received 13August 2010Received in revised form 8October 2010Accepted 8October 2010Available online 16October 2010Keywords:Reactive species Oxidative stress OxyR SoxRS NPR1/TGA Rap2.4a Keap1/Nrf2NF-κB AP-1Reactive oxygen species (ROS)are continuously produced and eliminated by living organisms normally maintaining ROS at certain steady-state levels.Under some circumstances,the balance between ROS generation and elimination is disturbed leading to enhanced ROS level called “oxidative stress ”.The primary goal of this review is to characterize two principal mechanisms of protection against oxidative stress —regulation of membrane permeability and antioxidant potential.The ancillary goals of this work are to describe up to date knowledge on the regulation of the previously mentioned mechanisms and to identify areas of prospective research and emerging directions in investigation of adaptation to oxidative stress.The ubiquity for challenges leading to oxidative stress development calls for identi ﬁcation of common mechanisms.They are cysteine residues and [Fe,S]-clusters of speci ﬁc regulatory proteins.The latter mechanism is realized via SoxR bacterial protein,whereas the former mechanism is involved in operation of bacterial OxyR regulon,yeast H 2O 2-stimulon,plant NPR1/TGA and Rap2.4a systems,and animal Keap1/Nrf2,NF-κB and AP-1,and others.Although hundreds of studies have been carried out in the ﬁeld with different taxa,the comparative analysis of adaptive response is quite incomplete and therefore,this work aims to cover a plethora of phylogenetic groups to delineate common mechanisms.In addition,this article raises some questions to be elucidated and points out future directions of this research.The comparative approach is used to shed light on fundamental principles and mechanisms of regulation of antioxidant systems.The idea is to provide starting points from which we can develop novel tools and hypothesis to facilitate meaningful investigations in the physiology and biochemistry of organismic response to oxidative stress.©2010Elsevier Inc.All rights reserved.Contents 1.Introduction ..............................................................1762.Regulation of membrane permeability for reactive oxygen species ....................................1773.Regulation of antioxidant potential ...................................................1783.1.Coordination of bacterial response to oxidative stress .......................................1783.1.1.SoxR —a sensor for superoxide anion ..........................................1783.1.2.OxyR —a sensor for hydrogen peroxide .........................................1793.2.Fungal response to oxidative stress................................................1803.2.1.Transcrition factor Yap1p ................................................1803.2.2.Transcription factors Msn2p and Msn4p and others....................................1813.3.Plant responses .........................................................1823.3.1.Coordination via NPR1/TGA system ...........................................1823.3.2.Coordination via Rap2.4a system.............................................183Comparative Biochemistry and Physiology,Part C 153(2011)175–190Abbreviations:AHP1,alkylhydroperoxide reductase;AHR,aryl hydrocarbon receptor;ARE,antioxidant response element;2CPA,2-Cys peroxiredoxin-A;CRD,cysteine rich domain;Cul3,Cullin 3;DPH,diphenylhexatriene;EpRE,electrophile response element;ERK,extracellular signal-regulated kinases;JA,jasmonic acid;G6PDH,glucose-6-phosphate dehydrogenase;GSH1,γ-glutamylcysteine synthase;GSH2,glutathione synthase;GPX2(3),glutathione peroxidases;GSH,glutathione;HIF-1α,hypoxia inducible factor alfa;HPI and HPII,hydroxyperoxidases;IkB,inhibitory protein of NF-κB;IkK,IkB kinase;Kelp1,Kelch-like ECH-associated Protein 1;MAP-kinases,mitogen-activated protein kinases;MDR,multidrug resistance;MPT,mitochondrial permeability transition pore;PR-1gene,pathogenesis-related gene;NES,nuclear exporting sequence (NES);NF-κB,nuclear factor-κB;NOS,NO-synthase;NPR1,nonoexpressor of pathogenesis-related gene 1;Nrf2,NF-E2-related factor 2;OmpF,outer membrane protein F;PERK,PKR-like endoplasmic reticulum kinase;PKC,protein kinase C;PPAR,peroxisome proliferator-activated receptor;RNS,reactive nitrogen species;ROS,reactive oxygen species;SA,salicylic acid;SAR,systemic acquired resistance;SOD,superoxide dismutases;soxR,superoxide response;TMA-DPH,trimethylammonium diphenylhexatriene;TRX2,thioredoxins 2;TRR1,thioredoxin reductase 1;TSA1,thioredoxin peroxidases 1;VCAM-1,vascular cell adhesion molecule 1;bZip,basic domain/leucine zipper transcription factors.⁎Tel./fax:+380342714683.E-mail address:lushchak@pu.if.ua.1532-0456/$–see front matter ©2010Elsevier Inc.All rights reserved.doi:10.1016/j.cbpc.2010.10.004Contents lists available at ScienceDirectComparative Biochemistry and Physiology,Part Cj o u r n a l h o me p a g e :w w w.e l s e vi e r.c o m /l oc a t e /c bp c3.4.Redox signaling in animals ....................................................1833.4.1.Keap1/Nrf2signaling ..................................................1843.4.2.NF-κB,AP-1,MAP-kinases and others...........................................1854.Questions to be elucidated and future directions .............................................1854.1.Bacteria .............................................................1864.2.Fungi ..............................................................1864.3.Plants ..............................................................1864.4.Animals parative approach ......................................................1865.Conclusions ..............................................................186Acknowledgements .............................................................187References ....................... (187)1.IntroductionThe generation of reactive oxygen species (ROS)is an inevitable aspect of life under aerobic conditions.ROS are continuously produced as side-products of certain metabolic pathways and also by some speci ﬁc systems under ﬁne cellular control.At the same time,ROS are degraded via several mechanisms,both,speci ﬁc and non-speci ﬁc.Two different processes,generation and degradation of ROS,usually are under delicate cellular control and very low (b 10−8M)steady-state ROS concentrations are maintained (Halliwell and Gutteridge,1989).However,under some circumstances,the balance between ROS production and elimination is disturbed leading to their enhanced steady-state level called “oxidative stress ”.Its development is either the reason,or common event of many pathological states,including aging.Therefore,it is clear why oxidative stress investiga-tion has become increasingly popular not only from basic points of view,but also from different applied aspects including medicine,sport science,toxicology and environmental science.Cell survival of oxidative stress challenge depends on physiological state,intensity and nature of stress.The possibility to enhance protective potential is among the critical issues.Therefore,this review will deal with two well-characterized mechanisms of increase of cellular tolerance to oxidative stress:(i )permeability of cellular membranes to reactive species and (ii )up-regulation of antioxidant and associated enzymes.However,before the analysis of enhancement of tolerance,I will brie ﬂy discuss the key aspects of ROS metabolisms,their effects on cells and their consequences.Over 90%of oxygen consumed by living organisms is used to produce energy by oxidative phosphorylation with operation of the electron-transport chain via a four-electron mechanism leading to ATP and water production.Consecutive addition of one by one electron to oxygen molecule also ﬁnally leads to water production (but no ATP is produced)via intermediate ROS forms (Fig.1).Thelatter include free radicals such as superoxide anion (O 2•−)and hydroxyl radical (•OH)and also non-radical reactive species,such as hydrogen peroxide (H 2O 2)and singlet oxygen.They seem are the beststudied among all reactive species.Although very important,reactive nitrogen species (RNS)such as nitric oxide (•NO),nitric peroxide (peroxynitrite,OONO •−)and their derivatives have garnered less attention.Along with ROS,•NO and other gaseous compounds such as carbon monoxide (CO)and hydrogen sul ﬁde (H 2S)play important regulatory functions (Li and Moore,2007).The main part of ROS (usually over 90%)in living organisms is produced by electron-transport chains —mitochondrial,endoplasmic reticulum,plasmatic and nuclear membranes,and photosynthetic system (Starkov,2008).In addition,minor ROS amounts are generated by some enzymes such as oxidases,through autooxidation of different molecules.The oxidases known producing ROS are NADPH oxidase,lipoxygenases,cyclooxygenases,and xanthine oxidase (Puddu et al.,2008),whereas molecules entering autooxidation may be exogenous and endogenous origination like neurotoxin 5-hydroxydecanoate (Rodriguez-Pallares et al.,2009)or catecholamines (Callaway et al.,2003)and many other compounds.Fig.1demonstrates ROS metabolism.One electron reduction of molecular oxygen ﬁnally yields water and can be realized spontane-ously.However,certain enzymes can enhance the speed of the transformations many-fold.Superoxide dismutases (SODs;EC 1.15.1.1)speed-up the dismutation of superoxide anion to molecular oxygen and hydrogen peroxide.Catalases (EC 1.11.1.6)decompose hydrogen peroxide,yielding water and molecular oxygen,and peroxidases (EC 1.11.1.x),using some reductants and peroxides as cosubstrates,produce water and reduced products.The hydroxyl radical is the most active oxidant among the previously mentioned ROS.It is short-lived and,therefore,has a short diffusion distance.Probably for this reason living organisms have not developed speci ﬁc enzymatic systems for its detoxi ﬁcation.The prevention of its production is the most ef ﬁcient way to protect cells against deleterious •OH effects.Low molecular mass compounds such as vitamins C and E,glutathione (GSH),uric acid and some others form a group of low molecular mass antioxidants some of which can directly neutralize •OH.Interaction of ROS with cellular components depends on many circumstances.However,place of formation,nature of ROS and target molecules are among main determinants.Chemical activity of ROS isenhanced in the order:O 2•−–H 2O 2–•OH.In fact,all cellular constituents can be modi ﬁed by ROS.However,several types are especially susceptible.The enzymes containing [Fe –S]-clusters such as aconitase (E.C.4.2.1.3),6-phosphogluconate dehydratase (E.C.4.2.1.12),and fumarate hydratase (E.C. 4.2.1.2),as well as ones possessing active thiol groups (e.g.glyceraldehyde-3-phosphate dehydrogenase (E.C.1.2.1.12))are particu-larly sensitive to ROS (Busi et al.,2006;Bouton,1999;Hancock et al.,2006).Most ROS-modi ﬁed molecules are subjected to degradation,but some may be accumulated in the cell.However,only few types of molecules modi ﬁed by ROS can be repaired:this includes DNA,and oxidized cysteine and methionine residues in proteins that may be reduced to their initial forms (Lushchak,2007).As mentioned previously,ROS are continuously produced and eliminated and,therefore,their concentration is a dynamicparameterFig.1.Different ways of oxygen reduction in biological systems.The upper part of the scheme shows four-electron reduction of oxygen molecule in electron transport chain.The bottom part demonstrates the consecutive addition of one electron to oxygen molecule with the formation of intermediate products –reactive oxygen species –superoxide anion radical,hedrogen peroxide and hydroxyl radical.To the end,partially reduced forms may accept four electrons and combining with protons give water molecule.In both cases the maximally reduced state of oxygen is reached in water molecule.176V.I.Lushchak /Comparative Biochemistry and Physiology,Part C 153(2011)175–190called “steady-state ”.Certain challenges may change it.The situation when due to some reasons the steady-state ROS concentration is acutely or chronically increased leading to oxidative modi ﬁcation of cellular constituents resulting in disturbance of cellular metabolism and regulatory pathways,particularly ROS-based has been called “oxidative stress ”.In some cases,the steady-state ROS concentration does not return to initial level,but stabilizes at new one called “quasi-stationary level ”(Fig.2).This can be found in certain pathologies,for example diabetes mellitus,atherosclerosis,cardiovascular and neurodegenerative dis-eases (Shen,2010;Runge et al.,2010).The increased ROS concentration may result from enhanced production,decreased degradation or both these changes simultaneously.The situation with decreased ROS steady-state level with followed consequences in opposite to oxidative stress is called “reductive stress ”.The latter is less studied than oxidative stress and the adaptations to it are yet to be characterized due to which it will not be covered in this review.However,this review will cover two main adaptations to oxidative stress —the decrease in permeability of cellular membranes and up-regulation of antioxidant and associated enzymes.2.Regulation of membrane permeability for reactive oxygen speciesIt has been long known that superoxide anion may penetrate biological membranes via anion channels,while hydrogen peroxide was thought to cross them freely (Chance et al.,1979).However,the latter issue was challenged at the beginning of this century,and evidence on the existence of a transmembrane H 2O 2gradient has been accumulating.Antunes and Cadenas (2000)using the concept of catalase latency,in Jurkat T-cells were able to evaluate the possible gradients of H 2O 2on cytoplasmic and peroxisomal membranes.Other evidence for the existence of H 2O 2gradients at biological membranes came from bacteria.Seaver and Imlay (2001)exploited enterobacteria Escherichia coli model and were able to demonstrate that the plasma membrane is not freely permeable for H 2O 2.The authors measured the activity of two hydroxyperoxidases responsible for H 2O 2decomposition in E.coli namely HPI and HPII (or Ahp).At micromolar external concentrations of the oxidant,the H 2O 2decomposition rate by intact cells did not correspond to one obtained with cellular extracts.This again showed the presence of latent activity which was interpreted as the existence of a barrier,representing actually cellular membrane.However,at millimolar levels of H 2O 2the difference was not substantial.We also independently measured the activity of catalase with millimolar H 2O 2concentrations in intact and disrupted E .coli cells and found virtually no difference in both cases (Semchyshyn et al.,2002).Therefore,it was concluded that under conditions used by us H 2O 2freely penetrated plasmatic membranes.Seaver and Imlay (2001)discussed the possibility of modi ﬁcation of membrane permeability and its possible involvement in regulation of sensitivity to externally induced oxidative stress.These authors also assumed that under certain conditions bacteria can excrete redox-cycling compounds to kill other cells,but do not experience the same H 2O 2-induced stress as their competitors.It means that the regulation of permeability for H 2O 2of cellular membranes may be bene ﬁcial to compete with other bacteria.The question on possible mechanisms of H 2O 2transport via biological membranes is very important.Henzler and Steudle (2000)investigated the transport and degradation of H 2O 2in isolated internodal cells of green algae Chara corallina and developed the ﬁrst model of this process.These cells were able to tolerate very high H 2O 2concentrations —as much as 100mM.High intensity of intracellular peroxide degradation was connected with operation of catalases and peroxidases.Henzler and Steudle (2000)established a physical/mathematical model predicting the osmotic reactions of internodes (water and salute ﬂows in the presence of catalase action),when H 2O 2was added to the external medium.The proposed model was tested experimentally.Interestingly,mercuric chloride (HgCl 2),a water channel inhibitor,reduced perme-ability for both,water and H 2O 2.Therefore,the authors suggested that some water channels in C.corrallina ,and probably in other organisms,operate as “peroxoporins ”rather than as “aquaporins ”(Henzler and Steudle,2000).The latter idea was further explored by Bienert et al.(2006,2007),who,using budding yeast Saccharomyces cerevisiae as a model,expressed 24aquaporins from plants and mammals in ﬁve yeast strains with different sensitivity toward oxidative stress.The expression of human AQP8and plant Arabidopsis thaliania TIP1;1and TIP1;2in yeast decreased growth and survival in the presence of H 2O 2.S .cerevisiae genome itself contains four open reading frames for aquaporins,but their deletion did not change yeast sensitivity to H 2O 2,making this yeast an excellent model to reveal a role of expressed foreign aquaporins.Interestingly,loading of yeast cells with ROS-sensitive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydro ﬂuorescein diacetate acetyl ester con ﬁrmed H 2O 2diffusion into the cells.Silver ions,blockers of aquaporin-mediated water diffusion,reversed both,aquaporin-depen-dent growth repression and H 2O 2-induced ﬂuorescence.The described data on S .cerevisiae along with ones on C .coralline show that aquaporins may be responsible for regulated transport of H 2O 2via biological membranes (Bienert et al.,2006,2007).In 2004,it was found that H 2O 2challenges in S.cerevisiae affected cellular permeability to H 2O 2(Sousa-Lopes et al.,2004).The yeast cells were treated with steady-state H 2O 2-delivery system using L -α-amino acid oxidase instead of the commonly applied glucose oxidase.The cellular permeability to H 2O 2in the stationary phase was 5-times lower,than in the exponential phase and cell survival at H 2O 2challenges correlated with H 2O 2-gradients.The limited H 2O 2diffusion into the cell was implicated to be a key protective mechanism against extracellular H 2O 2.The regulation of permeability was connected with membrane composition.Knocking out genes ERG3and ERG6,leading to defects in ergosterol biosynthesis,caused a lower H 2O 2gradient in yeast cells (Sousa-Lopes et al.,2004).The next question arising is whether the permeability of yeast cell plasma membrane can be regulated by H 2O 2.S .cerevisiae cells treatment with low concentrations of H 2O 2,clearly enhanced cell tolerance,which was connected with decreased permeability to H 2O 2(Branco etal.,Fig.2.The modern ideas on the dynamics of reactive oxygen species in biological systems.The steady-state level of reactive oxygen and nitrogen species under normal conditions and stresses leading to acute or chronic increase/decrease stationary levels of reactive species.If constitutive antioxidant potential is high enough to restore the initial levels of reactive species,after induction of the stress the level of reactive species level quickly returns to initial steady-state one.If the constitutive antioxidant potential is not high enough to eliminate excess of ROS,the system may either increase it and return to initial level,or reach a new steady-state level,which may be called “quasi-stationary level ”.The same logic can be applied to reductive stress –with the only oppist direction –decrerased ROS level.177V.I.Lushchak /Comparative Biochemistry and Physiology,Part C 153(2011)175–1902004).It became clear that,in addition to enhancement of antioxidant potential described earlier,the regulation of cell membrane permeabil-ity for H2O2is a mechanism responsible for yeast sensitivity to this compound.In addition,cycloheximide,an inhibitor of protein synthesis in eukaryotes,enhanced the tolerance.In this case,the up-regulation of antioxidant enzymes,activities of which are mainly increased via de novo synthesis,was not involved.Branco et al.(2004)suggested that this effect of cycloheximide could be connected with reduced steady-state level of HMG-CoA-reductase,the assumed rate-limiting enzyme in the ergosterol biosynthesis pathway.Because of the potential involvement of plasma membrane compo-sition in the adaptive regulation of H2O2-permeability,the role of fatty acid synthase,encoded by FAS1gene,was investigated(Matias et al., 2007).Very good inverse correlation was found between yeast survival under challenge with H2O2and the activity of fatty acid synthase.It was suggested that very-long-chain fatty acids through interdigitation or by modulating the formation of lipid rafts might decrease the overall or local plasma membrane permeability to H2O2,respectively,resulting in a higher resistance to H2O2(Matias et al.,2007).The involvement of modiﬁcation of cellular membrane composi-tion called for investigation of its physicochemical properties.Because it seemed that aquaporin was not involved in the regulation of plasma membrane permeability(revealed using inhibitor mercuric chloride), a simple diffusion was suggested to be responsible in this case(Folmer et al.,2008).The plasma membraneﬂuidity was evaluated with two ﬂuorescent probes,namely diphenylhexatriene(DPH)and trimethy-lammonium diphenylhexatriene(TMA-DPH).Both probes demon-strated the enhanced steady-stateﬂuorescence anisotropy after yeast incubation with H2O2which correlated with decreased plasma membrane permeability for H2O2.Disruption of ergosterol synthesis prevented the changes in anisotropy.Therefore,it was concluded that plasma membrane permeability might be regulated via modiﬁcation of its physicochemical properties(Folmer et al.,2008).A recent article on the regulation of plasma membrane permeability to H2O2of yeast cells(Pedroso et al.,2009)summarizes the known information on the topic.It shows that the adaptation to H2O2challenges rapidly modulates the expression of genes encoding enzymes involved in estrogen and lipid catabolism,leading to alterations of membrane lipid composition.The most prominent changes were:(i)decrease in the ratio between saturated and unsaturated long-chain fatty acids;(ii)decrease in content of oleic acid;(iii)increase in the ratio phosphatidylcholine/ phosphatidylethanolamine;(iv)enhancement in the level of sterol precursor squalene despite not changed sterol level;and(v)C26:0 became the major very-long-chain fatty acid with concominant decrease 2-hydroxy-C26:0and C20:0.It was concluded that H2O2led to reorganization of the plasma membrane structure as a result of rather quick changes in its chemical composition(Pedroso et al.,2009).The previous section shows that the active regulation of plasma membrane permeability may be a component of modiﬁcation of H2O2 diffusion.Cell culture transition from exponential to stationary phase results in decreased sensitivity for H2O2,as well as other challenges and this can be related with decreased permeability of the plasma membrane.However,under certain conditions an acute or chronic cell exposure to H2O2also may increase cell tolerance.Rapid changes in membrane permeability to H2O2may be responsible for the fast tolerance enhancement.Although at least two mechanisms,regulated transport via pores and simple diffusion,may be potentially responsible for the regulation of membrane permeability,at the moment we lack the mechanistic explanation of this phenomenon.3.Regulation of antioxidant potentialDespite the possibility to regulate ROS inﬂux into the cell,the regulation of cellular antioxidant potential seems more critical for survival under continuous exposure to externally induced oxidative stress. Therefore,the regulation of this potential will be described in some details.Several steps are important for this regulation,including sensing of reactive species and transduction of the signal to transcription and translation machineries.That is because the activity of antioxidant enzymes is mainly regulated at these levels.However,it should be noted,that posttranslational regulation can be involved in regulation of activity of these enzymes(Semchyshyn,2009),although the latter issue is still at an infant stage and needs further investigation.Therefore,in the following sections the best studied pathways of up-regulation of activities of antioxidant enzymes will be described.Being rather nonspeciﬁc,ROS interaction with speciﬁc sensors provides high speciﬁcity of ROS-induced pathways.The sensing and transduction of ROS-based signaling is mainly centered on sulfur chemistry,with the main player being the reversible oxidation of speciﬁc cysteine residues of certain proteins.It seems this is correct for bacterial OxyR system,yeast Yap1,plant NPR1/TGA and Rap2.4a systems,and animal Keap1/Nrf2,NF-κB and AP-1systems.In addition,the bacterial SoxR system uses another strategy—the protein possesses[Fe–S]-cluster capable of reversible oxidation.3.1.Coordination of bacterial response to oxidative stressThe adaptive response of bacterial antioxidant systems toward oxidative stress is coordinated mainly by two well studied systems, grouped in two regulons—SoxRS and OxyR.Theﬁrst responds to the stress induced by superoxide anion,while the second responds to the hydrogen peroxide-induced one.In both cases,the sensing step involves the oxidation of sensor molecules.Although other systems exist,we will focus on SoxRS and OxyR because molecular aspects of their operation are well characterized,with the enterobacterium Escherichia coli as the best studied model.3.1.1.SoxR—a sensor for superoxide anionIn E.coli cells,the genetic locus called soxR(superoxide response) positively regulates9of about40superoxide-activated proteins (Greenberg et al.,1990).The proteins are induced at the transcription level and include antioxidant manganese-containing superoxide dis-mutase(Mn-SOD),endonuclease IV(repairing radical-induced damage to DNA,EC3.1.21.2),and glucose-6-phosphate dehydrogenase(G6PDH, EC1.1.1.49).Induction of the SoxRS regulon also reduces the level of outer membrane protein(porin)F(OmpF)which is an integral membrane protein located in the outer membrane of the bacteria operating as a non-speciﬁc transport channel,and modiﬁes the level of small ribosomal protein S6(Storz and Imlay,1999;Lushchak,2001). Therefore,the described regulon operates as a part of the inducible protection system against xenobiotics(Greenberg et al.,1990).The soxRS regulon can be induced also by nitric oxide radical(Nunoshiba et al.,1993a,b;De Groote et al.,1997)and hypochlorous acid(Dukan et al.,1996),which may play a role in resistance to macrophages(Poole, 2005;Koutsolioutsou et al.,2005).A model of the operation of SoxRS system is given at Fig.3.The product of the soxR gene,SoxR protein,binds to E.coli DNA at speciﬁc site activating the expression of soxS gene,resulting in increased SoxS protein level(Demple and Amabile-Cuevas,1991;Hidalgo et al., 1997a,b).The latter protein activates genes which are a part of the general response of E.coli cells to increased generation of superoxide anion and nitric oxide.SoxS limits the transcription of soxS in vivo and binds to soxS promoter in vitro(Nunoshiba et al.,1993a,b).The protein binds promoters of target genes such as micF(encoding a non-coding RNA responsive to stress that post-transcriptionally controls expression of ompF),zwf(encoding glucose6-phosphate dehydroge-nase),nfo(encoding endonuclease IV),and sodA(encoding A or manganese superoxide dismutase—MnSOD).The binding of SoxS with micF and zwf promoters accelerates subsequent interaction between RNA-polymerase and the promoters(Li and Demple,1994; Ariza et al.,1995).Therefore,it was supposed that the small regulatory protein SoxS with a molecular mass of12.9kDa is a direct inducer of target genes of bacterial response to oxidative stress,although the178V.I.Lushchak/Comparative Biochemistry and Physiology,Part C153(2011)175–190。

