Stabilization of a Small Unmanned Aerial Vehicle Model without

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单核苷酸多态性与阿尔茨海默病的研究进展

目前唯一证实的一个与ＳＤ和晚发型家族性ＡＡＤ
（ａ —ｎｅｆｍｌｌＤ，ＯＡ１ｅｏｓｔａｉａＡＬＦＤ）ｔｉ发病有关的基因【，７】
它位于１ｑ３２９１．，该位点有３个等位基因（２，３￡，
ｓｏｅｅｋ等［首先研究了ＡｂｃＲ８１Ｄ患者中该基因多态
ＡＰ基因位于２号染色体上，表达ＡＰ它有Ｐ１Ｐ，两种降解途径：一是在ｐ和丫分泌酶的共同作用下一
生成Ｂ淀粉样多肽ＡＭ０和ＡＩ。Ａ１２是一种不一ＩＭ２３４
溶性多肽，比ＡＩ更易导致淀粉样物质生成，构Ｍ０是成Ａ沉积斑的核心蛋白；其二是在０分泌酶作用【一下，在ＡＢ１位和第１第６７位之间进行裂解，产生可
ＡＤｅｉｓｃｎｒｖｒａ．ｃａｄｖｒｉｎａｌｉｅｍｉｈｘｌｉｈｉａｒｅｎｆｖｒｏｓｒｓａｃｅ．ｈｒｆｒ，ｕｔｅｒｍａｎｏｔｏｅｓ１ＲａｉｌｉｅｓｔａｄｓｍｐｅｓｚｇｔｅｐａｎｔｅｄｓｇｅｍｅｔｏａｉｕｅｅｒｈｓＴｅｅｏｅｆｒｒｙｈ
ｔｎＡＰ）因、老素１ｐｅｅｉｎ１Ｐ１基因、ｅ，Ｐ基ｉ早（ｒｓｎｌ，Ｓ）ｉ早老素２（ｒｓｎｌ，Ｓｐｅｅｉｎ２Ｐ２）基因。ｉ
１１．ＡＰＰ
老年性痴呆，是以进行性痴呆为临床特征的神经退行性变性疾病，其神经病理特征为细胞外广泛的淀粉样斑块（ｍｙｏｄｐａｕｓＡ沉积和细胞内神经ａｌｉｌｑｅ，Ｐ）原纤维缠结。大约１％的Ａ为家族遗传性的０Ｄ

杨楚罗教授课题组：调控小分子受体烷基链的分支位置提升非富勒烯有机太阳能电池性能

杨楚罗教授课题组：调控小分子受体烷基链的分支位置提升非富勒烯有机太阳能电池性能深圳大学/武汉大学杨楚罗教授课题组和合作者以高效率的小分子受体Y6为基础，将烷基链分支位置离开Y6的中心核开发了两个新的SMA (Y6-C2和Y6-C3)。

在未经任何后处理的情况下，基于Y6-C2的器件效率高达15.89％，高于基于Y6（15.24％）和Y6-C3（13.76％）的器件，这是目前已报道的无需后处理的最高二元器件效率。

非富勒烯小分子受体（NFSMA）由于其易于调节的光学和电子特性引起了有机太阳能电池（OSC）领域研究者的极大兴趣和持续关注。

目前，基于NFSMA的器件效率（PCE）已经超过16％。

非富勒烯SMA的分子设计策略主要包括中心核修饰、末端基团修饰和侧链工程。

与中心核和末端单元修饰相比，“侧链工程”策略不仅可以节约合成时间，也可以微调分子特性，从而改善器件性能。

侧链的改变、包括对称性，尺寸和长度的变化，可以极大地影响SMA的分子间相互作用和结晶特性。

尽管侧链工程策略已被广泛研究，但烷基链分支位置对SMA的性质和性能的研究却很少。

调节烷基链的分支位置是增强有机场效应晶体管（OFET）中分子堆积和电荷载流子迁移率的有效方法，研究该方法对OSC性能的影响具有十分重要的意义。

图1 Y6, Y6-C2, Y6-C3和PC71BM的化学结构；二元，三元和大面积器件效率。

最近，深圳大学/武汉大学杨楚罗教授课题组和合作者以高效率的小分子受体Y6为基础，将烷基链分支位置离开Y6的中心核开发了两个新的SMA (Y6-C2和Y6-C3)。

与Y6相比，Y6-C2具有相似的吸收波长和电化学性质，但分子堆积更好，结晶度更高。

以PM6为给体分别与三个小分子受体共混制备器件，在未经任何后处理的情况下，基于Y6-C2的器件效率高达15.89％，高于基于Y6（15.24％）和Y6-C3（13.76％）的器件，这是目前已报道的无需后处理的最高二元器件效率。

非酒精性脂肪性肝病代谢组学研究进展

机制尚未完全明确，１９９８年Ｄａｙ等［１２］提出“二次打击”学说。开。同时ＮＡＦＬＤ肝硬化患者与酒精性肝硬化患者也可有效区
随后Ｔｉｌｇ等［１３－１４］提出“多重平行打击”理论，包括遗传因素、分开（ＡＵＣ＝０．８３）。他们认为此方法可作为区分ＮＡＦＬＤ纤维
ＩＲ、氧化应激、脂毒性、慢性炎症、纤维化、免疫和肠道菌群等，化程度及诊断的无创生物标志物，且可以显著减少对肝活检的
黄酯和１３－ｃｉｓＲＡ呈正相关。他们在人类组织中首次检测到验证；单不饱和ＴＡＧ的增加可能是ＮＡＦＬＤ和ＣＨＢ患者ＮＡＳＨ
ａｔＲＡ的活性代谢物４－ｏｘｏ－ａｔＲＡ，表明这种类维生素Ａ可能的特异性标志物。
有助于人体类维生素Ａ的信号传导。肝脏维生素Ａ的稳态平２．３代谢组学对ＮＡＦＬＤ药物作用与疗效研究的推动作用
录组学、蛋白质组学为代表的系统生物学技术提供了新的技术展的新学科，代谢组学较为全面的展示了机体的代谢结果，为
与思路。区别于其他组学技术，以内源性小分子代谢物为研究临床医学提供了新的技术和方法。
对象的代谢组学可以很好的揭示机体变化的最终代谢结果。因２非酒精性脂肪性肝病（ＮＡＦＬＤ）
收基作ＤＯ稿金者Ｉ：日项简１０期目介．３：：：９１２上栾研６７０９）２海究雨／０ｊ．中婷－ｉｓ医１ｓ（ｎ１药．１－１９大０９０００６学１—；修－附）５回，属２女５日第６，．期七主２０：人２要２１０民．２从００医４事－．院０１慢４２人７－性才１肝７培病养计的划基（础ＸＸ与２０临１９床－通信作者：顼志兵，ｘｚｂ６１６０＠１６３．ｃｏｍ
和遗传易感密切相关的代谢应激性肝损伤，包括非酒精性单纯１代谢组学概述
性肝脂肪变（ＮＡＦＬ）、非酒精性脂肪性肝炎（ＮＡＳＨ）、肝硬化和１．１代谢组学含义代谢组学最初于１９９９年由Ｎｉｃｈｏｌｓｏｎ

An increasing threat in hospitals_ multidrug-resistant Acinetobacter baumannii.