临床药师参与1例大剂量甲氨蝶呤排泄延迟的案例分析

临床药师参与1例大剂量甲氨蝶呤排泄延迟的案例分析张小丹，孙言才作者单位：安徽省肿瘤医院［中国科学技术大学附属第一医院（安徽省立医院）西区］药剂科，安徽合肥230031通信作者：孙言才，男，主任药师，硕士生导师，研究方向为医院药学与药事管理，Email：133****************摘要：目的通过临床药师参与救治大剂量甲氨蝶呤（HD-MTX）排泄延迟病人，促进临床HD-MTX的安全、合理使用。

方法安徽省肿瘤医院临床药师对2018年10月收治的1例HD-MTX排泄延迟病人实施全程化药学监护，积极分析甲氨蝶呤排泄延迟发生原因及血药浓度波动原因，提出合理性药学服务建议，协助医师制定个体化用药方案。

结果病人HD-MTX化疗后出现早期排泄延迟，临床药师分析认为其原因可能与尿pH值过低、药物相互作用、肾功能损伤、第三空间存在、低蛋白血症等因素相关；之后，病人甲氨蝶呤血药浓度出现波动现象，临床药师分析可能与利尿药的使用、肾功能进一步恶化及第三空间的存在相关。

通过以上分析，临床药师及时建议充分碱化尿液促进甲氨蝶呤排泄；停用可能导致甲氨蝶呤排泄延迟的药物泮托拉唑，改用雷尼替丁继续治疗；避免使用磺胺类药物以免排泄延迟及肾功能损伤进一步加重等。

在药师的积极参与下，病人成功救治，顺利出院。

结论HD-MTX排泄延迟的发生及血药浓度变化受多种因素影响，临床药师参与临床药物治疗，有助于促进临床合理用药，保障病人用药安全。

关键词：药学服务；药物相关性副作用和不良反应；临床药师；甲氨蝶呤；延迟排泄Clinical pharmacists participating in a case of delayed excretion of high-dose methotrexate:case analysisZHANG Xiaodan，SUN YancaiAuthor Affiliation:Department of Pharmacy,Anhui Provincial Cancer Hospital(West Branch of The First Affiliated Hospital of USTC),Hefei,Anhui230031,ChinaAbstract:Objective To promote safe and rational use of high-dose methotrexate(HD-MTX)in clinical by clinical pharmacists par⁃ticipating in the treatment of patients with delayed excretion of HD-MTX.Methods Clinical pharmacists of Anhui Provincial Cancer Hospital implemented whole-process pharmaceutical care on a patient with delayed excretion of HD-MTX admitted in October2018, analysed the reasons for delayed excretion of MTX and plasma concentration fluctuations,put forward reasonable pharmaceutical care advice,assisted physicians to make individualized medication.Results The patient had early delayed excretion after HD-MTX che⁃motherapy,clinical pharmacist analyzed that the cause might be related to factors such as low urine pH,drug interactions,renal dam⁃age,the existence of the third space,and hypoproteinemia.After that,the blood concentration of MTX fluctuated,clinical pharmacists analyzed that it might be related to the use of diuretics,further deterioration of renal function and the existence of the third space.Based on the above analyses,clinical pharmacists promptly suggested the following:fully alkalize urine to promote MTX excretion;discontinu⁃ation of pantoprazole,which may cause delayed excretion of MTX,and switch to ranitidine to continue treatment;avoid using sulfon⁃amides to avoid further delayed excretion and renal injury.With the active participation of pharmacists,the patient was rescued suc⁃cessfully and discharged smoothly.Conclusion The occurrence of delayed excretion of HD-MTX and the change of plasma concentra⁃tion are affected by many factors,clinical pharmacists participate in clinical drug therapy is helpful to promote rational clinical drug use and ensure the medication safety of patients.Key words:Pharmaceutical services;Drug-related side effects and adverse reactions;Clinical pharmacists;Methotrexate; Delayed excretion甲氨蝶呤是一种抗代谢类抗肿瘤药物，为二氢叶酸还原酶抑制剂，可与二氢叶酸还原酶形成不可逆的结合，阻止该酶将体内二氢叶酸还原成四氢叶酸，造成DNA合成障碍，从而阻止肿瘤细胞的生长和增殖［1］。

外胚层发育不良受体EDA2R的研究进展

肿瘤坏死因子受体超级家族（tumor necrosis fac⁃tor receptor superfamily，TNFRSF）的死亡受体（death receptor）以及它们的配体在胚胎正常发育及机体免疫和炎症反应过程中扮演了重要角色。

外胚层发育不良受体（ectodysplasin A2receptor，EDA2R）是一个在20年前被鉴定出来的TNFRSF成员（TNFRSF27）［1］，在肿瘤发生、雄激素性脱发等过程中起到重要的作用，但对于该受体作系统性介绍的综述文章尚未见报道。

本文就该受体的研究进展作一系统性的综述，旨在为相关研究提供新的思路。

1EDA2R的蛋白结构和配体1.1EDA2R的蛋白结构EDA2R基因位于人类染色体Xq12，全长约43kb，有6个外显子（GenBank登录号：NG_013271），外胚层发育不良受体EDA2R的研究进展蓝希钳1，2，肖海婷1，2，罗怀容1，2，陈建宁1，2（西南医科大学药学院：1衰老与再生医学实验室，2药理学教研室，四川泸州646000）【摘要】外胚层发育不良受体EDA2R（ectodysplasin A2receptor）是肿瘤坏死因子受体超级家族（tumor necrosis factor recep⁃tor superfamily，TNFRSF）中的一个较新的成员，在发育中的胚胎里有很高的表达，在成年人和动物的多个器官组织中也有表达。

与其它TNFRSF成员不同，尽管EDA2R蛋白在胞内没有死亡结构域（death domain，DD），但它仍可激活NF-κB和JNK通路，并介导细胞的凋亡。

本文广泛回顾了近年来与EDA2R有关的文献，就该蛋白分子的相关研究进展进行综述，以期为与该蛋白相关的分子功能或其介导的相关疾病的研究提供新的思路。

【关键词】EDA2R受体肿瘤坏死因子受体超级家族死亡结构域凋亡【中图分类号】R34文献标志码A doi：10.3969/j.issn.2096-3351.2021.03.018Research progress of ectodysplasin A2receptorLAN Xi-qian1，2，XIAO Hai-ting1，2，LUO Huai-rong1，2，CHEN Jian-ning1，2 1Key Laboratory for Aging and Regenerative Medicine；2Department of Pharmacology，School of Pharmac，South⁃west Medical University，Luzhou646000，Sichuan，China【Abstract】Ectodysplasin A2receptor（EDA2R）is a relatively new member of the tumor necrosis factor re⁃ceptor superfamily（TNFRSF），and it is highly expressed in developing embryos and is also expressed in multiple organs and tissues of adult human and animals.Different from other TNFRSF members，EDA2R protein does not contain the death domain in the intracellular region，but it can still activate the NF-κB and JNK pathways and medi⁃ate cell apoptosis.This article reviews related articles on EDA2R in recent years and related research advances in this protein，in order to provide new ideas for research on molecular functions associated with EDA2R or related dis⁃eases mediated by EDA2R.【Key words】Ectodysplasin A2receptor Tumor necrosis factor receptor superfamily Death domain Apoptosis基金项目：泸州市科技局－西南医科大学联合项目（2018LZXNYD-ZK12）；西南医科大学-泸州市中医医院基地项目（2019-LH005）第一作者简介：蓝希钳，博士。