Intensive care units (ICUs) of hospitals harbour critically ill patients who are extremely vulnerable to infections. These units, and their patients, pro-vide a niche for opportunistic microorganisms that are generally harmless for healthy individuals but that are often highly resistant to antibiotics and can spread epidemically among patients. Infections by such organisms are difficult to treat and can lead to an increase in morbidity and mortality. Furthermore, their eradication from the hospital environment can require targeted measures, such as the isolation of patients and temporary closure or even reconstruction of wards. The presence of these organisms, therefore, poses both a medical and an organizational burden to health-care facilities.One important group of bacteria that is associated with these problems is the heterogeneous group of organisms that belong to the genus Acinetobacter. This genus has a complex taxonomic history. Since the 1980s, in parallel with the emergence of acinetobacters as noso-comial pathogens, the taxonomy of the genus has been refined; 17 named species have been recognized and 15 genomic species (gen.sp.) have been delineated by DNA–DNA hybridization, but these do not yet have valid names (TABLE 1). The species that is most commonly involved in hospital infection is Acinetobacter bauman-nii, which causes a wide range of infections, including pneumonia and blood-stream infections. Numerous studies have reported the occurrence of multidrug-resistant (MDR) A. baumannii in hospitals, and at some locations pandrug-resistant strains have been identified. Currently, A. baumannii ranks among the most important nosocomial pathogens. Additionally, the number of reports of community-acquired A. baumannii infection has been steadily increasing, although overall this type of infection remains rare. Despite the numerous publi-cations that have commented on the epidemic spread of A. baumannii, little is known about the mechanisms that have favoured the evolution of this organism to multi-drug resistance and epidemicity. In this Review, we dis-cuss the current state of knowledge of the epidemiology, antimicrobial resistance and clinical significance of acinetobacters, with an emphasis on A. baumannii. The reader is also referred to previous reviews of this organism that have been written by pioneers in the field1,2.Identification of Acinetobacter speciesIn 1986, a phenotypic system for the identification of Acinetobacter species was described3, which together with a subsequent simplified version4 has proven useful for the identification of most, but not all, Acinetobacter species. In particular, Acinetobacter calcoaceticus, A. baumannii, gen.sp. 3 and gen.sp. 13TU cannot be separated well by this system4. These species are also highly similar by DNA–DNA hybridization5 and it has therefore been proposed that they should be grouped together into the so-called A. calcoaceticus–A. baumannii (Acb) complex4. From a clinical perspective this might not be appropriate, as the complex combines three of the most clinically relevant species (A. baumannii, gen.sp. 3 and gen.sp. 13TU) with an environmental spe-cies (A. calcoaceticus). It is noteworthy that the perform-ance of commercial systems for species identification that are used in diagnostic microbiology is also unsatisfactory.*Department of Infectious Diseases C5‑P, Leiden University Medical Centre, Albinusdreef 2, P.O.BOX 9600, 2300 RC Leiden, the Netherlands.‡Centre of Epidemiology and Microbiology, National Institute of Public Health, Srobarova 48, 10042 Prague, Czech Republic.§Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19‑21, 50935 Cologne, Germany. Correspondence to L.D.e‑mail: l.dijkshoorn@lumc.nl doi:10.1038/nrmicro1789An increasing threat in hospitals: multidrug-resistant Acinetobacter baumanniiLenie Dijkshoorn*, Alexandr Nemec‡ and Harald Seifert§Abstract | Since the 1970s, the spread of multidrug-resistant (MDR) Acinetobacter strains among critically ill, hospitalized patients, and subsequent epidemics, have become an increasing cause of concern. Reports of community-acquired Acinetobacter infections have also increased over the past decade. A recent manifestation of MDR Acinetobacter that has attracted public attention is its association with infections in severely injured soldiers. Here, we present an overview of the current knowledge of the genus Acinetobacter, with the emphasis on the clinically most important species, Acinetobacter baumannii.DNA–DNA hybridizationDetermines the degree ofsimilarity between the genomicDNA of two bacterial strains;the gold standard to assesswhether organisms belong tothe same species.Pandrug-resistantIn this Review, refers toA. baumannii that are resistantto all available systemicanti-A. baumannii antimicrobialagents, except for polymyxins.NATURe RevIewS |microbiology vOlUMe 5 | DeCeMbeR 2007 |939©2007Nature Publishing GroupIsolateA population of bacterial cells in pure culture that is derived from a single ing these systems, the clinically relevant species ofthe Acb complex are frequently uniformly identified asA. baumannii and many other species are not identified6–8.These problems have led to the development of genotypicmethods for Acinetobacter species identification, some ofwhich are discussed in BOX 1(also see fIg. 1). Currently,precise species identification is not feasible in most labora-tories, except for a few Acinetobacter reference laboratories.In light of the difficulties in distinguishing A. baumannii,gen.sp. 3 and gen.sp. 13TU, in this Review these specieswill be referred to as A. baumannii (in a broad sense)unless otherwise stated.Epidemiology of clinical acinetobactersThe natural habitat of Acinetobacter species.MostAcinetobacter species have been found in clinicalspecimens (TABLE 1), but not all are considered to beclinically significant. One important question is wheredoes A. baumannii come from? Furthermore, are thereenvironmental or community reservoirs? As mentionedearlier, A. baumannii, gen.sp. 3 and gen.sp. 13TU are themost frequent species that are found in human clinicalspecimens5,9,10. Of these, gen.sp. 3 was the most prevalentspecies among clinical isolates in a Swedish study5. In2 european studies, Acinetobacter lwoffii was the most Table 1 | Classification of the genusAcinetobacterto label these species by the initials of their respective authors, Tjernberg and Ursing (TU)5 or Bouvet and Jeanjean (BJ)108.940 | DeCeMbeR 2007 | vOlUMe 5 /reviews/micro©2007Nature Publishing GroupNature Reviews | Microbiology predominant species to be found on the skin of healthy individuals, with carrier rates of 29% and 58%, whereas other Acinetobacter species, including Acinetobacter junii , Acinetobacter johnsonii , Acinetobacter radioresistens and gen.sp. 15bJ, were detected at lower frequencies 11,12. The carrier rates for A. baumannii (including gen.sp. 13TU) in these studies ranged from 0.5 to 3%, whereas for gen.sp. 3 the rates ranged from 2 to 6%11,12. The faecal carriage of A. baumannii among non-hospitalized individuals in the United Kingdom and the Netherlands was 0.9%13. The most predominant species in faecal samples from the Netherlands were A. johnsonii (17.5%) and gen.sp. 11 (4%)13. A. baumannii was also recovered from the bodylice of homeless people 14 and it was proposed that the organisms were associated with transient bacteraemia in these individuals. In a study in Hong Kong, the car-rier rates of A. baumannii , gen.sp. 3 and gen.sp. 13TU on the skin of healthy individuals were 4, 32 and 14%, respectively 15. Thus, the carrier rates for gen.sp. 3 and gen.sp. 13TU in that study were strikingly higher than in the european studies. These findings indicate that, at least in europe, the carriage of A. baumannii in the community is relatively low. Apart from its occurrence in humans, A. baumannii has also been associated with infection and epidemic spread in animals at aveterinary clinic 16.NATURe RevIewS | microbiologyvOlUMe 5 | DeCeMbeR 2007 | 941© 2007Nature Publishing Group908070605040302010100Acinetobacter grimontiiGen.sp. 15TUAcinetobacter junii Acinetobacter haemolyticus Gen.sp. 14BJGen.sp. 13TUA. baumannii Gen.sp. 3Gen.sp.‘close to 13TU’Acinetobacter venetianusAcinetobacter calcoaceticusGen. sp.‘between 1 and 3’Acinetobacter tjernbergiaeAcinetobacter towneriAcinetobacter ursingiiGen.sp. 13BJ or 14TU Gen.sp. 15BJ Gen.sp. 17A. baylyiAcinetobacter lwoffiiAcinetobacter schindleriAcinetobacter bouvetiiAcinetobacter gerneriGen.sp. 10Gen.sp. 11Acinetobacter johnsoniiAcinetobacter radioresistens Acinetobacter parvusPearson correlation Species Gen.sp. 16Gen.sp. 6Nature Reviews | Microbiology Acinetobacter tandoii EndemicThe constant presence of aninfectious agent in a givengeographical area or hospital.There are few available data on the environmental occurrence of A. baumannii , gen.sp. 3 and gen.sp. 13TU,but these species have been found in varying percent-ages in vegetables, fish, meat and soil 17,18. A. baumannii has also recently been found in aquacultures of fish and shrimp farms in Southeast Asia 19. However, it is not yet clear to what extent these findings are attributable to an environmental niche or to contact with humans or animals. A. baumannii has been described as a soil organism, but without the support of appropriate references 20. It was probably assumed that the wide occurrence of unspeciated acinetobacters in soil and water 21 is also applicable to A. baumannii . However, in fact, there is little evidence that A. baumannii is a typical soil resident. Taken together, the existing data indicate that A. baumannii has a low prevalence in the community and that its occurrence in the environment is rare.A. baumannii in hospitals. The most striking mani-festation of A. baumannii is the endemic and epidemic occurrence of MDR strains in hospitals. The closely related gen.sp. 3 and gen.sp. 13TU might have a similar role 22–24, and their involvement could have been under-estimated as these species are phenotypically difficult to discriminate from A. baumannii . Most investigations ofA. baumannii in hospitals have been ad hoc studies thatwere triggered by an outbreak. More in-depth studies of the prevalence of this species in hospitals, including antibiotic-resistant and antibiotic-susceptible strains, are required to better understand its true importance.Depending on the local circumstances, and the strain in question, the pattern of an outbreak can vary. Therecan be a common source or multiple sources and somestrains have a greater tendency for epidemic spread than others. epidemiological typing — mostly by genotypic methods, such as amplified fragment length polymor-phism (AFlP) analysis (BOX 1) — is an important tool that can distinguish an outbreak strain from other, concurrent strains, and assess the sources and mode oftransmission of the outbreak strain.A scheme that depicts the dynamics of epidemic A. baumannii on a hospital ward is provided in fIg. 2. An epidemic strain is most commonly introduced by apatient who is colonized. Once on a ward, the strain can then spread to other patients and their environment. A. baumannii can survive in dry conditions 25 and during outbreaks has been recovered from various sites in thepatients’ environment, including bed curtains, furnitureand hospital equipment 26. These observations, and thesuccess that cleaning and disinfecting patients’ rooms has had in halting outbreaks, emphasize the role of thehospital environment as a reservoir for A. baumanniiduring outbreaks. The bacteria can be spread throughthe air over short distances in water droplets and inscales of skin from patients who are colonized 27, but the most common mode of transmission is from the handsof hospital staff. Patients who are colonized or infected by a particular A. baumannii strain can carry this strain at different body sites for periods of days to weeks 28, and colonization can go unnoticed if the epidemic strain isnot detected in clinical specimens 2,29.Population studies of A. baumannii . Comparative typ-ing of epidemic strains from different hospitals has indicated that there can be spread between hospitals. For example, during a period of outbreaks in the Netherlands that involved eight hospitals, one common strain was found in three of these hospitals and another common strain was found in two others 26. Similar observations of interhospital spread of MDR strains in particular geographical areas have been made in the Czech Republic 30, the United Kingdom 31, Portugal 32and the United States 33. Highly similar, but distinguishable, strains have been found at different locations and at different time points,Figure 1 | Amplified fragment length polymorphism (AFlP) analysis of Acinetobacter strains. A condensed dendrogram of the AFLP (described in BOX 1)fingerprints of 267 Acinetobacter reference strains of 32 described genomic species. Allspecies are well separated at the 50% cluster-cut-off level, which emphasizes the powerof this method for the delineation and identification of Acinetobacter species.942 | DeCeMbeR 2007 | vOlUMe 5/reviews/micro© 2007Nature Publishing GroupNature Reviews |MicrobiologyClonesA group of bacteria that wereisolated independently fromdifferent sources in time andspace, but share so manyidentical traits that it is likelythat they evolved from acommon ancestor.T yping methodA tool that differentiatesbacterial strains below the species level.RibotypingA typing method in which chromosomal DNA is digested by restriction enzymes, fragments are separated by electrophoresis and, finally, particular fragments are detected by labelled rRNA probes to generate DNA-banding patterns, which allows the differentiation of bacterial isolates.without a direct epidemiological link. It is assumed thatthese strains represent particular lineages of descent(clones). examples are european clones I–III34–36, whichhave been delineated by a range of genotypic typing meth-ods, such as AFlP analysis (BOX 1a; fIg. 1), ribotyping,macrorestriction analysis by pulsed-field gel electrophore-sis