IEC-61854架空线.隔离层的要求和检验

NORMEINTERNATIONALECEI IEC INTERNATIONALSTANDARD 61854Première éditionFirst edition1998-09Lignes aériennes –Exigences et essais applicables aux entretoisesOverhead lines –Requirements and tests for spacersCommission Electrotechnique InternationaleInternational Electrotechnical Commission Pour prix, voir catalogue en vigueurFor price, see current catalogue© IEC 1998 Droits de reproduction réservés Copyright - all rights reservedAucune partie de cette publication ne peut être reproduite niutilisée sous quelque forme que ce soit et par aucunprocédé, électronique ou mécanique, y compris la photo-copie et les microfilms, sans l'accord écrit de l'éditeur.No part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical,including photocopying and microfilm, without permission in writing from the publisher.International Electrotechnical Commission 3, rue de Varembé Geneva, SwitzerlandTelefax: +41 22 919 0300e-mail: inmail@iec.ch IEC web site http: //www.iec.chCODE PRIX PRICE CODE X– 2 –61854 © CEI:1998SOMMAIREPages AVANT-PROPOS (6)Articles1Domaine d'application (8)2Références normatives (8)3Définitions (12)4Exigences générales (12)4.1Conception (12)4.2Matériaux (14)4.2.1Généralités (14)4.2.2Matériaux non métalliques (14)4.3Masse, dimensions et tolérances (14)4.4Protection contre la corrosion (14)4.5Aspect et finition de fabrication (14)4.6Marquage (14)4.7Consignes d'installation (14)5Assurance de la qualité (16)6Classification des essais (16)6.1Essais de type (16)6.1.1Généralités (16)6.1.2Application (16)6.2Essais sur échantillon (16)6.2.1Généralités (16)6.2.2Application (16)6.2.3Echantillonnage et critères de réception (18)6.3Essais individuels de série (18)6.3.1Généralités (18)6.3.2Application et critères de réception (18)6.4Tableau des essais à effectuer (18)7Méthodes d'essai (22)7.1Contrôle visuel (22)7.2Vérification des dimensions, des matériaux et de la masse (22)7.3Essai de protection contre la corrosion (22)7.3.1Composants revêtus par galvanisation à chaud (autres queles fils d'acier galvanisés toronnés) (22)7.3.2Produits en fer protégés contre la corrosion par des méthodes autresque la galvanisation à chaud (24)7.3.3Fils d'acier galvanisé toronnés (24)7.3.4Corrosion causée par des composants non métalliques (24)7.4Essais non destructifs (24)61854 © IEC:1998– 3 –CONTENTSPage FOREWORD (7)Clause1Scope (9)2Normative references (9)3Definitions (13)4General requirements (13)4.1Design (13)4.2Materials (15)4.2.1General (15)4.2.2Non-metallic materials (15)4.3Mass, dimensions and tolerances (15)4.4Protection against corrosion (15)4.5Manufacturing appearance and finish (15)4.6Marking (15)4.7Installation instructions (15)5Quality assurance (17)6Classification of tests (17)6.1Type tests (17)6.1.1General (17)6.1.2Application (17)6.2Sample tests (17)6.2.1General (17)6.2.2Application (17)6.2.3Sampling and acceptance criteria (19)6.3Routine tests (19)6.3.1General (19)6.3.2Application and acceptance criteria (19)6.4Table of tests to be applied (19)7Test methods (23)7.1Visual examination (23)7.2Verification of dimensions, materials and mass (23)7.3Corrosion protection test (23)7.3.1Hot dip galvanized components (other than stranded galvanizedsteel wires) (23)7.3.2Ferrous components protected from corrosion by methods other thanhot dip galvanizing (25)7.3.3Stranded galvanized steel wires (25)7.3.4Corrosion caused by non-metallic components (25)7.4Non-destructive tests (25)– 4 –61854 © CEI:1998 Articles Pages7.5Essais mécaniques (26)7.5.1Essais de glissement des pinces (26)7.5.1.1Essai de glissement longitudinal (26)7.5.1.2Essai de glissement en torsion (28)7.5.2Essai de boulon fusible (28)7.5.3Essai de serrage des boulons de pince (30)7.5.4Essais de courant de court-circuit simulé et essais de compressionet de traction (30)7.5.4.1Essai de courant de court-circuit simulé (30)7.5.4.2Essai de compression et de traction (32)7.5.5Caractérisation des propriétés élastiques et d'amortissement (32)7.5.6Essais de flexibilité (38)7.5.7Essais de fatigue (38)7.5.7.1Généralités (38)7.5.7.2Oscillation de sous-portée (40)7.5.7.3Vibrations éoliennes (40)7.6Essais de caractérisation des élastomères (42)7.6.1Généralités (42)7.6.2Essais (42)7.6.3Essai de résistance à l'ozone (46)7.7Essais électriques (46)7.7.1Essais d'effet couronne et de tension de perturbations radioélectriques..467.7.2Essai de résistance électrique (46)7.8Vérification du comportement vibratoire du système faisceau/entretoise (48)Annexe A (normative) Informations techniques minimales à convenirentre acheteur et fournisseur (64)Annexe B (informative) Forces de compression dans l'essai de courantde court-circuit simulé (66)Annexe C (informative) Caractérisation des propriétés élastiques et d'amortissementMéthode de détermination de la rigidité et de l'amortissement (70)Annexe D (informative) Contrôle du comportement vibratoire du systèmefaisceau/entretoise (74)Bibliographie (80)Figures (50)Tableau 1 – Essais sur les entretoises (20)Tableau 2 – Essais sur les élastomères (44)61854 © IEC:1998– 5 –Clause Page7.5Mechanical tests (27)7.5.1Clamp slip tests (27)7.5.1.1Longitudinal slip test (27)7.5.1.2Torsional slip test (29)7.5.2Breakaway bolt test (29)7.5.3Clamp bolt tightening test (31)7.5.4Simulated short-circuit current test and compression and tension tests (31)7.5.4.1Simulated short-circuit current test (31)7.5.4.2Compression and tension test (33)7.5.5Characterisation of the elastic and damping properties (33)7.5.6Flexibility tests (39)7.5.7Fatigue tests (39)7.5.7.1General (39)7.5.7.2Subspan oscillation (41)7.5.7.3Aeolian vibration (41)7.6Tests to characterise elastomers (43)7.6.1General (43)7.6.2Tests (43)7.6.3Ozone resistance test (47)7.7Electrical tests (47)7.7.1Corona and radio interference voltage (RIV) tests (47)7.7.2Electrical resistance test (47)7.8Verification of vibration behaviour of the bundle-spacer system (49)Annex A (normative) Minimum technical details to be agreed betweenpurchaser and supplier (65)Annex B (informative) Compressive forces in the simulated short-circuit current test (67)Annex C (informative) Characterisation of the elastic and damping propertiesStiffness-Damping Method (71)Annex D (informative) Verification of vibration behaviour of the bundle/spacer system (75)Bibliography (81)Figures (51)Table 1 – Tests on spacers (21)Table 2 – Tests on elastomers (45)– 6 –61854 © CEI:1998 COMMISSION ÉLECTROTECHNIQUE INTERNATIONALE––––––––––LIGNES AÉRIENNES –EXIGENCES ET ESSAIS APPLICABLES AUX ENTRETOISESAVANT-PROPOS1)La CEI (Commission Electrotechnique Internationale) est une organisation mondiale de normalisation composéede l'ensemble des comités électrotechniques nationaux (Comités nationaux de la CEI). La CEI a pour objet de favoriser la coopération internationale pour toutes les questions de normalisation dans les domaines de l'électricité et de l'électronique. A cet effet, la CEI, entre autres activités, publie des Normes internationales.Leur élaboration est confiée à des comités d'études, aux travaux desquels tout Comité national intéressé par le sujet traité peut participer. Les organisations internationales, gouvernementales et non gouvernementales, en liaison avec la CEI, participent également aux travaux. La CEI collabore étroitement avec l'Organisation Internationale de Normalisation (ISO), selon des conditions fixées par accord entre les deux organisations.2)Les décisions ou accords officiels de la CEI concernant les questions techniques représentent, dans la mesuredu possible un accord international sur les sujets étudiés, étant donné que les Comités nationaux intéressés sont représentés dans chaque comité d’études.3)Les documents produits se présentent sous la forme de recommandations internationales. Ils sont publiéscomme normes, rapports techniques ou guides et agréés comme tels par les Comités nationaux.4)Dans le but d'encourager l'unification internationale, les Comités nationaux de la CEI s'engagent à appliquer defaçon transparente, dans toute la mesure possible, les Normes internationales de la CEI dans leurs normes nationales et régionales. Toute divergence entre la norme de la CEI et la norme nationale ou régionale correspondante doit être indiquée en termes clairs dans cette dernière.5)La CEI n’a fixé aucune procédure concernant le marquage comme indication d’approbation et sa responsabilitén’est pas engagée quand un matériel est déclaré conforme à l’une de ses normes.6) L’attention est attirée sur le fait que certains des éléments de la présente Norme internationale peuvent fairel’objet de droits de propriété intellectuelle ou de droits analogues. La CEI ne saurait être tenue pour responsable de ne pas avoir identifié de tels droits de propriété et de ne pas avoir signalé leur existence.La Norme internationale CEI 61854 a été établie par le comité d'études 11 de la CEI: Lignes aériennes.Le texte de cette norme est issu des documents suivants:FDIS Rapport de vote11/141/FDIS11/143/RVDLe rapport de vote indiqué dans le tableau ci-dessus donne toute information sur le vote ayant abouti à l'approbation de cette norme.L’annexe A fait partie intégrante de cette norme.Les annexes B, C et D sont données uniquement à titre d’information.61854 © IEC:1998– 7 –INTERNATIONAL ELECTROTECHNICAL COMMISSION––––––––––OVERHEAD LINES –REQUIREMENTS AND TESTS FOR SPACERSFOREWORD1)The IEC (International Electrotechnical Commission) is a worldwide organization for standardization comprisingall national electrotechnical committees (IEC National Committees). The object of the IEC is to promote international co-operation on all questions concerning standardization in the electrical and electronic fields. To this end and in addition to other activities, the IEC publishes International Standards. Their preparation is entrusted to technical committees; any IEC National Committee interested in the subject dealt with may participate in this preparatory work. International, governmental and non-governmental organizations liaising with the IEC also participate in this preparation. The IEC collaborates closely with the International Organization for Standardization (ISO) in accordance with conditions determined by agreement between the two organizations.2)The formal decisions or agreements of the IEC on technical matters express, as nearly as possible, aninternational consensus of opinion on the relevant subjects since each technical committee has representation from all interested National Committees.3)The documents produced have the form of recommendations for international use and are published in the formof standards, technical reports or guides and they are accepted by the National Committees in that sense.4)In order to promote international unification, IEC National Committees undertake to apply IEC InternationalStandards transparently to the maximum extent possible in their national and regional standards. Any divergence between the IEC Standard and the corresponding national or regional standard shall be clearly indicated in the latter.5)The IEC provides no marking procedure to indicate its approval and cannot be rendered responsible for anyequipment declared to be in conformity with one of its standards.6) Attention is drawn to the possibility that some of the elements of this International Standard may be the subjectof patent rights. The IEC shall not be held responsible for identifying any or all such patent rights. International Standard IEC 61854 has been prepared by IEC technical committee 11: Overhead lines.The text of this standard is based on the following documents:FDIS Report on voting11/141/FDIS11/143/RVDFull information on the voting for the approval of this standard can be found in the report on voting indicated in the above table.Annex A forms an integral part of this standard.Annexes B, C and D are for information only.– 8 –61854 © CEI:1998LIGNES AÉRIENNES –EXIGENCES ET ESSAIS APPLICABLES AUX ENTRETOISES1 Domaine d'applicationLa présente Norme internationale s'applique aux entretoises destinées aux faisceaux de conducteurs de lignes aériennes. Elle recouvre les entretoises rigides, les entretoises flexibles et les entretoises amortissantes.Elle ne s'applique pas aux espaceurs, aux écarteurs à anneaux et aux entretoises de mise à la terre.NOTE – La présente norme est applicable aux pratiques de conception de lignes et aux entretoises les plus couramment utilisées au moment de sa rédaction. Il peut exister d'autres entretoises auxquelles les essais spécifiques décrits dans la présente norme ne s'appliquent pas.Dans de nombreux cas, les procédures d'essai et les valeurs d'essai sont convenues entre l'acheteur et le fournisseur et sont énoncées dans le contrat d'approvisionnement. L'acheteur est le mieux à même d'évaluer les conditions de service prévues, qu'il convient d'utiliser comme base à la définition de la sévérité des essais.La liste des informations techniques minimales à convenir entre acheteur et fournisseur est fournie en annexe A.2 Références normativesLes documents normatifs suivants contiennent des dispositions qui, par suite de la référence qui y est faite, constituent des dispositions valables pour la présente Norme internationale. Au moment de la publication, les éditions indiquées étaient en vigueur. Tout document normatif est sujet à révision et les parties prenantes aux accords fondés sur la présente Norme internationale sont invitées à rechercher la possibilité d'appliquer les éditions les plus récentes des documents normatifs indiqués ci-après. Les membres de la CEI et de l'ISO possèdent le registre des Normes internationales en vigueur.CEI 60050(466):1990, Vocabulaire Electrotechnique International (VEI) – Chapitre 466: Lignes aériennesCEI 61284:1997, Lignes aériennes – Exigences et essais pour le matériel d'équipementCEI 60888:1987, Fils en acier zingué pour conducteurs câblésISO 34-1:1994, Caoutchouc vulcanisé ou thermoplastique – Détermination de la résistance au déchirement – Partie 1: Eprouvettes pantalon, angulaire et croissantISO 34-2:1996, Caoutchouc vulcanisé ou thermoplastique – Détermination de la résistance au déchirement – Partie 2: Petites éprouvettes (éprouvettes de Delft)ISO 37:1994, Caoutchouc vulcanisé ou thermoplastique – Détermination des caractéristiques de contrainte-déformation en traction61854 © IEC:1998– 9 –OVERHEAD LINES –REQUIREMENTS AND TESTS FOR SPACERS1 ScopeThis International Standard applies to spacers for conductor bundles of overhead lines. It covers rigid spacers, flexible spacers and spacer dampers.It does not apply to interphase spacers, hoop spacers and bonding spacers.NOTE – This standard is written to cover the line design practices and spacers most commonly used at the time of writing. There may be other spacers available for which the specific tests reported in this standard may not be applicable.In many cases, test procedures and test values are left to agreement between purchaser and supplier and are stated in the procurement contract. The purchaser is best able to evaluate the intended service conditions, which should be the basis for establishing the test severity.In annex A, the minimum technical details to be agreed between purchaser and supplier are listed.2 Normative referencesThe following normative documents contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication of this standard, the editions indicated were valid. All normative documents are subject to revision, and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the normative documents indicated below. Members of IEC and ISO maintain registers of currently valid International Standards.IEC 60050(466):1990, International Electrotechnical vocabulary (IEV) – Chapter 466: Overhead linesIEC 61284:1997, Overhead lines – Requirements and tests for fittingsIEC 60888:1987, Zinc-coated steel wires for stranded conductorsISO 34-1:1994, Rubber, vulcanized or thermoplastic – Determination of tear strength – Part 1: Trouser, angle and crescent test piecesISO 34-2:1996, Rubber, vulcanized or thermoplastic – Determination of tear strength – Part 2: Small (Delft) test piecesISO 37:1994, Rubber, vulcanized or thermoplastic – Determination of tensile stress-strain properties– 10 –61854 © CEI:1998 ISO 188:1982, Caoutchouc vulcanisé – Essais de résistance au vieillissement accéléré ou à la chaleurISO 812:1991, Caoutchouc vulcanisé – Détermination de la fragilité à basse températureISO 815:1991, Caoutchouc vulcanisé ou thermoplastique – Détermination de la déformation rémanente après compression aux températures ambiantes, élevées ou bassesISO 868:1985, Plastiques et ébonite – Détermination de la dureté par pénétration au moyen d'un duromètre (dureté Shore)ISO 1183:1987, Plastiques – Méthodes pour déterminer la masse volumique et la densitérelative des plastiques non alvéolairesISO 1431-1:1989, Caoutchouc vulcanisé ou thermoplastique – Résistance au craquelage par l'ozone – Partie 1: Essai sous allongement statiqueISO 1461,— Revêtements de galvanisation à chaud sur produits finis ferreux – Spécifications1) ISO 1817:1985, Caoutchouc vulcanisé – Détermination de l'action des liquidesISO 2781:1988, Caoutchouc vulcanisé – Détermination de la masse volumiqueISO 2859-1:1989, Règles d'échantillonnage pour les contrôles par attributs – Partie 1: Plans d'échantillonnage pour les contrôles lot par lot, indexés d'après le niveau de qualité acceptable (NQA)ISO 2859-2:1985, Règles d'échantillonnage pour les contrôles par attributs – Partie 2: Plans d'échantillonnage pour les contrôles de lots isolés, indexés d'après la qualité limite (QL)ISO 2921:1982, Caoutchouc vulcanisé – Détermination des caractéristiques à basse température – Méthode température-retrait (essai TR)ISO 3417:1991, Caoutchouc – Détermination des caractéristiques de vulcanisation à l'aide du rhéomètre à disque oscillantISO 3951:1989, Règles et tables d'échantillonnage pour les contrôles par mesures des pourcentages de non conformesISO 4649:1985, Caoutchouc – Détermination de la résistance à l'abrasion à l'aide d'un dispositif à tambour tournantISO 4662:1986, Caoutchouc – Détermination de la résilience de rebondissement des vulcanisats––––––––––1) A publierThis is a preview - click here to buy the full publication61854 © IEC:1998– 11 –ISO 188:1982, Rubber, vulcanized – Accelerated ageing or heat-resistance testsISO 812:1991, Rubber, vulcanized – Determination of low temperature brittlenessISO 815:1991, Rubber, vulcanized or thermoplastic – Determination of compression set at ambient, elevated or low temperaturesISO 868:1985, Plastics and ebonite – Determination of indentation hardness by means of a durometer (Shore hardness)ISO 1183:1987, Plastics – Methods for determining the density and relative density of non-cellular plasticsISO 1431-1:1989, Rubber, vulcanized or thermoplastic – Resistance to ozone cracking –Part 1: static strain testISO 1461, — Hot dip galvanized coatings on fabricated ferrous products – Specifications1)ISO 1817:1985, Rubber, vulcanized – Determination of the effect of liquidsISO 2781:1988, Rubber, vulcanized – Determination of densityISO 2859-1:1989, Sampling procedures for inspection by attributes – Part 1: Sampling plans indexed by acceptable quality level (AQL) for lot-by-lot inspectionISO 2859-2:1985, Sampling procedures for inspection by attributes – Part 2: Sampling plans indexed by limiting quality level (LQ) for isolated lot inspectionISO 2921:1982, Rubber, vulcanized – Determination of low temperature characteristics –Temperature-retraction procedure (TR test)ISO 3417:1991, Rubber – Measurement of vulcanization characteristics with the oscillating disc curemeterISO 3951:1989, Sampling procedures and charts for inspection by variables for percent nonconformingISO 4649:1985, Rubber – Determination of abrasion resistance using a rotating cylindrical drum deviceISO 4662:1986, Rubber – Determination of rebound resilience of vulcanizates–––––––––1) To be published.。

神经内镜微创术结合LCFD术对高血压脑出血患者症状、预后及影响术后生活质量的Logistics分析

DOI：10-3969/j.ien-1672-770.2021.01.017•临床研究-神经内镜微创术结合LCFD术对高血压脑出血患者症状、预后及影响术后生活质量的Logistics分析刘胜华，周政，唐协林,黎乾科，刘怡东，赵超，肖奎，羊飞龙,何绍勇，罗静，夏湘平，叶勇强!摘要】目的研究神经内镜微创术结合腰大池持续引流（LCFD）术治疗高血压脑出血（HICH）的预后及其影响因素。

方法66例HICH患者随机分为观察组及对照组，每组33例。

观察组患者采用神经内镜微创术结合LCFD治疗，对照组患者采用标准骨瓣开颅血肿清除术结合LCFD治疗。

比较两组患者的手术相关指标，以及手术前后格拉斯哥昏迷量表（GCS）评分、格拉斯哥预后量表（GOS）评分、日常生活活动能力（ADL）评分、Epwoth嗜睡量表（ESS）评分和预后生活质量SF-6评分;分析影响术后预后的因素。

结果观察组患者的手术时间及术中出血量显著低于对照组，血肿清除率显著高于对照组;术后的GCS、GOS、ADL、ESS评分及SF-6评分显著高于对照组（均P<0.001）。

多因素分析显示，与子女同住、吸烟、饮酒、脑出血量、脑中线移位、脑出血到手术时间、破入脑室为脑出血预后的独立影响因素。

结论神经内镜微创术结合LCFD对HICH具有较好的疗效，患者的预后较好。

同时应及时对患者是否与子女同住、吸烟、饮酒及脑出血量、脑中线移位、脑出血到手术时间、破入脑室进行了解与监测（并早期开展康复治疗干预$!关键词】高血压脑出血；神经内镜微创术（腰大池持续引流；预后；影响因素!中图分类号】R651；R743.2【文献标志码】B【文章编号】1672-770（2021%01异080-5Logistics analysis on effect of neuroendoscopic minimally invasive surgery combined with LCFD on the symptoms,prognosis and quality of life of patients with HICH LIE Sheng-Hua,ZHOU Zheng,TANG Xie-Ho,etal Depagmef O Neurosurgers,Third A fi Oated Hospital f NortS Sichuan MedOal Collefe,Miaayang621100,China Corresponding authos：ZHOU ZhengAbstract:Objective Tc explore the eOect of neuroendoscopic m inimallg invasive suryet combined with lumbar cerebrospinal fluig drainage（LCFD）on the symptoms,prognosis and qualitu of life of patients withhyao'Wnsiveintracerebralgemortayc（HICH）.Methods66patienW with hypertensive intracerebrcl hemorrhage wererandomlg divided into observation youp and contrci youp,33cases in each youp.Patients in thc observation groupwere treated with endoscopic minimallg invasive suryet combined with LCFD,whilo patienW in thc contrci group weretreated with standard craniotomy combined with LCFD.Thc perioperative indexes,GCS,GOS,ADL,ESS scores andprognosis of thc two groups were compaed,and thc factors influencing the qualitu of life were analyzed.Results Thcoperation Uec and intraoperative blood loss of the observation youp were siynificanUg gwcr than those of thc contrciyoup,and the hematoma clearance rats was siynificanUg higher than that of the contrci youp；the Gksyow coma scale（GCS）,Glasyow outcome score%GOS）,activities of daily living scale（ADL）,Epworth sleepiness scale%ESS）scoresand SF-6scores of the observation group were significanUg higher than those of the contrci group（ai P<0.001）.Multivaiato analysis showed that tiving with chiion,smoking,drinking,amount of cerebral hemorrhage,midiincshift,Uec from cerebral hemorrhage to operation,rd ventricular rupture were independent prognostic factors.Conclusion Neuroendoscopic minieallg invasive suryet combined with LCFD has good curative eOect and goodprognosis in patients with hich.At the same tiee,we shouk tieelg understand and monitor whether the patients livewith theic children,smoking,drinking and the amount of inWacerebrai hemorrhage,midiinc shift of brain,the timefrom cerebral hemorrhage to operation,and break into ventricles,and cairy out earlg rehabiliition intervention.Key wo+s:hypertensive intracerebral hemorrhage；neuroendoscopic minieallg invasive suryet;LCFD；prognosis基金项目：四川省医学会科研课题计划（S15056）；绵阳市卫生健康委科技计划项目（201953）作者单位：621100绵阳#川北医学院附属三台医院神经外科（刘胜华，唐协林#黎乾科，刘怡东，赵超，肖奎，羊飞龙,何绍勇，罗静）；陆军军医大学第二附属医院神经外科（周政）；遵义医学院附属医院神经外科（夏湘平）；四川省资阳市第一人民医院神经外科（叶勇强）通信作者:周政,E-mail:biybrain126@高血压脑出血（hypeOensiveintyceybymemowhaye, HICH）主要是由于血压升高造成的局部脑血管壁破裂所致；随着发病后脑组织受压迫,局部缺血缺氧，造成脑组织及神经细胞损伤，甚至凋亡，进而导致相关运动区域的功能障碍，严重影响患者的生命质量⑴$超早期神经内镜微创术可对高血压脑出血灶的大部分%80%）血肿进行清除［2］;术后通过腰大池持续引流％lumbar cerebrospinal Iuid drainage, LCFD），对于患者的神经功能恢复具有重要的意义。

Effects of culture condition on mycelium biomass and intracellular cordycepin production of Cordycep