and, most recently, multilocus sequence typing (seeboth MlST systems in Further information). Strainsthat belong to these clones are usually highly resistantto antibiotics, although within a clone there can bevariation in antibiotic susceptibility. Apparently, theseclones are genetically stable strains that are particularlysuccessful in the hospital environment and evolveslowly during their spread. whether these strains haveparticular virulence attributes or an enhanced ability tocolonize particular patients (discussed below) remainsto be established. Their wide spread might be explainedby the transfer of patients between hospitals and regionsover the course of time, although in many cases there isno evidence for this. It is also possible that they circulateat low rates in the community and are able to expand inhospitals under selective pressure from antibiotics. Sofar, their resistance to antimicrobial agents is the onlyknown selectively advantageous trait.Figure 2 | overview of the dynamics between patients, bacteria and the hospital environment. The possible modes of Acinetobacter baumannii entry into a ward are shown. Entrance through a colonized patient is the most likely mode. However, introduction through contaminated materials (such as pillows104) has also been documented. Notably, introduction by healthy carriers is also conceivable, although it is not known whether the rare strains that circulate inthe community have epidemic potential. Once on a ward, A. baumannii can spread from the colonized patient to the environment and other susceptible patients. The direct environment of the patient can become contaminated by excreta, air droplets and scales of skin. Interestingly, A. baumannii can survive well in the dry environment25, a feature it shares with staphylococci. Hence, the contaminated environment can become a reservoir from which the organism can spread. The acquisition of A. baumannii by susceptible patients can occur through various routes, of which the hands of hospital staff are thought to be the most common, although the precise mode of transmission is usually difficult to assess.NATURe RevIewS |microbiology vOlUMe 5 | DeCeMbeR 2007 |943©2007Nature Publishing GroupMacrorestriction analysisA typing method in which chromosomal DNA is digested with rare-cutting enzymes, so creating large fragments that are separated in an alternating electric field (pulsed-field electrophoresis) according to their size.OsteomyelitisAn infection of bone or bone marrow.Clinical impact of Acinetobacter infectionsNosocomial infections. Acinetobacters are opportun-istic pathogens that have been implicated in variousinfections that mainly affect critically ill patients inICUs. Hospital-acquired Acinetobacter spp.infectionsinclude: ventilator-associated pneumonia; skin andsoft-tissue infections; wound infections; urinary-tractinfections; secondary meningitis; and bloodstreaminfections. These infections are mainly attributed toA. baumannii, although gen.sp. 3 and gen.sp. 13TUhave also been implicated. Nosocomial infectionsthat are caused by other Acinetobacter species, suchas A. johnsonii, A. junii, A. lwoffii, Acinetobacterparvus, A. radioresistens, Acinetobacter schindleriand Acinetobacter ursingii, are rare and are mainlyrestricted to catheter-related bloodstream infec-tions8,37–40. These infections cause minimal mortalityand their clinical course is usually benign, althoughlife-threatening sepsis has been observed occasion-ally41. The rare outbreaks of some of these species (forexample, A. junii) have been found to be related tocontaminated infusion fluids41.The risk factors that predispose individuals to theacquisition of, and infection with, A. baumannii aresimilar to those that have been identified for otherMDR organisms. These include: host factors suchas major surgery, major trauma (in particular, burntrauma) and prematurity in newborns; exposure-related factors such as a previous stay in an ICU, thelength of stay in a hospital or ICU, residence in a unitin which A. baumannii is endemic and exposure tocontaminated medical equipment; and factors thatare related to medical treatment such as mechani-cal ventilation, the presence of indwelling devices(such as intravascular catheters, urinary cathetersand drainage tubes), the number of invasive proce-dures that are performed and previous antimicrobialtherapy42. Risk factors that are specific for a par-ticular setting have also been identified, such as thehydrotherapy that is used to treat burn patients andthe pulsatile lavage treatment that is used for wounddébridement43,44.The most frequent clinical manifestations of noso-comial A. baumannii infection are ventilator-associatedpneumonia and bloodstream infection, both of whichare associated with considerable morbidity and mor-tality, which can be as high as 52%45,46. Risk factorsfor a fatal outcome are severity-of-illness markers, anultimately fatal underlying disease and septic shockat the onset of infection. bacteraemic A. baumanniipneumonia has a particularly poor prognosis46. Acharacteristic clinical manifestation is cerebrospinal-shunt-related meningitis, caused by A. baumannii inpatients who have had neurosurgery47. wound infec-tions have been reported mainly in patients who havesevere burns or trauma, for example, soldiers who havebeen injured during military operations43,48. Urinary-tract infections related to indwelling urinary-tractcatheters usually run a more benign clinical courseand are more frequent in rehabilitation centres thanin ICUs49.The clinical impact of nosocomial A. baumanniiinfection has been a matter of continuing debate. Manystudies report high overall mortality rates in patientsthat have A. baumannii bacteraemia or pneumonia45,46.However, A. baumannii mainly affects patients withsevere underlying disease and a poor prognosis. It hastherefore been argued that the mortality that is observedin patients with A. baumannii infections is caused bytheir underlying disease, rather than as a consequenceof A. baumannii infection. In a case-control study, blotand colleagues50 addressed whether A. baumannii con-tributes independently to mortality and concluded thatA. baumannii bacteraemia is not associated with a sig-nificant increase in attributable mortality. Similar find-ings for A. baumannii pneumonia have been reportedby Garnacho and colleagues51. by contrast, in recentreviews of matched cohort and case-control studies,Falagas and colleagues52,53 concluded that A. baumanniiinfection was associated with an increase in attributablemortality, ranging from 7.8 to 23%. These contradictoryconclusions show that the debate on the clinical impactof A. baumannii is still ongoing.Community-acquired infections.A. baumannii isincreasingly recognized as an uncommon but impor-tant cause of community-acquired pneumonia. Mostof the reported cases have been associated withunderlying conditions, such as alcoholism, smoking,chronic obstructive pulmonary disease and diabetesmellitus. Community-acquired A. baumannii pneu-monia appears