Annals of Microbiology, 59 (2) 293-299 (2009)Effects of culture conditions on mycelium biomass and intracellular cordycepin production of Cordyceps militaris in natural mediumChunyan XIE, Gaixia LIU, Zhenxin GU*, Gongjian FAN, Lei ZHANG, Yingjuan GUCollege of Food Science and Technology, Nanjing Agricultural University, Jiangsu, 210095, ChinaReceived 28 October 2008 / Accepted 6 March 2009Abstract - Effects of culture conditions on the growth of mycelium and the production of intracellular cordycepin of Cordyceps militaris were investigated in this study. The natural medium which is a mixture of 53% brown rice paste, 6% beerwort and 42% soybean meal juice was used for the fermentation in shaking flasks. Fermentation temperature, pH and medium capacity, which had been proved to be significant factors, were optimized by Box-Behnken design. Results showed that dry mycelia weight (DMW) and cordycepin yield (CY) was varied with the transformation of culture conditions. The highest DMW (19.1 g/L) and CY (1.8 mg/g) would be obtained at the condition of fermentation temperature 28 °C, pH 6.2 and medium capacity 57 mL.Key words: Cordyceps militaris; dry mycelia weight; intracellular cordycepin; culture conditions; Box-Behnken design.INTRODUCTIONCordyceps militaris is a kind of famous traditional medici-nal mushroom in China, which belongs to the Ascomycete s, Clavicepitacea, Cordyceps and parasites on the larvae of Lepidoptera (Shih et al., 2007). It has extensively been used as tonic food and herb remedy material in China, Japan, and other Asian countries as it has many functional components which had been separated, such as cordycepin, ergosterol, adenosine and polysaccharides, etc. (Cunningham et al., 1950; Hubbell et al., 1985).Cordycepin is a nucleoside derivative extracted from C. militaris (Cunningham et al., 1950). It performs anti-tumour, anti-bacterial, and anti-fungal functions (Kim et al., 2002; Zhou et al., 2002). It can not only inhibit the production of inflamma-tory mediators and the activity of adenylate cyclase in platelets (Won and Park, 2005), but also increase the intracellular levels of cAMP and cGMP in collagen-induced human platelet aggregation (Cho et al., 2007). In the former studies, workers had searched effective methods in order to obtain more cordycepin. And bio-fermentation was regarded as the optimal method, since the transformation in this course was fairly mild (Shih et al., 2007). Comparing the solid-state fermentation with the liquid-state fermentation, the latter was prior to the former for its shorter fermentation period.In the liquid-state fermentation of C. militaris, complex organic compounds were considered as the optimum medium for the production of mycelium and cordycepin (Mao et al., 2005). Lee et al. (2004), K im et al. (2005), Shih et al. (2007), had reported that different culture conditions had notable effects on the yield of mycelium and metabolic substance. To our knowl-edge, most of the fermentation of C. militaris used biochemi-cal components as the culture medium, and the extracellular metabolic substances had been taken lots of attention in the researches (Mao et al., 2005; Masuda et al., 2006). However, cordycepin and other metabolic substances from mushrooms not only excrete to culture broth, but also exist in mycelia.At the same time, it is well known that natural medium con-tains more nutrition for the growth of microorganisms (Lu et al., 2008) and the complex components in the natural medium would make it difficult to purify the metabolic substances in the culture broth. Therefore, it would be more significant to study the effects of culture conditions on the production of intracellular cordycepin in natural culture medium.In this study, we used complex organic compounds as the medium materials, including brown rice paste, beerwort and soybean meal juice, and we attempted to elucidate the effects of culture conditions on the growth of mycelium and the produc-tion of intracellular cordycepin in natural culture medium. To our knowledge, there were few reports concerning the optimal cul-ture conditions of intracellular cordycepin by submerged culture of C. militaris.MATERIALS AND METHODSMaterials. Brown rice, malt and soybean meal were purchased294 C. XIE et al.Culture Medium Products Factory in Beijing, China. Glucoamylase (10000 IU/mg) and papain (2000 IU/mg) were purchased from Wuxi Boli Bio-Products Co Ltd, and Nanjing Scigene Technology Co Ltd, Jiangsu Province, China, respectively. Cordiceps milita-ris strain was purchased from the Institute of Edible Fungus in Academy of Agricultural Science, Jiangsu Province, China.Preparation of brown rice paste, beerwort and soybean meals juice. Brown rice was pulverized by a disintegrator (FSD-100A, T aizhou city, Zhejiang Province, China) and sifted through a 60 mesh sieve. Brown rice powder were mixed with distilled water at a ratio of 1:4 (w:v) and gelatinized at 90-95 °C for 30 min. The mixture was cooled to 60 °C, and then it was liquefied by adding A-amylase of 10000 IU/g rice powder and incubating at 60 °C for 1 h. After, the mixture was homogenized by colloid mill (DJM, Shanghai province, China) and then glucoamylase, 20000 IU/g rice powder, was added, mixed thoroughly and incu-bated at 60 °C up to 2 h. After maceration, the paste was sifted through a 150 mesh sieve; the soluble solid of the filtrate was adjusted to 6.0 °Brix.Malt sprout was pulverized by a disintegrator and sifted through a 60 mesh sieve. The powder was mixed with distilled water at a ratio of 1:4 (w:v) and stayed at 35-37 °C for 30 min, at 50-55 °C for 60 min, and at 65 °C for 3 h, respectively. After maceration, the mixture was centrifuged at 4000 rpm for 10 min, its supernatant was collected and its soluble solid was adjusted to 6.0 °Brix.Soybean meal was pulverized by a disintegrator and sifted through a 60 mesh sieve. The crushed soybean meal was mixed with distill water at a ratio of 1:10 (w:v) and boiled for 10-15 min. Then the mixture was cooled to 60 °C, after adding papain at a quantity of 4000 IU/g soybean meal powder, it was mixed thoroughly and incubated at 60 °C up to 2 h. After maceration, the mixture was centrifugated at 4000 rpm for 10 min, its super-natant was collected and its soluble solid was adjusted to 6.0 °Brix.Maintenance and inoculum preparation. The stock culture of C. militaris was maintained on Potato Dextrose agar (PDA) slants and sub-cultured every month. Slants were inoculated with mycelia and incubated at 25 °C for 7 days, and then were used to prepare inoculum culture. The inoculum medium consisted of brown rice paste, beerwort and soybean meal juice (1/1/1, v/v/v). From PDA plates four discs of 6 mm in diameter were punched out and the mycelium of C. militaris discs was trans-ferred to 250 mL flask containing 50 mL of the inoculum medium. The culture was incubated at 25 °C for 5 days in a reciprocating shaker at 100 rpm.Fermentation of Cordiceps militaris. The natural medium was made up of 53% brown rice paste, 6% beerwort and 42% soybean meal juice, which was the optimal combination for myc-elium growth and cordycepin production of C. militaris. The inoc-ulation quantity was 6% (v/v), which had been determined by former studies (data not shown). After inoculum, the fermenta-tion medium was incubated in a reciprocating shaker at 100 rpm. For optimizing the culture conditions, fermentation temperature, pH and medium capacity were designed and shown in T able 1.TABLE 1 - Independent variables and their levels in the Box-Behnken designVariables Parameter Unit Coded and actual level-10+1X1Fermentation temperature°C222630X2pH 4.5 6.07.5X3Medium capacity ml4080120TABLE 2 - Observed and predicted values of intracellular cordycepin and DMW by Box-Behnken design Run X1 (°C)X2X3 (ml)Intarcellular cordycepin (mg/g)DMW (g/L)Observed value Predicted value Observed value Predicted value 122 4.5800.820.7215.7116.08 230 4.580 1.63 1.5515.9215.77 3227.5800.97 1.0516.7216.42 4307.5800.97 1.0616.0216.11 522 6.0400.340.3319.8218.93 630 6.040 1.61 1.5818.7918.62 722 6.0120 1.19 1.2211.1511.97 830 6.01200.790.8011.4311.66 926 4.5400.440.5517.2617.68 10267.540 1.14 1.0819.0719.71 1126 4.5120 1.15 1.2113.0312.40 12267.51200.640.5311.4711.05 1326 6.080 1.68 1.6517.5617.87 1426 6.080 1.63 1.6517.5617.87 1526 6.080 1.73 1.6518.4417.87 1626 6.080 1.53 1.6517.5617.87 1726 6.080 1.68 1.6518.2117.87Ann. Microbiol., 59 (2), 293-299 (2009) 295Analytical method. For the measurement of dry mycelium weight (DMW), samples were centrifuged at 4000 rpm for 10 min. The mycelia were washed with distilled water and centri-fuged again. Then they were transferred to pre-weighed culture dishes and dried in vacuum at 60 °C to a constant weight.For the analysis of yield of intracellular cordycepin (YIC), 0.50 g of dried mycelia was processed ultrasonically (40 kHz 250 W) for 1 h at 50 °C with 8 mL ethanol (50%, v:v) (Wang et al ., 2005b) . The mixture was centrifugated at 10000 rpm for 20 min. The supernatant was diluted to 10 mL with ethanol (50%, v:v), and then filtered with a 0.45 μm membrane. The filtrate was analyzed by High Performance Liquid Chromatography (HPLC) (Zhong et al ., 2002; Masuda et al ., 2006). Cordycepin was sepa-rated by reverse-phase HPLC using Agilent 1200 (Agilent, USA) with a Prodigy C 18 reverse-phase column (5 μm), 4.6 x 250 mm i.d. The mobile phase was a mixture of methanol and 0.02 M potassium dihydrogenphosphate (15:85). The injection volume was 20 μL, the flow-rate was 1.0 mL/min, the working tempera-ture was 40 °C, and the detection wavelength was 260 nm.Experimental design. In our previous experiments, results showed that fermentation temperature, pH and medium capac-ity influenced the yield of mycelium and intracellular cordycepin significantly (data were not shown). Therefore, the three factors were chosen as the independent variables for the optimization by response surface methodology (RSM).The RSM used in this study is a three-level three-factor Box-Behnken design (T able 2). The design consisted of 17 experi-ments, including 5 replicates. The software of Design Expert ver-sion 6.0.10 (Stat-Ease, Inc) was applied to analyze the experi-mental design data. In order to be correlated to the independent variables, the response variable fitted by a second order model. The general form of the second degree polynomial equation is:(1)where Y is the predicted response, a 0, a i , a ii , a ij are the constant coefficients, and X i , X j are the coded independent variables.Statistical analysis. Analysis of variance (ANOVA) and Duncan’s multiple range tests were performed in order to determine the significant difference in the production of mycelia and intracel-lular cordycepin by C. militaris under different culture conditions. A second-order polynomial regressed equation was established by analysis of Box-Behnken experimental data, and the optimum conditions for fermentation were found using the software of Design Expert version 6.0.10. All trials were carried out in tripli-cate and the averages were taken as responses.RESULTS AND DISCUSSIONDry mycelia weight and cordycepin in the fermentationAs shown in T able 3, the production of mycelia and cordycepin was increased first and decreased later with the prolonging of fermenta-tion time. The highest yield of mycelia (15.29 g/L) and cordycepin (1.73 mg/g) was obtained at 7 days fermentation. Therefore, in our later experiment the fermentation was lasted for 7 days.Optimization of the yield of myceliaThe RSM design for the different culture condition and fermenta-tion results were shown in T able 2. Results showed that DMW var-ied considerably with the change of culture condition. Analyzing by the software of Design Expert version 6.0.10, a polynomial model (Equation 2) describing the correlation between DMW and the three variables was obtained as follows:2221231231239.04 2.70 6.200.170.050.41 1.080.01DMW Y =-+X +X +X -X -X -X -X Xwith R 2 = 0.971(2)The analysis of variance for Equation 2 was checked by the coefficient of determination R 2 (R 2 = 0.971), it indicated that 97.1% of the variability of DMW in the response could be explained by the model. The test statistics of P values showed that model 1 was very significant (P < 0.001). As shown in T able 4, medium capacity had significant linear and quadratic effects on the growth of C. militaris (P < 0.01), and the quadratic effects 33320111i i ii i i jij i i i i ja X a X X X a a Y ¤¤¤¤TABLE 3 - Yield of mycelia and cordycepin in different fermen-tation timeFermentation time (d)DMW (g/l)YIC (mg/g)1 1.91 ± 0.11e 0.30 ± 0.02e 29.45 ± 0.65d 0.31 ± 0.03e 311.04 ± 0.43cd 0.75 ± 0.04d 413.08 ± 0.88bc 1.15 ± 0.09c 514.71 ± 0.37ab 1.07 ± 0.06c 616.08 ± 0.66a 1.45 ± 0.09b 715.29 ± 1.86a 1.73 ± 0.10a 814.00 ± 0.65ab 1.48 ± 0.02b 911.08 ± 1.21cd1.14 ± 0.07cTABLE 4 - Analysis of Variance (ANOVA) for the regression equation (2)Source Sum of Squares DF Mean SquareF Value Prob > F Model 121.42717.3542.67< 0.0001X 10.1910.190.470.5092X 20.5910.59 1.460.2582X 366.52166.52163.64< 0.0001X 12 3.001 3.007.380.0237X 22 3.641 3.648.940.0152X 3212.58112.5830.950.0004X 2 X 3 2.851 2.857.020.0265Residual 3.66790.41Lack of Fit 2.9450.59 3.250.1384Pure Error 0.7240.18Cor Total125.0816296 C. XIE et al.of fermentation temperature and pH were also significant(P < 0.05); the interaction effect of pH with medium capacity was significant (P < 0.05). However, both interaction effect of fer-mentation temperature with pH and the effect of fermentation temperature with medium capacity on the growth of C. militaris were not significant (P > 0.10). The above optimum parameters for the growth of C. militaris were evaluated by non-linear opti-mization algorithm and a maximum DMW of 19.69 g/L would be achieved at a temperature of 25 °C, at pH of 6.7, and a medium capacity of 50 mL.Figure 1 shows the interaction effect of pH with medium capacity on the growth of C. militaris. When pH was about 6.0, medium capacity became the critical factor for the growing of C. militaris, DMW was reducing with the increasing of the medium capacity. When medium capacity was about 50 mL, pH became the critical factor for the growth of C. militaris. The DMW was found to increase at the beginning and reduce later with the elevation of pH; and when the medium capacity exceeded 75 mL, it showed a downward trend. It could be observed that the optimum pH and medium capacity for the growth of C. militaris were about 6.5 and 50 mL, respectively. Optimization of the production of intracellular cordycepin By applying the software of Design Expert version 6.0.10, a poly-nomial model (Equation 3) describing the correlation between intracellular cordycepin production and the three variables was obtained as follows:22-42-3-3=-29.01 +1.15+3.12 +0.14 -0.01 -0.15 -2.89E -0.03-2.61E-5.03EYIC123123121 323Y X X X X X X X X X X X X(3) with R2 = 0.942The determinate coefficient R2 indicated the goodness of the model. Here, it is 0.942, which indicated that 94.2% of the variability in the response could be explained by the model. The statistical significance of Equation 3 was checked by F-test, and the analysis of variance for the fitted quadratic polynomial model is summarized in T able 5. It was found that the model is highly sig-nificant, as is evident from the model F-value or a very low prob-ability value (P < 0.0001). Both fermentation temperature and medium capacity had extremely significant linear and quadratic effects on the production of intracellular cordycepin (P < 0.01); pH had extremely significant quadratic effect on it (P < 0.01). The interactions of any two of the three factors were highly sig-nificant (P < 0.01), and the relation of fermentation temperature and medium capacity was more significant than the association between pH and medium capacity. The interaction effect of fer-mentation temperature with pH was the least significant among them (P = 0.0081). In addition, the optimal condition for the production of intracellular cordycepin was calculated using math-ematical method of extreme of multiple variables function. The optimal condition was as follows: fermentation temperature 29 ºC, pH 5.7 and medium capacity 67.4 mL. Correspondingly, the highest yield of intracellular cordycepin was 1.8 mg/g.The fitted response surface for the production of intracellular cordycepin by the above model was generated using the Design Expert software and was given in Fig. 2 to Fig. 4.Figure 2 showed the interaction effect of fermentation tempera-ture with pH on the production of intracellular cordycepin. It was evident that YIC increased with the elevation of fermentation temperature, when pH was kept invariant. On the other hand, YIC enhanced gradually begin and then decreased with the increase of pH, and the turning point was 6.0. Therefore, pH 6.0 is the optimal condition. Similar results were obtained by Shih etTABLE 5 - Analysis of Variance (ANOVA) for the regression equation (3)Source Sum of Squares DF Mean Square F Value Probability > F Model 3.35790.37330.049< 0.0001 X10.53310.53342.9600.0003 X20.00010.0000.0020.9636 X30.14710.14711.8530.0108 X120.18410.18414.8590.0063 X220.51010.51041.0690.0004 X320.89810.89872.341< 0.0001 X1 X20.16610.16613.4080.0081 X1 X30.69710.69756.1470.0001 X2 X30.36510.36529.4040.0010 Residual0.08770.012Pure Error0.02340.006Cor Total 3.44416X1: fermentation temperature, X2: pH, X3: medium capacity.Ann. Microbiol., 59 (2), 293-299 (2009) 297The effect of fermentation temperature and medium capac-ity on the formation of cordycepin was enumerated in Figure 3. As it is shown, the yield of cordycepin raised with the increase of medium capacity when fermentation temperature was fixed, and the augment tendency became slower with the addition of medium capacity. When medium capacity was kept invariant, YIC increased with the elevation of fermentation temperature, and the scope of optimum temperature for the formation of intracel-lular was at about 26 to 30 °C.Figure 4 is the plot for variation of cordycepin production, as functions of pH and medium capacity when fermentation tem-perature was kept at 26 °C. The yield of intracellular cordycepin was influenced by pH and medium capacity. It was observed that pH was the key factor to enhance the production of cordycepin when medium capacity was 80 mL. And the yield rose firstly and then decreased with pH increasing. The fluctuation of YIC caused by the variation of medium capacity was similar with the trend that induced by pH. Based on an overall consideration of various factors, the optimum pH and medium capacity were about 6.0and 80 mL.From the above analysis, the highest yield of mycelia and intracellular cordycepin could be achieved at appropriate fer-mentation temperature, pH and medium capacity (Fig. 1 to Fig. 4). By analyzing the plots, the optimal values of the fermen-tation conditions to obtain approximately 19.1 g/L DMW and 1.8 mg/g intracellular cordycepin was determined as follows: fermentation temperature 28 °C; pH 6.2 and medium capacity 57 mL.Verification experimentsVerification experiment which was performed under the predicted culture condition by analysis of design expert 6.0 demonstrated that experimental values were generally close to the predicted values (T able 6). The result confirmed the validity and adequacy of the predicted models.DISCUSSIONIn this work, the effects of fermentation temperature, pH and medium capacity on the production of mycelium and intracellular cordycepin of C .militaris were studied . Importantly, the mixture of brown rice paste, beerwort and soybean meal juice was used as the fermentation medium.As previously shown (T able 3), DMW was higher than 11.0 g/L in any fermentation conditions, which indicated that C. militaris grew well in the natural medium. Formerly, workers reported that C. militaris grew well in ample carbon and nitrogen source (K im et al ., 2003a, 2003b). Other researchers showed that most kinds of basidiomycete prefer to the medium of com-plex organic nitrogen sources in submerged fermentation (Mao and Zhong, 2006), and others reported that C. militaris species such as C. militaris C738 and C. militaris NG3 grew poorly in inorganic nitrogen sources (Mao et al ., 2005; Kim et al . 2003a, 2003b). Brown rice, malt and soybean meal, which contain lots of complex organic substances, were regarded as food materials for human and some animals. The brown rice paste after enzy-molysis contained lots of reducing sugar besides phytic acids, vitamin E and vitamin B, and so on (Standard T ables of Food Composition in Japan, 2000). Beerwort had various vitamins, such as biotin, pantothenate, thiamine, pyridoxine, riboflavin, folic acid, nicotinic acid, etc (Guan, 1997). Sometimes, soybean meal was used as nitrogen source for microorganisms (Wang et FIG. 3 - Response surface graphs for YIC of Cordyceps militaris as afunction of medium capacity and fermentation temperature.FIG. 2 - Response surface graphs for YIC of Cordyceps militarisas a function of pH and fermentation temperature.298 C. XIE et al.Fermentation temperature, pH and medium capacity influ-enced the growth and the production of C. militaris, respectively. Because fermentation temperature had close relationship with the growth of mushroom fungus, it must be controlled at a cer-tain value. Zhang (1988) reported that the befitting temperature for the growth of mushroom fungus was in the interval of 20-30 °C, and the optimum was 25 °C. In the former studies, workers always cultivated C. militaris at the steady temperature of 25 °C. Our results showed that C. militaris could grow well during 25 and 26 °C, and the production of intracellular cordycepin was elevated with the increasing of temperature in the limited scope.pH could affect mycelial cell membrane function, cell mor-phology and structure, the uptake of various nutriments, and product biosynthesis (Gerlach et al., 1998). The optimum val-ues of pH for various edible mushrooms were different. Some reported that acidic condition was more suitable for the mycelial growth and the metabolites production (Park et al., 2001; Hsieh et al., 2005; K im et al., 2005; Shu and Lung, 2004). In this work, C. militaris could grow when pH was in the scope of 4.5 to 7.5, and its growth rate rose firstly with the increasing of pH, but decreased beyond pH 6.0. That was similar with the growth rhythm of Coriolus hirsutus (Emelyanova, 2005).Most edible mushrooms are aerobic (Zhang, 1988) and aeration is one of the most important cultivation conditions for aerobes. In our experiments, shaking flask was used for the fermentation, so medium capacity became the important factor for obtaining dissolved oxygen. In the former studies, many have reported that oxygen supply could evidently influence the forma-tion and accumulation of bioactive metabolites in the submerged cultivation of medicinal fungus (Zhong et al., 2002). Our results indicated that with the increasing of medium capacity DMW decreased but cordycepin production enhanced. Therefore, high dissolved oxygen suitable for the growth but bad for cordycepin produced.In conclusion, the best culture condition for the growth of C. militaris and the production of intracellular cordycepin in natural medium was placed as follows: fermentation temperature 28 °C, pH 6.2 and medium capacity 57 mL. Under this condition, the yield of mycelium and intracellular cordycepin were 7 and 20% higher than the initial production, respectively. The information obtained in this work was helpful for the further study and the utilization of C. militaris.REFERENCESin a cyclic AMP- and cyclic GMP-dependent manner. Eur. J.Pharmacol., 58: 43-51.Cunningham K.G., Manson W., Spring F.S. (1950). Cordycepin, a metabolic product from cultures of Cordyceps militaris (Linn.) Link. Nature, 166: 949.Emelyanova E.V., (2005). Effects of cultivation conditions on the growth of the basidiomycete Coriolus hirsutus in a medium with pentose wood hydrolyzate. Process Biochem., 40: 1119-1124. Guan D.Y. (1997). Beer Industrial Handbook, China’s Light Industry Press, Beijing, China.Gerlach S.R., Siedenberg D., Gerlach D., Schtigerl K., Giuseppin M.L.F., Hunik J. (1998). Influence of reactor systems on the morphology of Asperg illus awamori. Application of neural network and cluster analysis for characterization of fungal morphology. Process Biochem.,33: 601-615.Hsieh C., Tsai M.J., Hsu T.H., Chang D.M., Lo C.T. (2005). Medium optimization for polysaccharide production of Cordyceps sin-ensis. Appl. Biochem. Biotechnol.,120: 145-157.Hubbell H.R., Pequignot E.C., Willis D.H., Lee C., Suhadolnik R.J.(1985). Differential antiproliferative actions of 2’, 5’ oligo A trimer core and its cordycepin analogue on human tumor cells. Int. J. Cancer.,36: 389-394.Kim J.R., Yeon S.H., Kim H.S., Ahn Y.J. (2002). Larvicidal activity against Plutella xylostella of cordycepin from the fruiting body of Cordyceps militaris. Pest Manag. Sci.,58: 713-717.K im S.W., Xu C.P., Hwang H.J., Choi J.W., K im C.W., Yun J.W.(2003a). Production and characterization of exopolysaccha-rides from an enthomopathogenic fungus Cordyceps militaris NG3. Biotechnol. Progr., 19: 428-435.K im S.W., Hwang H.J., Xu C.P., Sung J.M., Choi J.W., Yun J.W.(2003b). Optimization of submerged culture process for the production of biomass and exo-polysaccharides by Cordyceps militaris C738. J. Appl. Microbiol., 94: 120-126.K im H.O., Lim J.M., Joo J.H., K im S.W., Hwang H.J., Choi J.W., Yun J.W. (2005). Optimization of submerged culture condi-tion for the production of mycelial biomass and exopolysac-charides by Ag rocybe cylindracea. Bioresour. Technol., 96: 1175-1182.Lee B.C., Bae J.T., Pyo H.B., Choe T.B., Kim S.W., Hwang H.J., Yun J.W. (2004). Submerged culture conditions for the production of mycelial biomass and exopolysaccharides by the edible basidiomycete Grifola frondosa. Enzyme Microb. Technol., 35: 369-376.Lu X.X., Chen Z.G., Gu Z.X., Han Y.B. (2008). Isolation ofTABLE 6 - Results of the verified experimentsStandard order*Fermentationtemperature(°C)pH Mediumcapacity(ml)128.0 6.257.016.68 ± 0.6219.11 1.81 ± 0.09 1.79 225.0 6.050.015.84 ± 0.4517.89 1.51 ± 0.14 1.27 329.5 5.767.414.98 ± 0.2517.29 1.71 ± 0.07 1.84 430.07.580.014.13 ± 0.3716.110.96 ± 0.10 1.01 * 1: optimal culture condition, 2: other’s reported condition, 3: optimal condition for YIC, 4: stochastic condition.Ann. Microbiol., 59 (2), 293-299 (2009) 299Mao X.B., Eksriwong T., Chauvatcharin S., Zhong J.J. (2005).Optimization of carbon source and carbon/nitrogen ratio for cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris. Process Biochem., 40: 1667-1672.Mao X.B., Zhong J.J. (2006). Significant effect of NH4+ on cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris. Enzyme Microb. Technol., 38: 343-350.Masuda M., Urabe E., Sakurai A., Sakakibara M. (2006).Production of cordycepin by surface culture using the medici-nal mushroom Cordyceps militaris. Enzyme Microb. Technol., 39: 641-646.Park J.P., Kim S.W., Hwang H.J., Yun J.W. (2001). Optimization of submerged culture conditions for the mycelia growth and exo-biopolymer production by Cordyceps militaris. Lett. Appl.Microbiol., 33: 76-81.Shih I.L., Tsai K.L., Hsieh C. (2007). Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris. Biochem. Eng.J., 33: 193-201.Shu C.H., Lung M.Y. (2004). Effect of pH on the production and molecular weight distribution of exopolysaccharide byAntrodia camphorata in batch cultures. Process Biochem., 39: 931-935.Standard T ables of Food Composition in Japan (2000). 5th edn., Resources Council, Science and Technology Agency, Japan.Wang R.H., Law R.C.S., Webb C. (2005a). Protease production and conidiation by Aspergillus oryzae in flour fermentation.Process Biochem., 40: 217-227.Wang Y.J., Li D.W., Wang Y.C., Zheng T.T. (2005b). Integrated extracting technology of cordycepin and polysaccharides in Cordyceps militaris. Acta Bot. Boreal.,25: 1863-1867 (in Chinese).Won S.Y., Park E.H. (2005). Anti-inflammatory and related phar-macological activities of cultured mycelia and fruiting bodies of Cordyceps militaris. J. Ethnopharmacol., 96: 551-561.Zhang X.Y. (1988). Edible Fugus, Press of Chongqing University, Chongqing, China.Zhong J.J., Fang Q.H., T ang Y.J. (2002). Enhanced production of valuable bioactive metabolites in submerged cultures of medicinal mushroom Ganoderma lucidum by manipulation of oxygen supply. J. Plant Biochem. Biotechnol., 4: 109-115.Zhou X.X., Meyer C.U., Schmidtke P., Zeep F. (2002). Effect of cordycepin on interleukin-10 production of human peripheral blood mononuclear cells. Eur. J. Pharmacol., 453: 309-317.。

转录组学揭示慢性炎症性关节炎的关键调控因子

转录组学揭示慢性炎症性关节炎的关键调控因子导读目前对于慢性炎症性关节炎的治疗尽管已经取得了明显的进展，但还没有进行全面的分析来系统研究治疗慢性炎症性疾病过程中基因或通路的动态变化。

研究人员依据胶原诱导性关节炎(CIA)的进展和消退对小鼠滑膜组织进行动态基因表达谱分析，鉴定出与炎症消退相关的基因。

通过网络分析预测出3个治疗CIA模型的分泌型细胞因子：Itgb1、Rps3、Ywhaz。

这些因子主要由Treg和M2巨噬细胞表达，它们能够抑制促炎细胞因子的产生。

特别是Ywhaz在关节炎消退的小鼠血清和具有良好治疗反应的类风湿关节炎(RA)患者的尿液中升高。

此外，腺病毒介导的Ywhaz基因转导到受影响的关节显著抑制CIA小鼠关节炎的进展，并使关节组织、淋巴结和脾脏中促炎细胞因子的表达受到抑制，这表明Ywhaz是治疗RA的重要靶点。

因此，研究人员通过转录组学对慢性炎症性关节炎过程中的滑膜组织进行综合分析，发现了可以作为预测疾病消退的分子标志物：Itgb1、Rps3、Ywhaz，这三个生物标志物同时也可以作为慢性炎症性关节炎的治疗靶点。

实验设计1.建立CIA模型，对不同病理时期关节组织进行基因表达谱分析，并通过qRT-PCR检测不同病理时期促炎、抗炎细胞因子mRNA表达；2. qRT-PCR、IHC、Western blotting检测滑膜组织中Itgb1、Ywhaz、Rps3 mRNA、蛋白表达；3. qRT-PCR、流式、Western blotting检测T细胞、巨噬细胞亚群中Itgb1、Ywhaz、Rps3 mRNA、蛋白表达；4. qRT-PCR检测tgb1、Ywhaz、Rps3对FLSs促炎细胞因子的影响；5.ELISA分别检测有无抗风湿药干预CIA小鼠血清中Ywhaz水平；6. qRT-PCR等检测Ywhaz对CIA小鼠的治疗作用。

结果1 疾病进展过程中CIA模型滑膜组织转录组学分析为了描述CIA模型的炎症反应，研究人员向老鼠尾部注射CFA和CII后进行关节炎评分，来监测随时间变化CIA模型关节炎的临床严重程度。

蓝舌病毒通过诱导巨噬细胞产生干扰素抑制HBV增殖

蓝舌病毒通过诱导巨噬细胞产生干扰素抑制HBV增殖高云飞, 高凤成, 李清峰（榆林市第一医院感染科，陕西榆林 719000）摘要：目的探讨蓝舌病毒（bluetongue virus，BTV）16激活巨噬细胞天然免疫的机制及BTV16刺激后的巨噬细胞培养上清对HBV的抑制作用。

方法采用Real-time PCR分别检测BTV未预处理或预处理12 h后巨噬细胞中GAPDH、IFN-α、IFN-β、IRF-3、IRF-5和IRF-7的mRNA表达水平，采用Western blot分别检测IRF3、IRF5、IRF7蛋白的表达。

使用ELISA法检测巨噬细胞培养基上清中HBsAg及HBeAg的表达。

结果 BTV16能够激活TLR3/干扰素信号转导通路，诱导巨噬细胞高表达Ⅰ型干扰素并诱导干扰素调节因子高表达，20%体积比的BTV刺激后的巨噬细胞培养上清对HBV复制即有较强的抑制作用（P＜0.05），Ⅰ型干扰素受体抗体预处理后，BTV16刺激巨噬细胞的上清对HBV释放的抑制作用显著减弱（t = 14.031，P = 0.003）。

结论 BTV16能够激活巨噬细胞的天然免疫反应，显著抑制HBV在肝细胞的复制，为临床治疗乙型肝炎提供了新思路。

关键词：巨噬细胞；天然免疫；肝炎病毒，乙型；干扰素Bluetongue virus inhibits HBV proliferation by inducing macrophages to produce interferonGAO Yun-fei, GAO Feng-cheng, LI Qing-feng (Department of Infectious Diseases, Yulin First Hospital, Yulin 719000, Shaanxi Province, China)Abstract: Objective To investigate the mechanism of bluetongue virus (BTV) 16 activating macrophage innate immunity and the inhibitory effect of macrophage culture supernatant stimulated by BTV16 on HBV. Methods The expression levels of GAPDH, IFN-α, IFN-β, IRF-3, IRF-5 and IRF-7 mRNA in macrophages which is pretreated by BTV for 12 hours or not were detected by Real-time PCR, respectively. The expression of IRF3, IRF5 and IRF7 protein were detected by Western blot. The expression of HBsAg and HBeAg in macrophage culture medium were detected by ELISA. Results BTV16 could activate TLR3/IFN signaling pathway and induce macrophages to overexpress interferon Ⅰ, and sufficient to induce high expression of interferon regulatory factor. Twenty percent volume ratio of macrophage culture medium stimulated BTV had a strong inhibitory effect on HBV replication (P ＜ 0.05). After pretreatment of interferon Ⅰreceptor antibodies, the inhibitory effect of macrophage culture medium stimulated BTV on HBV releasing significantly decreased (t = 14.031, P = 0.003). Conclusion BTV16 can activate the innate immune response of macrophages and significantly inhibit the replication of HBV in hepatocytes, which provides a new idea for clinical treatment of hepatitis B.Key words: Macrophage; Innate immunity; Hepatitis B virus; Interferon乙型肝炎病毒（hepatitis B virus，HBV）属于嗜肝DNA病毒科[1]，HBV感染可引起肝脏炎症、纤维化及肝硬化，且为肝细胞癌的主要致病因素[2,3]。

银耳多糖对人软骨细胞的增殖效应和抗炎作用

谭敏颖，戴川景，卢学敏，等. 银耳多糖对人软骨细胞的增殖效应和抗炎作用[J]. 食品工业科技，2024，45（1）：1−8. doi:10.13386/j.issn1002-0306.2023060077TAN Minying, DAI Chuanjing, LU Xuemin, et al. Proliferation and Anti-inflammatory Effects of Tremella fuciformis Polysaccharide on Human Chondrocytes[J]. Science and Technology of Food Industry, 2024, 45(1): 1−8. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023060077· 特邀主编专栏—食品中天然产物提取分离、结构表征和生物活性（客座主编：杨栩、彭鑫） ·银耳多糖对人软骨细胞的增殖效应和抗炎作用谭敏颖1,2，戴川景1，卢学敏1，王毅刚1，关　磊2，程　勇2, *（1.浙江理工大学生命科学与医药学院，浙江杭州 310018；2.浙江天草生物科技股份有限公司，浙江湖州 313399）摘　要：目的：骨关节炎（Osteoarthritis ，OA ）是一种常见的慢性关节性疾病，本研究旨在探究银耳多糖对骨关节炎细胞模型人软骨细胞T/C-28a2的增殖效应和抗炎作用。

方法：通过MTT （3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide ）和结晶紫染色实验检测银耳多糖对T/C-28a2细胞增殖活力和细胞毒性的影响；用脂多糖（Lipopolysaccharide ，LPS ）处理T/C-28a2细胞建立骨炎症模型，酶联免疫吸附测定（Enzyme-linked immunosorbent assay ，ELISA ）检测药物处理后细胞白介素-6（Interleukin-6，IL-6）的表达；利用蛋白免疫印迹（Western blot ）检测药物处理后相关骨保护因子和炎症因子的表达；通过ROS 活性氧释放实验检测药物对细胞的氧化应激水平和抗炎症反应。

2024年江苏新高考一卷英语试题.doc

2024年江苏新高考一卷英语试题2024年江苏新高考一卷英语试题及答案例：How much is the shirt?A.E19.15.B.E9.18.C.E9.15.答案是C.1.What is Kate doing?A.Boarding a flight.B.Arranging a tripC.Seeing a friend off.2.What are the speakers talking about?A.pop star.B.An old songC.A radio program3.What will the speakers do today?A.Goto an art show.B.Meet the mans aunt.C.Eat out with Mark4.What does the man want to do?A.Cancel an order.B.Ask for a receipt.C.Reschedule a delivery5.When will the next train to Bedford leave?A.At 9:45.B.At 10:15C.At 11:00.第二节 (共15小题;每小题1.5分，满分22.5分)听下面5段对话或独白。

每段对话或独白后有几个小题，从题中所给的 A 、B 、C 三个选项中选出最佳选项。

听每段对话或独白前，你将有时间阅读各个小题，每小题5秒钟;听完后，各小题将给出5秒钟的作答时间。

每段对话或独白读两遍。

听第6段材料，回答第6、7题。

6.What will the weather be like today?A.StormyB.SunnyC.Foggy7.What is the man going to do?A.Plant a tree.B.Move his carC.Check the map听第7段材料，回答第8至10题。

镉或铬胁迫对拟穴青蟹血细胞总数和酚氧化酶活性的影响

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细胞凋亡的线粒体途径

Mitochondrial Pathways in physiological cell death
If mitochondria are pivotal in controlling cell life and death, then how do these organelles kill? At least three general mechanisms are known, and their effects may be interrelated, including (i) disruption of electron transport, oxidative phosphorylation, and adenosine triphosphate (ATP) production; (ii) release of proteins that trigger activation of caspase family proteases; and (iii) alteration of cellular reduction-oxidation (redox) potential
disruptionofelectrontransportandenergymetabolism线粒体跨膜电位的耗散与细胞凋亡有密切关系近年来陆续有报道说明线粒体跨膜电位的耗散早于核酸酶的激活也早于磷酯酰丝氨酸暴露于细胞表面
细胞凋亡的线粒体途径
蒋舜媛董霞程在全
2002-12-17
细胞死亡损伤性死亡 Nhomakorabea生化特征:
染色质降解, 核小体间连接DNA部位被降解,产生寡聚核小体DNA片段,即180-200DP 整数倍的不同长度的DNA片断.
Fig.1. Schematic summary of biochemical mechanisms of apoptosis.