to be a unique clinical entity thathas a high incidence of bacteraemia, a fulminantclinical course and a high mortality that ranges from40 to 64%. It has been observed almost exclusivelyin tropical climates, in particular in Southeast Asiaand tropical Australia54,55. It is currently unclear, how-ever, if host factors or particular virulence factors areresponsible for these severe infections. Multidrugresistance in these organisms is uncommon55. Othermanifestations of community-acquired A. baumanniiinfections are rare.Infections associated with natural disasters and warcasualties. A characteristic manifestation of nosoco-mial A. baumannii is wound infection that is associ-ated with natural or man-made disasters, such as theMarmara earthquake that occurred in 1999 in Turkey,the 2002 bali bombing and military operations48,56,57.A strikingly high number of deep-wound infections,burn-wound infections and osteomyelitis cases havebeen reported to be associated with repatriated casual-ties of the Iraq conflict48. Isolates often had multidrugresistance. based on the common misconception thatA. baumannii is ubiquitous, it has been argued that theorganism might have been inoculated at the time ofinjury, either from previously colonized skin or fromcontaminated soil. However, recent data clearly indi-cate that contamination of the environment of fieldhospitals and infection transmission in health-carefacilities have had a major role in the acquisition ofA. baumannii58.944 | DeCeMbeR 2007 | vOlUMe 5 /reviews/micro©2007Nature Publishing GroupNature Reviews |MicrobiologyQuorum sensingThe phenomenon whereby the accumulation of signalling molecules enables a single cell to sense the number of bacteria (cell density) that are present, which allows bacteria to coordinate certain behaviours or actions.Epidemicity and pathogenicityThe fact that colonization with A. baumannii is morecommon than infection, even in susceptible patients,emphasizes that the pathogenicity of this species is gen-erally low. However, once an infection develops, it canbe severe. Studies on the epidemicity and pathogenicityfactors of A. baumannii are still at an elementary stage.A number of putative mechanisms that might have a rolein colonization, infection and epidemic spread are sum-marized in fIg. 3. Genetic, molecular and experimentalstudies are required to elucidate these mechanisms inmore detail.Recent DNA sequencing of a single A. baumanniistrain identified 16 genomic islands that carry putativevirulence genes that are associated with, for example,cell-envelope biogenesis, antibiotic resistance, autoin-ducer production, pilus biogenesis and lipid metabo-lism59. Resistance to desiccation, disinfectants25,60 andantibiotics is important for environmental survival. Theextraordinary metabolic versatility3 of A. baumanniicould contribute to its proliferation on a ward and inpatients. Pilus-mediated biofilm formation on glass andplastics has been demonstrated61. If formed on medicaldevices, such as endotracheal tubes or intravascularcatheters, these biofilms would probably provide a nichefor the bacteria, from which they might colonize patientsand give rise to respiratory-tract or bloodstream infec-tions. electron microscopy studies have demonstratedthat pili on the surface of acinetobacters interact withhuman epithelial cells62. In addition, thread-like connec-tions between these bacteria were suggestive of an earlyphase of biofilm formation. The pili and hydrophobicsugars in the O-side-chain moiety of lipopolysaccharide(lPS)63 might promote adherence to host cells as a firststep in the colonization of patients. Quorum sensing, thepresence of which has been inferred from the detectionof a gene that is involved in autoinducer production59,could control the various metabolic processes, includingbiofilm formation.Resistance to antibiotics, as well as the protectiveconditions of the skin (such as dryness, low pH, theresident normal flora and toxic lipids) and those ofthe mucous membranes (such as the presence of mucus,lactoferrin and lactoperoxidase and the sloughing ofcells) are prerequisites for bacterial survival in a host thatis receiving antibiotics. In vitro and animal experimentshave identified various factors that could have a rolein A. baumannii infection. For example, A. baumanniiouter membrane protein A (AbOmpA, previouslycalled Omp38) has been associated with the inductionof cytotoxicity64. Iron-acquisition mechanisms65 andserum resistance66 are attributes that enable the organ-ism to survive in the bloodstream. The lPS and lipid Aof one strain, at the time named A. calcoaceticus, hadbiological activities in animals that were similar to thoseof other enterobacteria67. These included lethal toxicity,pyrogenicity and mitogenicity for mouse-spleen b cells.More recently, A. baumannii lPS was found to be themajor immunostimulatory component that leads toa proinflammatory response during A. baumanniipneumonia68 in a mouse model.Taken together, the chain of events from environmen-tal presence to the colonization and infection of patientsdemonstrates the extraordinary ability of A. baumannii toadapt to variable conditions. This ability suggests that theorganism must possess, in addition to other factors, effec-tive stress-response mechanisms. Together with its resist-ance to antibiotics, these mechanisms might explain thesuccess of particular A. baumannii strains in hospitals.Figure 3 | The factors that contribute to Acinetobacter baumannii environmentalpersistence and host infection and colonization. Adherence to host cells, asdemonstrated in an in vitro model using bronchial epithelial cells62, is considered to be afirst step in the colonization process. Survival and growth on host skin and mucosalsurfaces require that the organisms can resist antibiotics and inhibitory agents and theconditions that are exerted by these surfaces. Outgrowth on mucosal surfaces andmedical devices, such as intravascular catheters and endotracheal tubes61, can result inbiofilm formation, which enhances the risk of infection of the bloodstream and airways.Quorum sensing59 might have a regulatory role in biofilm formation. Experimentalstudies have identified various factors that could have a role in A. baumannii infection, forexample, lipopolysaccharide has been shown to elicit a proinflammatory response inanimal models67,68. Furthermore, the A. baumannii outer membrane protein A has beendemonstrated to cause cell death in vitro64. Iron-acquisition mechanisms65 and resistanceto the bactericidal activity of human serum66 are considered to be important for survivalin the blood during bloodstream infections. Environmental survival and growth requireattributes such as resistance to desiccation25,60, versatility in growth requirements3, biofilm-forming capacity61 and, probably, quorum-sensing activity59. Finally, adequate stress-response mechanisms are thought to be required for adaptation to different conditions.NATURe RevIewS |microbiology vOlUMe 5 | DeCeMbeR 2007 |945©2007Nature Publishing Group。