敲减Wingless_Wnt1基因表达对赤拟谷盗发育中的影响_英文_

ISSN 1007-7626CN 11-3870/Q中国生物化学与分子生物学报http ：//cjbmb．bjmu．edu．cnChinese Journal of Biochemistry and Molecular Biology2012年8月28（8）：733 738Received ：March 26，2012；Accepted ：June 6，2012Supported by National Natural Science Foundation of China （No．31040018and No．31172146），Shanxi Scholarship Council of China （2010-2012）and International Cooperation Projects of Shanxi Province*Corresponding author Tel ：86-25-85891763；E-mail ：libin@njnu．edu．cn ；Tel ：0086-351-7018268，E-mail ：lzy@sxu．edu．cn收稿日期：2012-03-26；接受日期：2012-06-06国家自然科学基金（（No．31040018；No．31172146）和山西省国际合作项目资助*联系人Tel ：86-25-85891763；E-mail ：libin@njnu．edu．cn ；Tel ：0086-351-7018268，E-mail ：lzy@sxu．edu．cnKnocking-down of Wingless /Wnt1Influences the Development of Tribolium castaneumPENG Ya-Nan 1），LI Cheng-Jun 2），Li Bin 2）*，LI Zhuo-Yu 1）*（1）Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education ，Institute of Biotechnology ，Shanxi University ，Taiyuan030006，China ；2）Jiangsu Key Laboratory for Biodiversity and Biotechnology ，College of Life Sciences ，Nanjing Normal University ，Nanjing210046，China ）Abstract Known as highly conserved during evolution ，the Wnt signaling pathway plays a vital role in regulating animal embryonic axis ，embryonic differentiation ，and deciding cell polarity and maintaining adult dynamic equilibrium．Mutations or deregulations of its components might cause the occurrence of carcinoma．We studied the role of Wingless /Wnt1during larva-adult development of the red flour beetle ，Tribolium castaneum ，with dsRNA-mediated Wingless （Wg ）/Wnt 1gene knocked down．The treated late larvae metamorphosed into pupae with drastically increased wing interval and decreased wing width （P ＜0.01）．The pursuant pupa-adult eclosion was also severely affected and most of pupae died during this period．The qPCR result showed that the mRNA level of Cadherin-like and Smoothened （Smo ）geneswere up-regulated greatly ，and that of armadillo-2was slightly higher ，after Wingless /Wnt 1gene was knocked down．We drew the conclusion that Wnt-1signaling pathway is closely related to the proper wingdevelopment and adult metamorphosis of Tribolium ．In addition ，the elevated expression of Cadherin-like and Armadillo-2may be accountable for the reduced wing width and enlarged wing interval caused by wggene silencing ，because those alterations can either enhance cell adhesion or change cell morphology．Importantly ，the up-regulation of smo gene indicates that Hedgehog signaling pathway may be affected by the RNAi of Wg and involved in the abnormal or lethal phenotypes observed in our experiment．Key words Tribolium castaneum ；Wg RNAi ；Wingless /Wnt1protein ；Wnt signaling pathway ；eclosion敲减Wingless /Wnt1基因表达对赤拟谷盗发育中的影响彭亚男1），李承军2），李斌2）*，李卓玉1）*（1）化学生物学与分子工程教育部重点实验室山西大学生物技术研究所，太原030006；2）南京师范大学生命科学学院，南京210046）摘要Wnt 信号通路是进化中高度保守的一条信号转导途径，在调控动物的胚胎轴向正常发育、胚胎分化、决定细胞极性、维持成体动态平衡等方面发挥重要作用．该信号通路的异常激活还与肿瘤的发生密切相关．本实验将体外人工合成的Wingless （Wg ）/Wnt 1基因dsRNA 显微注射入赤拟谷盗晚期幼虫体内，研究Wingless /Wnt1蛋白在赤拟谷盗发育过程中发挥的作用．实验结果显示，注射Wingless （wg ）/Wnt 1基因dsRNA 后，赤拟谷盗发育形成的蛹，翅膀宽度减小，翅间距明显增大，且羽化过程也受到严重影响．此外，qPCR 结果表明，赤拟谷盗Wingless （Wg ）/Wnt 1基因被沉默后，Cadherin-like 和Smoothened （Smo ）基因的表达显著上调，A rmadillo -2基因略上调．这些结果揭示，Chinese Journal of Biochemistry and Molecular Biology Vol．28Wnt-1信号通路和赤拟谷盗翅膀发育以及成虫羽化过程密切相关．蛹翅宽减小，翅间距增大，可能是由于调控细胞粘连及细胞形态的Cadherin-like和Armadillo-2基因的上调所引起．更重要的是，Smo基因的上调，表明了Wnt信号通路和Hedgehog信号通路在赤拟谷盗发育过程中有交互作用．关键词赤拟谷盗；Wg RNAi；Wingless/Wnt1蛋白；Wnt信号通路；羽化中图分类号Q966Wnt gene was first reported by Nusse and his colleagues in1982，and named as int-1．When this gene was abnormally activated，it would induce tumor［1］．The subsequent name‘wnt’was derived from a combination of int-1and wingless（a developmental patterning gene in Drosophila），because these two genes were shown to be homologous［2］．Later，the family of wnt genes was confirmed to exist in many species from nematodes to vertebrates：nineteen in human［3］，seven in Drosophila，seven in Apis，six in Anopheles，and nine in Tribolium castaneum［4，5］．Wnt genes encode a large family of secreted，cysteine-rich proteins that play a key role in animal development，as intercellular signaling molecules．Most Wnt proteins are comprised of350to380amino acids，with more than100conserved residues scattering across the entire sequence．The initiation of these proteins is a sequence of hydrophobic signal followed by a site that can be recognized by signal peptides．Each protein has one or more sites for N-linked glycosylation and up to 24conservative cysteine residues that form disulfide bonds［6］．Wnt ligands（Wnts）bind to various transmembrane receptors by autocrine or paracrine and drive the Wnt signaling pathway，thereby triggering intercellular cascades to regulate transcription in target genes．Wnt signaling pathway activated by Wnt proteins is involved in embryonic development，as well as cell proliferation and differentiation in adult development．It is required in the processes of regulating the establishment of head-to-tail axis，the differentiation of neural crest，the correct formation of brain and heart，kidney morphogenesis and sex determination［7］．The disruption of this precise system will induce developmental disabilities．A total of nine Wnt genes have been reported in Tribolium genome，including Wnt A，Wnt8，orthologs of the vertebrate Wnt5-7and Wnt9-11genes，in addition to Wg or Wnt1［5］．The expression patterns of these nine Wnt genes in embryogenesis have been discussed at length in existing studies，especially their segment polarity function of the canonical Wg/Wnt1 gene．However，less is known about the role of Wnt signaling pathway in postembroyonic developmental process of Tribolium．An investigation had been carried out about the role of Wg/Wnt1protein during the larval-adult development of Tribolium．1Materials and Methods1.1Beetle strain and maintenanceThe Georgia-1（GA-1）strain of Tribolium castaneum used in this study was reared at30ħand all experiments were performed at room temperature 25ħ［8］．1.2Analysis of Wg gene transcript levels in different developmental stages by RT-PCREarly and late eggs，larvae，pupae and adults were collected for RNA preparation，followed byRT-PCR．The first day of the embryonic period was taken for early egg stage，the initial1-7days of larval stage for early larval stage，and the starting1-3days of pupa/adult stage for early pupa/adult stage．Sequence of designed primers was Wg-F1：5'-GTGCCAATA ATGCGATTCAC-3'，Wg-R1：5'-TTCCTTTGTAGT GCGTTTCG-3'．Amplification conditions of RT-PCR were：94ħ5min，94ħ30s，60ħ30s，72ħ30s，35cycles，25ħ10min．The housekeeping gene Rps3（Tribolium ribosomal protein3）was used as an internal control．Amplification conditions of RT-PCR were：94ħ5min，94ħ30s，60ħ30s，72ħ30s，28cycles，25ħ10min．1.3RNA interferenceDouble-stranded RNA was produced from a714 bp fragment of the T．castaneum Wingless gene （fragment positions493 1206）．Template for dsRNA synthesis was amplified by using gene-specific primers that have T7promoter sequences at the end．The sequences of primers were as follows：Wg-F2：5'-TAATACGACTCACTATAGGGATAG ATACGTGCAAC TGCGA-3'，Wg-R2：5'-TAATACGACTCACTATAGG GCTCGAATACGACGACTTCCT-3'．Amplification conditions of RT-PCR were：94ħ5min，94ħ30s，60ħ30s，72ħ1min，35cycles，25ħ10min．Double-stranded RNA was synthesized and purified using the Transcript Aid TM T7High Yield Transcription kit．dsRNA was diluted to1μg/μL with1ˑinjection buffer（5mmol/L KCl，0.1mmol/L K3PO4，pH6.8）prior to injection．For injection，T．castaneum late larvae were affixed to microscope slides with tweezers at their posterior abdomen．Approximately0.2μL of dsRNA solution was injected into each pupa through a micromanipulator set-up，at a ventrolateral position between abdominal segments three and four．Untreated late larvae of the same number were selected as437No．8PENG Ya-Nan et al：Knocking-down of Wingless/Wnt1Influences the Development of Tribolium castaneum controls．Beetles of group IB were injected with0.2μL of injection buffer（1ʒ10）．Larva RNAi wasperformed with WPI Nanoliter microscopic injectionsystem．After，these beetles were reared in the sameconditions mentioned above．Their development statuswas observed every24hours．Five days after injection，three insects from each treatment were collected forRT-PCR to confirm Wg gene was silenced．Anotherreaction with a pair of primers for rps3gene was usedas the internal loading control．Sequences of primersfor Rps3g ene was：Rps3-F5'-TCAAATTGATCGGAGGTTTG-3'，Rps3-R5'-GTCCCACGGCAACATAATCT-3'，and Wg-F1and Wg-F2were used for Wggene．1.4Detection of mRNA level of other relatedgenes by qPCRTc-Armadillo-2，NCBI mRNA accession numberXM_966892，is located on LG9；smo，XM_966834，is on LG8；cadherin-like，XM_966295．Primers ofqPCR were designed and synthesized by TakaraBiotechnology CO．（Dalian，China）．The detailedinformation of primer sequences was described in Table1．Three cDNA templates were prepared with threepupae from each group．Technical triplicates of eachreaction mix were prepared in20μL final volumecontaining：10μL of2ˑSYBR Green PCR Mix，0.4μL of Rox，3μL of water-diluted cDNA（1︰20），which corresponds to1μg of total RNA，0.8μL ofeach primer（10pmol）．Finally，5μL DEPC H2Owas introduced to raise the system to20μL．Thefollowing cycling protocol was used：40cycles of30sat95ħ，5s at95ħ，34s at60ħ．To verify theapplicant’s consistency，the product was tested in amelting point analysis conducted directly afteramplification．Relative expression level of each genewas determined versus a constitutively expressed generps3．The results were displayed by meansʃstandarderrors with three independent experiments．Table1Details of primers used for the three evaluatedgenesGene Sequences（5'→3'）Product length（bp）Armadillo-2For：caatcacggtagtcagccttttc81Rev：tgtgtgccaatctccagtccSmo For：atcggttactgcgtcctggt95Rev：aaggcggggtatttgttggCadherin-like For：gacttcaaagatgctcagtcgaaa118Rev：taacaactaaaacggcaaccacac2Results2.1Knockdown of Wg gene in Tribolium late larvaeWe first examined expression pattern of Wg gene by reverse transcription-PCR（RT-PCR）．The results indicated that wg gene showed a high expression from late larval stage to late pupa stage，and the expression was declined in adult stage（Fig．1A）．It is likely that Wg gene plays a crucial role in larva-pupa-adult transitions．To study the role of Wg/Wnt1protein during these transitions，we injected Wg-dsRNA into late larvae．Five days later，we verified the mRNA level of Wg gene by using RT-PCR．The results showed that Wg gene of beetles was silenced as expected （Fig．1B）．Fig．1Analysis of wg gene expression by reversetranscription-PCR（A）The mRNA level of Wg ineight developmental stages．1：early egg；2：late egg；3：early larva；4：late larva；5：early pupa；6：late pupa；7：early adult；8：late adult．（B）Knocking-down of Wgtranscript by RNAi．Control：no injecting；IB：injectinginjection buffer；RNAi：injecting Wg-dsRNA．PCRanalysis of Rps3with the same cDNA template served asinternal control2.2Wnt1protein is required for wing develop-ment of Tribolium pupaeBecause a fraction of late larvae died as a result of injury caused by dsRNA injections，we observed phenotypes of the remaining bettles：n（group RNAi）=27；n（group IB）=25；n（group Control）=30．All of the remaining larvae survived on the pupa stage，but the wing interval of pupae from group RNAi was bigger than that of control beetles（Fig．2A）．Furthermore，body size，the length，width and interval of each pupa wing were measured using OLYMPUS microscope．The experiments showed that the wing width of Wg RNAi pupae decreased，and the wing interval expanded distinctly （Fig．2B）．However，the body size and wing length did not appear to be affected in comparison to the two control groups（Fig．2B）．2.3Wnt1protein contributes to pupa-adult moltingAlthough the wg RNAi larvae had metamorphosed into pupae，three defective phenotypes were revealed at the ensuing adult eclosion（Fig．3）．Ultimately，these abnormal insects died during the eclosion process．The proportion of three cases in each group was calculated：n（group RNAi）=27；n（group IB）=25；n（group Control）=30．And the result was indicated in Table 2．Most insects in group Control and IB showed normal phenotype．However，the majority of insects in group537Chinese Journal of Biochemistry and Molecular Biology Vol．28Fig．2RNAi results of wg on Tribolium pupae（A）Phenotype deficiency of pupae by RNAi．（a）Pupae in group of control；（b）Pupae in group of IB；（c）and（d）RNAi treated pupae．Arrow indicates the wing interval．These indexes were measured under OLYMPUS microscope．The photo was magnified by2.5times．（B）The statistical results of body size，wing length，wing width and wing interval of Tribolium pupae in group of control，IB and RNAi．Data processing was made usingsoftware SPSS．Star markers（＊＊）indicates a highly significant difference between control and treated insects（P＜0.01）Fig．3RNAi effects of wg on Tribolium adult eclosion（A）and（E）：normal adult；（B）and（F）：deficiency adult stopped at the prophase of emergence；（C）and（G）：deficiency adult emerged incompletely；（D）and（H）：defective adult exhibiting abnormal phenotypes（dorsal is on top row；ventral is on bottom row）637No．8PENG Ya-Nan et al ：Knocking-down of Wingless /Wnt1Influences the Development of Tribolium castaneum Table 2Statistical results of three kinds of emergence phenotypes in each groupGroup Normal eclosion Stop at beginning eclosion Incomplete eclosion Finish eclosion yet with phenotype defects Control 94%0%0%6%IB 97%0%0%3%RNAi7%41%22%30%The percentage in theTable 2is the ratio of Tribolium with certain emergence phenotype in corresponding groupRNAi did not finish the adult eclosion completely．Some of the pupae stopped at the beginning eclosion ；some went further ；and others metamorphosed into pharate adults with phenotype defects．2.4Knock-down of Wg has effects on a few genesat transcript levelIn order to study the molecular mechanism of Wnt1/Wingless protein in regulating wing development and pupa-adult molting of Tribolium ，the isolated total RNA of pupae from each group was screened to test whether the observed effects of Wg gene knock-down were caused by those factors ：Armadillo -2，Smo a ndCadherin-like ，which have been demonstrated to play some roles in the wing development of Drosophila ［9-11］．The result of qPCR indicated that the expression of Cadherin-like and Smo were up-regulated markedly ，the level of Armadillo-2with a slightincrease．Fig．4The mRNA level of Cadherin-like ，Smo ，andArmadillo-2affected by the RNAi of Wg The Y-axis denoted mRNA relative expression levels（normalized by Tcrps 3）．Mean ʃSE of mRNA relative expression levels of three genes mentioned above were showed．Star markers （*）indicated a significant difference between control and treated insects （P ＜0.05）．Double star markers （＊＊）indicated a highly significant difference between control and treated insects （P ＜0.01）3DiscussionStudies have suggested that Wnts can signal through three different pathways ：the canonical β-catenin pathway ，the non-canonical planar cell polarity （PCP ）and Wnt /Ca 2+pathways．The β-catenin pathway is required for growth and cell fate specification ［12］，whereas the two non-canonical pathways are implicated in cell polarity and cellmovement ［13］．The classic Wingles s /Wnt signaling pathway has been demonstrated to play a key role in the wing development of Drosophila ．The functional mechanism is also clear．The pathway is involved in the growth ，specification ，and morphogenesis of the wing pouch of Drosophila ［11］，one region of the wing disc that will be transformed to the adult wing blade during pupa development ［14］．Further studies suggested that Wingless promoted the wing disc pouch growth mainly through the inhibition of apoptosis ［15］．Meanwhile ，the regulation of wingless for vestigial ，one of its targets which defines the wing primordium and is required for its growth ，contributes partly to the increase of thewing pouch size ［16，17］．Wingless /Wnt signaling also participates in the cell specification of the wing disc．The depletion of wingless gene during the development of Drosophila early larvae will lead to the result of losing normal wing structures．Meanwhile ，wingless can induce the expression of genes along dorsal-ventral boundary during late larval development ，including senseless regulating cell fate specification at the wing margin ［18］．The latest studies have revealed the role of wingless in controlling the cell shape of late larval wing disc ［11］．In addition ，Wingless /Wnt signaling is implicated in cell adhesion by directing the graded expression of Shotgun ，which encodes E-cadherin in Drosophila ．For cells along the proximodistal axis of the developing wing epithelium ，those further away from the source of Wg signaling in the wing imaginal discs display lower levels of DE-Cadherin expression when compared with those receiving high threshold of Wg signaling ［19］．The model insect Tribolium used in our experiment is one of the well-known holometabolous pests for stored products ，mainly in tropical or warmer regions around the world ［20］．It belongs to Tenebrionidae ，Coleopteran ，and the genome sequence of the Tribolium is now available ［21］．Although Tribolium belongs to holometabolous taxa as well as Drosophila ，its developmental pattern differs from that of Drosophila ．In Tribolium ，primordia of anterior segments appears first ，then primordia of more posterior segments are generated from a posterior growth zone．However ，all segments in Drosophila occur nearly simultaneously ［22］．Our data therefore complement the Drosophila on functional genetic737Chinese Journal of Biochemistry and Molecular Biology Vol．28analysis of basic biological questions．In this study，the results showed that the disruption of canonical Wingless/Wnt signaling pathway in Tribolium also resulted in abnormal wing phenotype，along with severely affected emergence process，indicating that Wnt1protein is necessary for the wing development of Tribolium．This result also shows that Tribolium is an excellent model for studying genetic regulation．Furthermore，the qPCR result shows that Cadherin-like，Armadillo-2and Smo genes of T．castaneum pupae with abnormal wings were upregulated．Because E-cadherin and Armadillo-2play some roles in wing cell adhesion and shape［23］，we believe that Cadherin-like and Armadillo-2may influence the wing growth of Tribolium by strengthening cell adhesion or changing cell shapes．In addition，the abnormal activation of Smo implies that smo gene is possibly related to the effects of wing development and adult emergence．This finding points to a conclusion that Wnt1protein may play an additional role independent of Wnt signaling pathway in postembryonic development of Tribolium．Importantly，it may mediate the cross-talk of Wnt signaling pathway and Hedgehog pathway．Further studies need to be completed in the future．参考文献（References）［1］Nusse R，Varmus H E．Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the sameregion of the host genome［J］．Cell，1982，31（1）：99-109［2］Rijsewijk F，Schuermann M，Wagenaar E，et al．The Drosophila homolog of the mouse mammary oncogene int-1is identical to thesegment polarity gene wingless［J］．Cell，1987，50（4）：649-657［3］Garriock R J，Warkman A S，Meadows S M，et al．Census of vertebrate Wnt genes：isolation and developmental expression ofXenopus Wnt2，Wnt3，Wnt9a，Wnt9b，Wnt10a，and Wnt16［J］．Dev Dyn，2007，236（5）：1249-1258［4］Murat S，Hopfen C，McGregor A P．The function and evolution of Wnt genes in arthropods［J］．Arthropod Struct Dev，2010，39（6）：446-452［5］Bolognesi R，Beermann A，Farzana L，et al．Tribolium Wnts：evidence for a larger repertoire in insects with overlappingexpression patterns that suggest multiple redundant functions inembryogenesis［J］．Dev Genes Evol，2008，218（3-4）：193-202［6］Cho SJ，Valles Y，Giani V C，et al．Evolutionary dynamics of the Wnt gene family：a lophotrochozoan perspective［J］．MolBiol Evol，2010，27（7）：1645-1658［7］Agholme F，Aspenberg P．Wnt signaling and orthopedics，anoverview［J］．Acta Orthop，2011，82（2）：125-130［8］Haliscak J P，Beeman R W．Status of Malathion Resistance in5 Genera of Beetles Infesting Farm-Stored Corn，Wheat，and Oatsin the United-States［J］．J Econ Entomol，1983，76（6）：717-722［9］Terriente-Félix A，López-Varea A，de Celis J F．Identification of genes affecting wing patterning through a loss-of-functionmutagenesis screen and characterization of med15function duringwing development［J］．Genetics，2010，185（2）：671-684［10］Blair S S，Ralston A．Smoothened-mediated Hedgehog signaling is required for the maintenance of the anterior-posterior lineagerestriction in the developing wing of Drosophila［J］．Development，1997，124（20）：4053-4063［11］Logan C Y，Nusse R．The Wnt signaling pathway in development and disease［J］．Annu Rev Cell Dev Biol，2004，20：781-810［12］Kohn A D，Moon R T．Wnt and calcium signaling：beta-catenin-independent pathways［J］．Cell Calcium，2005，38（3-4）：439-446［13］Widmann T J，Dahmann C．Wingless signaling and the control of cell shape in Drosophila wing imaginal discs［J］．Dev Biol，2009，334（1）：161-173［14］Gonsalves F C，DasGupta R．Function of the wingless signaling pathway in Drosophila［J］．Methods Mol Biol，2008，469：115-125［15］Johnston L A，Sanders A L．Wingless promotes cell survival but constrains growth during Drosophila wing development［J］．NatCell Biol，2003，5（9）：827-833［16］Kim J，Sebring A，Esch JJ，et al．Integration of positional signals and regulation of wing formation and identity byDrosophila vestigial gene［J］．Nature，1996，382（6587）：133-138［17］Zecca M，Struhl G．Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial auto regulation［J］．Development，2007，134（16）：3001-3010［18］Jafar-Nejad H，Tien A C，Acar M，et al．Senseless and Daughterless confer neuronal identity to epithelial cells in theDrosophila wing margin［J］．Development，2006，133（9）：1683-1692［19］Jaiswal M，Agrawal N，Sinha P．Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wingdevelopment in Drosophila［J］．Development，2006，133（5）：925-935［20］Brown S J，Shippy T D，Miller S，et al．The red flour beetle，Tribolium castaneum（Coleoptera）：a model for studies ofdevelopment and pest biology［J］．Cold Spring Harb Protoc，2009，2009（8）：pdb．emo126［21］Tribolium Genome Sequencing Consortium，Richards S，Gibbs R A，et al．The genome of the model beetle and pest Triboliumcastaneum［J］．Nature，2008，452（7190）：949-955［22］Ober K A，Jockusch E L．The roles of wingless and decapentaplegic in axis and appendage development in the redflour beetle，Tribolium castaneum［J］．Dev Biol，2006，294（2）：391-405［23］Menzel N，Melzer J，Waschke J，et al．The Drosophila p21-activated kinase Mbt modulates E-cadherin-mediated celladhesion by phosphorylation of Armadillo［J］．Biochem J，2008，416（2）：231-241837。

Lp-PLA2心血管事件新的独立预测因子

circulating LDL and plaque formation
The LDL Oxidation Process
PUFA
Oxidation
O
O
O
long chain
O
O
+
O
P O
O -
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Phosphatidylcholine (PC)
O
O O OOH O
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long chain
ox-Fatty Acid
• Incidence: Over 1 million MI cases each year, up to 44%
result in death
• Prevalence: Over 12 million cases of angina - Broad range of ages - Men and women - All socioeconomic sectors
CHD
• The Role of Lp-PLA2 in Screening and
Managing Patients with CHD
Cardiovascular Diseases
Many advances, but far from conquered
Cardiovascular diseases are the prime cause of mortality in the United States and around the world
Limitations of the Current Approach to CHD
Identifying patients at risk – need better ways to identify patients at risk

把一个实验写成英语作文

把一个实验写成英语作文The Impact of Caffeine on Plant Growth.Introduction.Caffeine is a central nervous system stimulant that is found in coffee, tea, and energy drinks. It is the most widely consumed psychoactive drug in the world. Caffeine has been shown to have a variety of effects on humans, including increased alertness, wakefulness, and physical performance. However, its effects on plants are not as well known.Some studies have shown that caffeine can have a positive effect on plant growth. For example, one study found that caffeine increased the growth of Arabidopsis thaliana seedlings. Another study found that caffeine increased the yield of tomatoes. However, other studies have shown that caffeine can have a negative effect on plant growth. For example, one study found that caffeinedecreased the growth of rice seedlings.The purpose of this study was to investigate the effects of caffeine on the growth of mung bean seedlings. We hypothesized that caffeine would have a positive effect on plant growth.Materials and Methods.Mung bean seeds were planted in pots filled with soil. The pots were placed in a growth chamber with a 16-hour light/8-hour dark cycle. The temperature in the growth chamber was 25 degrees Celsius.The seedlings were watered with a solution of either caffeine or water. The caffeine solution was made by dissolving caffeine powder in water. The concentration of caffeine in the solution was 100 ppm.The seedlings were grown for 10 days. After 10 days, the seedlings were harvested and their height and weight were measured.Results.The results of the study showed that caffeine had a significant effect on the growth of mung bean seedlings.The seedlings that were watered with the caffeine solution were taller and heavier than the seedlings that werewatered with water.The average height of the seedlings that were watered with the caffeine solution was 10.2 cm. The average heightof the seedlings that were watered with water was 8.5 cm.The average weight of the seedlings that were watered with the caffeine solution was 0.25 g. The average weightof the seedlings that were watered with water was 0.20 g.Discussion.The results of this study suggest that caffeine canhave a positive effect on the growth of mung bean seedlings. This finding is consistent with the results of otherstudies that have shown that caffeine can increase the growth of plants.The mechanism by which caffeine promotes plant growth is not fully understood. However, it is thought that caffeine may increase the production of hormones that stimulate plant growth. Caffeine may also increase the uptake of nutrients by plants.The findings of this study have implications for the use of caffeine in agriculture. Caffeine could be used as a fertilizer to increase the yield of crops. However, more research is needed to determine the optimal concentration of caffeine for use in agriculture.Conclusion.Caffeine has a positive effect on the growth of mung bean seedlings. This finding suggests that caffeine could be used as a fertilizer to increase the yield of crops. However, more research is needed to determine the optimal concentration of caffeine for use in agriculture.。

大脑中动脉狭窄或闭塞的急性脑梗死斑块特征及侧枝代偿评估与预后的相关因素分析

World Latest Medicne Information (Electronic Version) 2021 Vo1.21 No.212投稿邮箱：zuixinyixue@·论著·大脑中动脉狭窄或闭塞的急性脑梗死斑块特征及侧枝代偿评估与预后的相关因素分析赵静1，常文龙2（通信作者），刘洋3，邢晓明1，张超1，刘永刚1，王宁1（1.保定市第一中心医院神经内一科，河北　保定　071000；2.保定市第一中心医院内分泌实验室，河北　保定　071000；3.保定市第一中心医院医学影像科，河北　保定　071000）0　引言脑卒中在临床心脑血管疾病中较为常见，同时其也是导致病患残疾的重要因素。

颈动脉狭窄或闭塞是导致脑卒中的常见因素，其中以大脑中动脉最为常见。

大脑中动脉主要是为两侧大脑半球提供血液，当大脑中动脉发生狭窄或闭塞时，病患临床表现存在一定差异性。

通过大量研究发现出现上述情况主要是与侧枝循环建立有关。

临床有研究指出[1]，侧枝循环是否建立受到多种心脑血管危险因素影响。

但目前临床对这一说法尚未形成统一结论。

为了解大脑中动脉出现狭窄或闭塞时形成侧枝循环的原因，本次研究对大脑中动脉狭窄或闭塞病患临床资料进行分析，现将研究过程以及研究结果汇报如下。

1　资料与方法1.1　一般资料。

选择2018年12月至2020年12月在本院实施大脑中动脉M 1段重度狭窄或闭塞治疗基金项目：本文系保定市社发类项目（项目编号：NO.1951ZF070）作者简介：赵静（1981-），女，汉族，河北保定，硕士研究生，副主任医师，研究方向：脑血管病；常文龙（1981-），男，汉族，河北邯郸，本科，副主任检验师，研究方向：医学检验。

摘要：目的探究大脑中动脉狭窄或闭塞的病患脑梗死斑块特点和侧枝循环同疾病预后之间关系。

方法选择2018年12月至2020年12月在本院实施大脑中动脉M1段重度狭窄或闭塞治疗的40例病患为实验组，另选择同时期在本院进行健康体检的40例体检者为常规组。

捕获因果关系（2）

捕获因果关系（2）摘要：个体之间的差异不仅体现在那些通常被称为“处理前异质性”（pretreatment heterogeneity）的背景特征上，还体现在个体对特定处理、事件或干预的反应中。

处理与处理倾向（treatment and the propensity of treatment）之间的交互作用是社会科学中关于选入处理组和因果推断问题的一个主要研究兴趣。

尽管“处理效应异质性”（treatment-effect heterogeneity）的重要性在因果推断的文献中已经得到了广泛的认可，但社会科学定量实证研究（empirical quantitative social science research）还没有将其完全纳入。

本文（1）描述了异质性处理效应研究的主要估计策略；（2）讨论了近期关注因果效应异质性的研究，关注点在教育带来的效应，以及我们由此得到的启示；（3）利用上大学对公民参与的影响对该方法进行了说明。

本文的主要目的是鼓励研究人员用对检验处理前异质性一样的严格程度对处理效应异质性进行例行检验。

捕获因果关系(1)直接点击蓝色标题查看上期推送研究者可能希望从观测数据中决定，在一系列协变量给定的情况下，是否是等于零的。

该差分表明了是否进入处理组所存在的处理效应异质性。

如果大于，更有可能进入处理组的个体的处理效应更大，这种情况有时候被称作正向选择（positiveselection）。

类似地，如果大于，更有可能进入处理组的个体的处理效应更小，这种情况有时候被称作负向选择（negative selection）。

不同的参数可以通过加权回归进行估计，其中总体的权重是个体预测的进入处理组的可能性pi（Morgan and T odd 2008）：我们的目标是表示总体组成的权重，和的使用类似于调查权重的使用。

使得控制组成为处理组的一个代表性样本而处理组本身不受改变；的作用刚好相反。

这些参数还可以通过匹配方法进行估计，对于特定处理效应来说不重要的个体被赋予零权重（如在最近邻匹配中被丢弃）或其他权重（如在核匹配）（Abadie and Imbens 2006; Morgan and Harding 2006; Rubin 1974）。