细胞器之间相互作用在非酒精性脂肪性肝病发生发展中的作用

2 ＤＯＩ：１０．３９６９／ｊ．ｉｓｓｎ．１００１－５２５６．２０２３．０１．０２８细胞器之间相互作用在非酒精性脂肪性肝病发生发展中的作用刘天会首都医科大学附属北京友谊医院肝病中心，北京１０００５０通信作者：刘天会，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）摘要：细胞器除了具有各自特定的功能外，还可与其他细胞器相互作用完成重要的生理功能。

细胞器之间相互作用的异常与疾病的发生发展密切相关。

近年来，细胞器之间相互作用在非酒精性脂肪性肝病（ＮＡＦＬＤ）发生发展中的作用受到关注，特别是线粒体、脂滴与其他细胞器之间的相互作用。

关键词：非酒精性脂肪性肝病；细胞器；线粒体；脂肪滴基金项目：国家自然科学基金面上项目（８２０７０６１８）ＲｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｉｎｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅＬＩＵＴｉａｎｈｕｉ．（ＬｉｖｅｒＲｅｓｅａｒｃｈＣｅｎｔｅｒ，ＢｅｉｊｉｎｇＦｒｉｅｎｄｓｈｉｐＨｏｓｐｉｔａｌ，ＣａｐｉｔａｌＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｂｅｉｊｉｎｇ１０００５０，Ｃｈｉｎａ）Ｃｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ：ＬＩＵＴｉａｎｈｕｉ，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）Ａｂｓｔｒａｃｔ：Ｉｎａｄｄｉｔｉｏｎｔｏｉｔｓｏｗｎｓｐｅｃｉｆｉｃｆｕｎｃｔｉｏｎｓ，ａｎｏｒｇａｎｅｌｌｅｃａｎａｌｓｏｉｎｔｅｒａｃｔｗｉｔｈｏｔｈｅｒｏｒｇａｎｅｌｌｅｓｔｏｃｏｍｐｌｅｔｅｉｍｐｏｒｔａｎｔｐｈｙｓｉｏｌｏｇｉｃａｌｆｕｎｃｔｉｏｎｓ．Ｔｈｅｄｉｓｏｒｄｅｒｓｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓａｒｅｃｌｏｓｅｌｙａｓｓｏｃｉａｔｅｄｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｖａｒｉｏｕｓｄｉｓｅａｓｅｓ．Ｉｎｒｅｃｅｎｔｙｅａｒｓ，ｔｈｅｒｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓｈａｓａｔｔｒａｃｔｅｄｍｏｒｅａｔｔｅｎｔｉｏｎｉｎｔｈｅｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅ，ｅｓｐｅｃｉａｌｌｙｔｈｅｉｎｔｅｒａｃｔｉｏｎｓｂｅｔｗｅｅｎｍｉｔｏｃｈｏｎｄｒｉａ，ｌｉｐｉｄｄｒｏｐｌｅｔｓ，ａｎｄｏｔｈｅｒｏｒｇａｎｅｌｌｅｓ．Ｋｅｙｗｏｒｄｓ：Ｎｏｎ－ａｌｃｏｈｏｌｉｃＦａｔｔｙＬｉｖｅｒＤｉｓｅａｓｅ；Ｏｒｇａｎｅｌｌｅｓ；Ｍｉｔｏｃｈｏｎｄｒｉａ；ＬｉｐｉｄＤｒｏｐｌｅｔｓＲｅｓｅａｒｃｈｆｕｎｄｉｎｇ：ＮａｔｉｏｎａｌＮａｔｕｒａｌＳｃｉｅｎｃｅＦｏｕｎｄａｔｉｏｎｏｆＣｈｉｎａ（８２０７０６１８）细胞器可以通过膜接触位点与其他细胞器相互作用，完成物质与信息的交换，形成互作网络［１］。