第二讲雌雄配子体发育

LRR-RLK Controls PMC & Tapetum Cell Fate
PMC fate is a default for tapetal cells
Meiosis-specific gene expression
What is the ligand for EMS1 ?
ems1功能缺失造成原本发育成绒毡层的细胞层变成了Msc
Pollen & Pollen Tube
10μm
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Genetic Control on Pollen Development
rhf1/2
Liu and Qu, Mol. Plant (2008)
Ovule Development: A SEM/CLSM view
Chalaza
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• Why do megaspores have different fates? • What’s the role of programmed cell death ? • What’s the underlying mechanism? • What’re the genes involved?
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Effect of task-related extracerebral circulation on diffuse optical tomography: experimental data and simulations on the foreheadTiina Näsi,1,2,* Hanna Mäki,1,2 Petri Hiltunen,1 Juha Heiskala,3 Ilkka Nissilä,1,2Kalle Kotilahti,1,2 and Risto J. Ilmoniemi1,21Department of Biomedical Engineering and Computational Science (BECS), Aalto University School of Science,P.O. Box 12200, FI-00076 AALTO, Espoo, Finland2BioMag Laboratory, HUS Medical Imaging Center, Helsinki University Central Hospital, P.O. Box 340, FI-00029HUS, Finland3Department of Computer Science, University College London, Gower Street, London WC1E 6BT, United Kingdom*tiina.nasi@aalto.fiAbstract: The effect of task-related extracerebral circulatory changes ondiffuse optical tomography (DOT) of brain activation was evaluated usingexperimental data from 14 healthy human subjects and computersimulations. Total hemoglobin responses to weekday-recitation, verbal-fluency, and hand-motor tasks were measured with a high-density optodegrid placed on the forehead. The tasks caused varying levels of mental andphysical stress, eliciting extracerebral circulatory changes that thereconstruction algorithm was unable to fully distinguish from cerebralhemodynamic changes, resulting in artifacts in the brain activation images.Crosstalk between intra- and extracranial layers was confirmed by thesimulations. The extracerebral effects were attenuated by superficial signalregression and depended to some extent on the heart rate, thus allowingidentification of hemodynamic changes related to brain activation during theverbal-fluency task. During the hand-motor task, the extracerebralcomponent was stronger, making the separation less clear. DOT provides atool for distinguishing extracerebral components from signals of cerebralorigin. Especially in the case of strong task-related extracerebral circulatorychanges, however, sophisticated reconstruction methods are needed toeliminate crosstalk artifacts.©2013 Optical Society of AmericaOCIS codes: (170.0110) Imaging systems; (170.1470) Blood or tissue constituent monitoring;(170.3880) Medical and biological imaging; (170.6960) Tomography.References and links1. E. Kirilina, A. Jelzow, A. Heine, M. Niessing, H. Wabnitz, R. Brühl, B. Ittermann, A. M. Jacobs, and I.Tachtsidis, “The physiological origin of task-evoked systemic artefacts in functional near infrared spectroscopy,”Neuroimage 61(1), 70–81 (2012).2. P. D. Drummond, “Adrenergic receptors in the forehead microcirculation,” Clin. Auton. Res. 6(1), 23–27 (1996).3. P. D. Drummond, “The effect of adrenergic blockade on blushing and facial flushing,” Psychophysiology 34(2),163–168 (1997).4. L. Minati, I. U. Kress, E. Visani, N. Medford, and H. D. Critchley, “Intra- and extra-cranial effects of transientblood pressure changes on brain near-infrared spectroscopy (NIRS) measurements,” J. Neurosci. Methods197(2), 283–288 (2011).5. I. Tachtsidis, T. S. Leung, A. Chopra, P. H. Koh, C. B. Reid, and C. E. Elwell, “False positives in functionalnear-infrared topography,” Adv. Exp. Med. Biol. 645, 307–314 (2009).6. T. Takahashi, Y. Takikawa, R. Kawagoe, S. Shibuya, T. Iwano, and S. Kitazawa, “Influence of skin blood flowon near-infrared spectroscopy signals measured on the forehead during a verbal fluency task,” Neuroimage 57(3), 991–1002 (2011).7. M. A. Franceschini, D. K. Joseph, T. J. Huppert, S. G. Diamond, and D. A. Boas, “Diffuse optical imaging of thewhole head,” J. Biomed. Opt. 11(5), 054007 (2006).8. S. R. Arridge, “Optical tomography in medical imaging,” Inverse Probl. 15(2), R41–R93 (1999).9. A. Gibson and H. Dehghani, “Diffuse optical imaging,” Philos. Transact. A Math. Phys. Eng. Sci. 367(1900),3055–3072 (2009).#178634 - $15.00 USD Received 26 Oct 2012; revised 28 Jan 2013; accepted 7 Feb 2013; published 13 Feb 2013(C) 2013 OSA 1 March 2013 / Vol. 4, No. 3 / BIOMEDICAL OPTICS EXPRESS 41210. B. W. Zeff, B. R. White, H. Dehghani, B. L. Schlaggar, and J. P. Culver, “Retinotopic mapping of adult humanvisual cortex with high-density diffuse optical tomography,” Proc. Natl. Acad. Sci. U.S.A. 104(29), 12169–12174 (2007).11. N. M. Gregg, B. R. White, B. W. Zeff, A. J. Berger, and J. P. Culver, “Brain specificity of diffuse opticalimaging: improvements from superficial signal regression and tomography,” Front Neuroenergetics 2, 14 (2010).12. S. P. Koch, C. Habermehl, J. Mehnert, C. H. Schmitz, S. Holtze, A. Villringer, J. Steinbrink, and H. Obrig,“High-resolution optical functional mapping of the human somatosensory cortex,” Front Neuroenergetics 2, 12 (2010).13. C. Habermehl, S. Holtze, J. Steinbrink, S. P. Koch, H. Obrig, J. Mehnert, and C. H. Schmitz, “Somatosensoryactivation of two fingers can be discriminated with ultrahigh-density diffuse optical tomography,” Neuroimage 59(4), 3201–3211 (2012).14. I. Nissilä, T. Noponen, K. Kotilahti, T. Katila, L. Lipiäinen, T. Tarvainen, M. Schweiger, and S. Arridge,“Instrumentation and calibration methods for the multichannel measurement of phase and amplitude in optical tomography,” Rev. Sci. Instrum. 76(4), 044302 (2005).15. H. Dehghani, B. R. White, B. W. Zeff, A. Tizzard, and J. P. Culver, “Depth sensitivity and image reconstructionanalysis of dense imaging arrays for mapping brain function with diffuse optical tomography,” Appl. Opt.48(10), D137–D143 (2009).16. J. Heiskala, M. Pollari, M. Metsäranta, P. E. Grant, and I. Nissilä, “Probabilistic atlas can improve reconstructionfrom optical imaging of the neonatal brain,” Opt. Express 17(17), 14977–14992 (2009).17. M. Cope, “The application of near infrared spectroscopy to non invasive monitoring of cerebral oxygenation inthe newborn infant,” Ph.D. Thesis (University College London, Department of Medical Physics andBioengineering, 1991).18. Y. Yamashita, A. Maki, and H. Koizumi, “Wavelength dependence of the precision of noninvasive opticalmeasurement of oxy-, deoxy-, and total-hemoglobin concentration,” Med. Phys. 28(6), 1108–1114 (2001).19. C. R. Genovese, N. A. Lazar, and T. Nichols, “Thresholding of statistical maps in functional neuroimaging usingthe false discovery rate,” Neuroimage 15(4), 870–878 (2002).20. J. C. Hebden, F. M. Gonzalez, A. Gibson, E. M. C. Hillman, R. M. Yusof, N. Everdell, D. T. Delpy, G. Zaccanti,and F. Martelli, “Assessment of an in situ temporal calibration method for time-resolved optical tomography,” J.Biomed. Opt. 8(1), 87–92 (2003).21. D. A. Boas, G. Strangman, J. P. Culver, R. D. Hoge, G. Jasdzewski, R. A. Poldrack, B. R. Rosen, and J. B.Mandeville, “Can the cerebral metabolic rate of oxygen be estimated with near-infrared spectroscopy?” Phys.Med. Biol. 48(15), 2405–2418 (2003).22. J. P. Kuhtz-Buschbeck, R. Gilster, S. Wolff, S. Ulmer, H. Siebner, and O. Jansen, “Brain activity is similarduring precision and power gripping with light force: an fMRI study,” Neuroimage 40(4), 1469–1481 (2008). 23. S. G. Costafreda, C. H. Y. Fu, L. Lee, B. Everitt, M. J. Brammer, and A. S. David, “A systematic review andquantitative appraisal of fMRI studies of verbal fluency: role of the left inferior frontal gyrus,” Hum. BrainMapp. 27(10), 799–810 (2006).24. S. Heim, S. B. Eickhoff, and K. Amunts, “Specialisation in Broca’s region for semantic, phonological, andsyntactic fluency?” Neuroimage 40(3), 1362–1368 (2008).25. P. Hiltunen, S. Särkkä, I. Nissilä, A. Lajunen, and J. Lampinen, “State space regularization in the nonstationaryinverse problem for diffuse optical tomography,” Inverse Probl. 27(2), 025009 (2011).26. J. Heiskala, P. Hiltunen, and I. Nissilä, “Significance of background optical properties, time-resolved informationand optode arrangement in diffuse optical imaging of term neonates,” Phys. Med. Biol. 54(3), 535–554 (2009). 27. M. A. Franceschini, S. Fantini, J. H. Thompson, J. P. Culver, and D. A. Boas, “Hemodynamic evoked responseof the sensorimotor cortex measured noninvasively with near-infrared optical imaging,” Psychophysiology 40(4), 548–560 (2003).1. IntroductionNear-infrared spectroscopy (NIRS) utilizes light for noninvasively recording blood oxygenation and volume changes in tissue. It is often applied to measure, through the scalp and skull, cerebral hemodynamic changes related to neuronal activity. Its sensitivity to physiologically important parameters, high temporal resolution, portability, inexpensiveness, and safety make the method a compelling alternative to other hemodynamic imaging modalities such as functional magnetic resonance imaging (fMRI) and positron emission tomography. However, besides cerebral hemodynamics, the technique is also sensitive to scalp circulation [1], which is controlled by the autonomic nervous system directly [2,3] and through heart rate and blood pressure indirectly [4]. As stress-related cardiovascular and extracerebral changes are related, the extracerebral effects are expected to show a dependency on heart rate or other cardiovascular parameters. Supporting this view, changes in mean arterial blood pressure and heart rate, as well as pulse magnitude in the scalp measured with laser Doppler flowmetry have been shown to correlate with NIRS signals and the NIRS signals have been shown to contain contribution from extracerebral layers [4–7]. Thus, the#178634 - $15.00 USD Received 26 Oct 2012; revised 28 Jan 2013; accepted 7 Feb 2013; published 13 Feb 2013 (C) 2013 OSA 1 March 2013 / Vol. 4, No. 3 / BIOMEDICAL OPTICS EXPRESS 413interpretation of NIRS is complicated by scalp and systemic circulation, especially in tasks with increased mental or physical effort.NIRS can be extended to three-dimensional (3D) imaging with an image reconstruction algorithm if the tissue is probed with overlapping measurements of multiple source-to-detector separations [8]. This technique is known as diffuse optical tomography (DOT). DOT reconstructs images that are technically 3D and can thus, in theory, provide separation between extracerebral physiology and cerebral hemodynamic responses [9]. In practice, the ability to discriminate between changes occurring at different depths is limited. To improve the depth discrimination of DOT, high-density probes and coaxial fibers operating simultaneously as sources and detectors have been used in recent studies [10–13]. Furthermore, signal-processing methods, such as the superficial signal regression (SSR), have been proposed to reduce extracerebral and global systemic variation in the data [11].In this study, we examine how extracerebral changes triggered by mental and physical stress during task performance affect DOT reconstructions. We investigate and compare experimental and simulated data of cerebral and extracerebral changes to elucidate the effect of scalp circulation on reconstructions of cerebral hemodynamics. We also evaluate the performance of SSR for reducing extracerebral contribution. Furthermore, we use simulations to test the potential benefits of increased measurement density by replacing each source and detector fiber with a coaxial fiber in enhancing the sensitivity and specificity of the reconstructions to cerebral hemodynamics.2. Methods2.1 Subjects and tasksFourteen healthy, right-handed, Finnish-speaking subjects (ages: 23–34, mean 28; 3 female) participated in two similar measurement sessions on two separate days subsequent to signing an informed consent. One of the two sessions of one subject was rejected because of technical difficulties with the NIRS recording. The study was accepted by the Ethics Committee of Helsinki University Central Hospital and was in compliance with the Declaration of Helsinki.To induce different levels of mental and physical stress, the subjects performed weekday-recitation, verbal-fluency, and hand-motor tasks during two similar sessions (Fig. 1(a)). Each session included also transcranial magnetic stimulation (TMS) which was related to another study. The subjects performed first alternating hand-motor and weekday-recitation tasks (performed 5 times in total; see Fig. 1(a)) and then verbal-fluency tasks (6 repetitions) in each session. Thereafter, TMS pulses were delivered to the right prefrontal cortex (real TMS) or to the vertex (sham TMS, induced current direction along the interhemispheric fissure) at 1 Hz for about 20 min. Subsequent to TMS, the subjects performed verbal-fluency tasks (6 repetitions) and finally alternating hand-motor and weekday-recitation tasks (5 times in total; see Fig. 1(a)). The post-TMS hand-motor and weekday-recitation tasks were included in the analysis to maximize the amount of data. The post-TMS verbal-fluency tasks were excluded in order to keep the number of repetitions for each task approximately the same. This set was selected not to be analyzed because the TMS protocol was designed to have an effect on the verbal-fluency task even though no significant changes in task performance were observed (the effect of TMS on the verbal-fluency data is beyond the scope of this study).The hand-motor task consisted of repeatedly and strongly squeezing a hand gripper for 30 s in a self-paced manner (Fig. 1(b)). The verbal-fluency task consisted of a 15-s pre-task period, a 30-s task period, and a 30-s post-task period. During the pre- and post-task periods, the subjects recited weekdays in a consecutive manner. During the task period, they had to name as many words as they could starting with a given letter. The order of the letters was randomized separately for each measurement session. The weekday-recitation task was similar to the verbal-fluency task except during the task period that consisted of reciting weekdays.Tasks were performed in Finnish, and cues were given visually on a computer screen. Between tasks, resting periods of 42–64 s (verbal fluency), 42–48 s (weekday recitation) or#178634 - $15.00 USD Received 26 Oct 2012; revised 28 Jan 2013; accepted 7 Feb 2013; published 13 Feb 2013 (C) 2013 OSA 1 March 2013 / Vol. 4, No. 3 / BIOMEDICAL OPTICS EXPRESS 41450–60 s (hand motor) were held. The subjects were instructed to move as little as possible during the tasks in order to minimize the contribution of motion artifacts.Fig. 1. (a) Task protocol of one measurement session, (b) task description, and (c) positioningof the DOT probe (digitized optode locations of one subject overlaid over the surface renderingof the subject’s MRI). Sources are marked with blue crosses and detectors with red circles. Themidline and the 10% perimeter according to the International 10–20 system are depicted withdashed lines. The area presented in the results is shaded.2.2 MeasurementsFrequency-domain NIRS data were obtained from the subjects sitting on a chair in a dimly lit room. The NIRS instrument guided intensity-modulated light from two time-multiplexed laser diodes (785 and 824 nm) through optical fibers into the tissue and recorded the attenuation of the modulation amplitude and the phase delay arising in the tissue [14]. The high-density fiber-optic DOT probe was attached on the left side of the forehead according to the International 10–20 system so that the lowest medial source was at Fpz and the lowest row of sources followed the 10% perimeter (Fig. 1(c)). The probe consisted of three rows of five detector fiber bundles and three rows of five source fibers interleaved in a regular pattern and cast in silicone to keep the interoptode distances and terminal angles constant, yet giving the probe flexibility. The signals up to the fourth nearest-neighbor channels (12-, 27-, 36-, and 43-mm source-to-detector separation) were analyzed. The shortest channels record photons travelled mainly in the extracranial layer, whereas in the longer channels relatively more photons have reached the brain tissue as well [15]. The signal-to-noise ratio of longer than 43-mm channels was low; these were therefore not included in the analysis.Anatomical MRIs of subjects were acquired to digitize and coregister the optode locations in the individual anatomy with MRI-guided eXimia Navigated Brain Stimulation system (Nexstim Ltd., Helsinki, Finland). In the second session, the probe was placed at the same location as in the first session with the help of the navigation system.To monitor systemic cardiovascular changes, beat-to-beat heart rate was recorded from one subject with an electrocardiogram in one session and with a photoplethysmograph in the other sessions (S/5 patient monitor, Datex-Ohmeda, Finland).An accelerometer attached to the DOT probe was used for movement detection. In five sessions, the accelerometer data could not be recorded because of technical issues.2.3 Signal processingThe modulation amplitude detected after light propagation through the tissue was resampled from the original sampling rate of approximately 0.5 Hz to a sampling rate of 1 Hz, high-pass filtered (cutoff: 0.005 Hz) to reduce instrumental drift, and low-pass filtered (0.22 Hz) to#178634 - $15.00 USD Received 26 Oct 2012; revised 28 Jan 2013; accepted 7 Feb 2013; published 13 Feb 2013 (C) 2013 OSA 1 March 2013 / Vol. 4, No. 3 / BIOMEDICAL OPTICS EXPRESS 415attenuate high-frequency noise. Data corresponding to individual task repetitions were baseline-corrected by setting the mean of the resting data in the time window −10…−1 s prior to task onset to zero (prior to task period in hand motor task and prior to pre-task period in weekday-recitation and verbal-fluency tasks). Artifacts were rejected from the analysis by visual inspection of the NIRS and accelerometer data. Channels were rejected from the analysis if the natural logarithm of their mean variance over all subjects exceeded the value −4, as they most likely had a poor contact (total of 28 out of 162 channels were rejected; 22 of these were 43-mm channels).The attenuation time series were analyzed with and without SSR. In the SSR algorithm, the average time course over the first nearest-neighbor channels was fitted to the time course of each channel and subtracted prior to calculating DOT reconstructions to reduce global and extracerebral contribution [11].The NIRS data during separate task repetitions were sorted by the mean heart-rate change at the end of the task period (15…30 s) and grouped into three equal numbered groups within each task. Group “small” consisted of repetitions with the smallest heart-rate changes, group “large” of repetitions with the largest changes, and group “intermediate” of the remaining repetitions.2.4 Diffuse optical tomographyDOT images of absorption changes in the tissue were reconstructed from the light-attenuation data separately for each task repetition. The sensitivity relation between the measurement and the change in the absorption coefficient in the tissue for each voxel was obtained from a Monte Carlo simulation in a homogeneous head model. The external shape of the head model and the optode positions were derived from the MRI and digitized optode locations of one subject. The following background optical properties were used in the simulation: absorption coefficient ȝa = 0.017 mm −1, reduced scattering coefficient ȝs ' = 1.1 mm −1, anisotropy factor g = 0.8, and refractive index n = 1.4. In addition to this homogeneous head model, also a 5-layered head model (scalp ȝa = 0.016 mm −1, ȝs ' = 1.4 mm −1; skull ȝa = 0.024 mm −1, ȝs ' = 1.25 mm −1; cerebrospinal fluid ȝa = 0.004 mm −1, ȝs ' = 0.025 mm −1; gray matter ȝa = 0.0186 mm −1, ȝs ' = 0.61 mm −1; white matter ȝa = 0.014 mm −1, ȝs ' = 1.1 mm −1; g = 0.8 and n = 1.4 for all layers) was created from the same MRI. As reconstructions with this 5-layered model were qualitatively similar to those obtained with the homogeneous model, results only with the homogeneous model are presented in this article. The Monte Carlo method is explained in detail elsewhere [16]. The reconstruction was performed with Tikhonov regularization by minimizing, separately for each time point, the functional 22y J x L x αΔ−Δ+Δ (1) where ǻy is the measurement data, ǻx the differential absorption coefficient to be reconstructed, J the sensitivity matrix, Į the regularization parameter (Įlow = 5 and Įhigh = 103 used in this study) and L the discrete Laplace operator. Phase data were not utilized because of their low signal-to-noise ratio when switching detector gains rapidly as in this study.Reconstructed changes in the absorption coefficient at the two wavelengths were converted into hemoglobin concentration changes with the specific extinction coefficients of oxy- and deoxyhemoglobin [17]. The sum of oxy- and deoxyhemoglobin concentrations ([HbO 2] and [HbR]), i.e., the total hemoglobin concentration ([HbT]), is presented in the results section, since the light wavelengths were not optimal for separating [HbO 2] and [HbR] because of crosstalk [18]. The results for [HbO 2] and [HbR] are included in the Appendix. To separately visualize the reconstructed changes of [HbT] (ǻ[HbT]) in the brain and in the scalp, the voxel data were projected into the intra- and extracranial layers. For the intracranial projection, ǻ[HbT] was averaged from a depth of 0 to 5 mm below the skull, and for the extracranial projection, it was averaged from the head surface to a depth of 4 mm. The #178634 - $15.00 USD Received 26 Oct 2012; revised 28 Jan 2013; accepted 7 Feb 2013; published 13 Feb 2013(C) 2013 OSA 1 March 2013 / Vol. 4, No. 3 / BIOMEDICAL OPTICS EXPRESS 416projections were averaged over task repetitions and over time steps in the following three time windows: baseline (resting data from −10 to −1 s prior to task onset), task (15…30 s with respect to task period onset), and post-task (45…60 s) periods.2.5 Statistical testingProjections and time courses are presented as averages over task repetitions; the reliability of the averages is depicted with the standard error of the mean (SEM) or the 95% confidence interval of the mean obtained from t-statistics. The uncorrected significance level of all statistical tests was set at 0.05.To test the effect of TMS on the DOT projections, projections calculated from repetitions of sham and real TMS sessions were compared pixel by pixel with two-sample t-tests. The p-values were adjusted for multiple comparisons by controlling the false discovery rate (FDR) over the pixels of the intra- and extracranial projections [19].Task-related heart-rate changes (mean over 15…30 s of the task period) were tested with two-way analysis of variance (ANOVA) for dependence on factors ‘task’ (levels: weekday recitation, verbal fluency, hand motor) and ‘heart-rate group’ (small, intermediate, large). Post-hoc testing of significant two-way interaction was performed with one-way ANOVAs. Significant one-way ANOVAs were further followed up with Tukey–Kramer post-hoc tests. The post-hoc one-way ANOVAs were Bonferroni-corrected for multiple comparisons with factor three.To test the statistical significance of ǻ[HbT] in the projections during the task period, they were compared with baseline projections pixel by pixel with paired t-tests. The p-values were adjusted for pixelwise multiple comparisons by controlling the FDR over the pixels of the intra- and extracranial projections. The results of these t-tests are presented as statistical t-maps, where t-values exceeding the threshold for statistical significance are colored.To quantify the heart-rate dependency of ¨[HbT], the projections of the task period were tested pixel by pixel with one-way ANOVA for the continuous factor average heart-rate change. The p-values were adjusted for multiple comparisons by controlling the FDR over pixels of the intra- and extracranial projections. The results are presented as F-maps, where F-values exceeding the level of statistical significance are colored.2.6 SimulationsTo understand our observations and the limitations of DOT in separating cerebral and extracerebral changes, we simulated measurements with cerebral and extracerebral perturbations. We applied two virtual DOT probes: a probe similar to the one used in the measurements (“measurement probe”) and another probe with an identical fiber array, but each source and detector replaced with a hybrid optode functioning both as a source and a detector (“hybrid probe”) [20]. The measurement probe had 162 and the hybrid probe 642 active channels, as all channels with source-to-detector separations from 0 to 43 mm were included in the analysis.Five simulated data sets were generated with the following details: (1) a local transient ǻ[HbT] in the gray matter, representing cerebral activity (step function in time and space; size 10 mm × 10 mm × depth of gray matter ~3 mm; magnitude 4 μM, Fig. 2(a)); (2) a homogeneous ǻ[HbT] in the scalp covering the whole measurement area, representing an extracerebral change (step function in time; depth ~3 mm; activation at the same time as for the local simulated ǻ[HbT]; magnitude 4 μM); (3) combined (1) and (2) to simulate simultaneous cerebral and extracerebral changes; (4) as (3), but with a homogeneous extracerebral change of only 0.8 μM; (5) as (4), but with a negative extracerebral change of 0.8 μM.The simulated data were generated by multiplying a pre-calculated sensitivity matrix with the absorption changes corresponding to the above-introduced simulated changes. The sensitivity matrix was obtained from a Monte Carlo simulation in the 5-layered head model created from the MRI of one subject [16]. Zero-mean white Gaussian noise was added to the simulations prior to calculating the reconstructions. The standard deviation (SD) of the noise#178634 - $15.00 USD Received 26 Oct 2012; revised 28 Jan 2013; accepted 7 Feb 2013; published 13 Feb 2013 (C) 2013 OSA 1 March 2013 / Vol. 4, No. 3 / BIOMEDICAL OPTICS EXPRESS 417was obtained from the experimental signals: All values of the resting data −10…0 s prior to the task onset were pooled together. The SD was calculated separately for each source-to-detector separation over the pooled values and an exponential function was fitted to the SD estimates (Fig. 2(b)). The SD for the simulated noise was taken from the fitted function separately for each source-to-detector separation and divided by the square root of the number of repetitions to obtain a noise level similar to the averaged responses in the measured data (the SD was calculated over non-averaged data).The reconstructions of the simulated data were calculated in the same manner as for the measured physiological data using the homogeneous head model. Separate head models were applied for generating the simulated data and performing the reconstructions.Fig. 2. (a) Simulated cerebral change (intracranial projection; corresponds to the shaded area inFig. 1(c) on the brain surface) and (b) SD of the experimental resting data as a function of thesource-to-detector separation (circle: 824 nm, cross: 785 nm) and the fitted exponentialfunction which was utilized for calculating the SD of the simulated noise.3. Results3.1 Effect of TMS on the hand-motor and weekday-recitation tasksThe DOT projections in hand-motor and weekday-recitation tasks did not show statistically significant differences between sham and real TMS sessions (see Appendix). Even without any adjustment to the p-values, less than 1% of the pixels exceeded the limit of statistical significance. In the following analysis we assume that the effect of TMS on the DOT data was negligible.3.2 Heart rateThe heart rate increased during all tasks (Fig. 3); the average increase differed significantly between the heart-rate groups (Fig. 4). The weekday-recitation task showed the lowest and the hand-motor task the highest heart-rate changes, except in the heart-rate group “small” where the verbal-fluency and hand-motor task did not differ significantly. The average heart-rate increases were 6 ± 4 beats per minute (bpm) in the weekday-recitation task (mean over repetitions ± SD), 11 ± 6 bpm in the verbal-fluency task, and 15 ± 9 bpm in the hand-motor task.Fig. 3. Averaged heart-rate time series in separate tasks and heart-rate groups. Vertical dashedlines indicate start of the pre-task, start and end of the task, and end of the post-task period.Shading depicts the 95% confidence interval of the mean.#178634 - $15.00 USD Received 26 Oct 2012; revised 28 Jan 2013; accepted 7 Feb 2013; published 13 Feb 2013 (C) 2013 OSA 1 March 2013 / Vol. 4, No. 3 / BIOMEDICAL OPTICS EXPRESS 418。