伯杰氏细菌鉴定手册最新版弧菌科部分

GENUS XIII.SHEWANELLA491M NaCl.Growth occurs at4–30ЊC.Optimum growth tem-perature is25ЊC.Grows between pH6–8.Optimum pH for growth is7.0.Chemoheterotrophic facultative anaerobe.Can grow an-aerobically using nitrate,fumarate,iron,manganese, TMAO,thiosulfate,and elemental sulfur as alternative elec-tron acceptors with lactate acting as the carbon source.Cat-alase,oxidase,and lipase positive.Amylase and gelatinase negative.Glucose,galactose,lactate,acetate,pyruvate,cit-rate,succinate,glutamate,Casamino acids,yeast extract, and peptone are used aerobically as energy sources.Fruc-tose,glycerol,sorbitol,arabinose,formate,and ethanol are not utilized.Isolated from the accessory nidamental glands of female adults of the squid species Loligo pealei.The mol%GםC of the DNA is:45.0(HPLC).Type strain:ANG-SQ1,ATCC700345.GenBank accession number(16S rRNA):AF011335.12.Shewanella violacea Nogi,Kato and Horikoshi1999,341VP(Effective publication:Nogi,Kato and Horikoshi1998b, 337.)Јce.a.L.gen.n.violacea of violet.Cells are straight or slightly curved rods,0.8–1.0ן2–4l m.Colonies on marine agar are circular,smooth,convex with entire edges,and butyrous in consistency.After2–3d, colonies are nonpigmented;after more than7d,colonies appear violet.NaCl is required for growth;optimal levels for growth are2–3%.No growth with6%NaCl.Psychrophilic.Grows optimally between4–10ЊC.Baro-philic.Optimal pressure for growth is30MPa.Other characteristics are as given in the genus descrip-tion and in Table BXII.c.148.Acid is produced from cel-lobiose and d-galactose,but not from dl-arabinose,d-fruc-tose,glycerol,inositol,lactose,maltose,d-mannitol,d-man-nose,d-rafﬁnose,l-rhamnose,d-sorbitol,sucrose,d-treha-lose,or d-xylose.Isolated from the Ryukyu Trench,northwest Paciﬁc Ocean,at a depth of5110m.The mol%GםC of the DNA is:47(T m).Type strain:DSS12,JCM10179.GenBank accession number(16S rRNA):D21225.13.Shewanella woodyi Makemson,Fulayﬁl,Landry,Van Ert,Wimpee,Widder and Case1997,1039VPwoodЈy.i.M.L.gen.n.woodyi of Woody,in honor of the American biologist J.Woodland Hastings.Cells are rod-shaped,0.4–1.0ן1.4–2.0l m.Colonies on marine agar are pink-orange due to the accumulation of cytochromes.NaCl is required for growth.Growth factors are not required.Psychrophilic.Growth occurs between4and25ЊC;op-timum temperature,20–25ЊC;no growth at30ЊC.Other characteristics are as given in the genus descrip-tion and in Tables BXII.c.148and BXII.c.151.Isolated from squid ink,seawater and marine snow(col-lected from the Alboran Sea).The mol%GםC of the DNA is:39(by measurement of the relative binding of DNA-binding-ﬂuorescent dyes bis-benzimide and chromomycin A3).Type strain:MS32,ATCC51908,DSM12036.GenBank accession number(16S rRNA):AF003549.Order XI.“Vibrionales”Vib.ri.o.naЈles.M.L.masc.n.Vibrio type genus of the order;-ales ending to denote order;M.L.fem.pl.n.Vibrionales the order of bacteria whose circumscription is based on thegenus Vibrio.Description is the same as for the family Vibrionaceae.Type genus:Vibrio Pacini1854,411.Family I.Vibrionaceae Ve´ron1965,5245ALJ.J.F ARMER III AND J.M ICHAEL J ANDAVib.ri.o.naЈce.ae.M.L.masc.n.Vibrio type genus of the family;-aceae ending to denote family;M.L.fem.pl.n.Vibrionaceae the family of bacteria whose circumscription is based on the genusVibrio.Gram-negative straight or curved rods.Motile by means of polar ﬂagella.Additional lateralﬂagella may be produced when grown on solid media;these differ in wavelength and antigenicity from the polarﬂagellum and may number from a few to over100ﬂagella/cell.Do not form endospores or microcysts.Chemoor-ganotrophs.Facultative anaerobes,having both a respiratory and a fermentative metabolism.Oxygen is a universal electron ac-ceptor.Do not denitrify.Most strains:are oxidase positive,reduce nitrate to nitrite,ferment d-glucose and utilize it as a sole or principal source of carbon and energy,grow in minimal media with d-glucose or other compounds as the sole source of carbon and energy and use NH4םas the sole nitrogen source.A few species require vitamins and amino acids.Ferment and utilize a wide variety of simple and complex carbohydrates and utilize a wide variety of other carbon sources.Most species require Naםor a seawater base for growth and require0.5–3%NaCl for op-timum growth.Several species are bioluminescent;other species include a few bioluminescent strains.Primarily aquatic.Found in fresh,brackish,and sea water,often in association with aquatic animals and plants.Several species are pathogenic for humans.FAMILY I.VIBRIONACEAE 492Other species are pathogenic forﬁsh,eels,and other aquatic animals.The mol%GםC of the DNA is38–51%.The family is classiﬁed in the phylum Proteobacteria in the class Gammapro-teobacteria.Type genus:Vibrio Pacini1854,411.Historical overview A history of the family Vibrionaceae as it has appeared in Bergey’s Manual is given in Table BXII.c.152. Related families include Enterobacteriaceae,Aeromonadaceae,and Pasteurellaceae.The family Vibrionaceae has undergone intense study since theﬁrst edition of Bergey’s Manual of Systematic Bac-teriology(Krieg and Holt,1984)was published in1984.In their chapter on the family Vibrionaceae in that edition,Baumann and Schubert(1984)included the genera Vibrio,Photobacterium,Ae-romonas,and Plesiomonas.These are the same four genera in-cluded in the original classiﬁcation of the family proposed by Ve´ron almost twenty years earlier(Ve´ron,1965).In this Manual Aeromonas and Plesiomonas are classiﬁed in other families(Table BXII.c.152).For practical identiﬁcation schemes,it is still useful to consider Aeromonas and Plesiomonas together with other oxidase positive genera of fermentative bacteria such as Vibrio and Pho-tobacterium(Table BXII.c.153).A detailed history of changes in the classiﬁcation of Vibrio and related genera that occurred as new methods were introduced has been given by Farmer(1992). These methods include examinations of the structure,function, and regulation of proteins;comparison of mol%GםC content; DNA–DNA hybridization;rRNA–DNA hybridization;5S rRNA cataloging and sequence comparisons;and16S rRNA gene se-quence comparisons(Fig.BXII.c.157).The family Vibrionaceae presently includes three genera:Genus1.Vibrio(the type genus)Genus II.PhotobacteriumGenus III.SalinivibrioThe type strain of the type and only species of the genus Allomonas,Allomonas enterica,is very closely related to Vibrioﬂu-vialis by DNA–DNA hybridization studies and phenotypic analysis (Kalina et al.,1984).Allomonas and Allomonas enterica are not described separately in this edition of the Manual;the reader is referred to the description of V.ﬂuvialis.The two species of the genus Listonella,Listonella anguillarum(the type species)and Lis-tonella pelagia,are included in the genus Vibrio in this edition of the Manual as Vibrio anguillarum and Vibrio pelagius.*F URTHER DESCRIPTIVE INFORMATIONHabitats The ecological niches of members of the family Vibrionaceae have been described by Campbell(1957);Baumann and Baumann(1981a);Sakazaki and Balows(1981);and Simidu and Tsukamoto(1985)).In humans,some vibrios cause diarrhea, wound infections,and occasionally other extraintestinal infec-tions.In aquatic animals,vibrios cause wound and generalized infections.Many vibrios and related organisms are also widely distributed in aquatic environments.Many factors govern the distribution of these organisms,but the most important probably include:particular human,animal or plant hosts;inorganic nu-trients and carbon sources available;temperature;salinity;dis-solved oxygen;and depth below the surface for the species that are found in the ocean(Simidu and Tsukamoto,1985).A few species are adapted to particular hosts.For example,Vibrio chol-erae serogroup O1is adapted to humans and is the cause of cholera,a life-threatening diarrheal disease.Recent studies have shown that the ecology of this organism is more complex than originally thought.Photobacterium leiognathi is usually isolated fromﬁsh in shallow tropical water,and P.phosphoreum is usually found in the luminous organs ofﬁsh that live at depths of200–1200meters(Hastings and Nealson,1981).Isolation Most members of the Vibrionaceae grow well on ordinary complex media.Samples are spread onto solid medium or diluted in an enrichment broth.NaCl concentrations of0.5–0.85%satisfy the requirements of most species,although a few require greater concentrations of NaCl.Incubation temperatures are also important.A few species grow only at temperatures Ͻ25ЊC;others grow at25ЊC but not at35–37ЊC.General and selective media for Vibrionaceae are described in the chapter on the genus Vibrio.Identiﬁcation Methods for the isolation and identiﬁcation of Vibrio spp.from clinical specimens and non-clinical samples are discussed in detail in the chapter describing the genus Vibrio. Assignment of non-clinical isolates to a species can be problem-atic because over50species of Vibrio and Photobacterium must be considered and because comparative data for these organisms are sparse relative to data available for clinically important spe-cies.The US Centers for Disease Control and Prevention maintain computer programs and databases for the identiﬁcation of iso-lates subjected to a battery of45–60phenotypic tests;for details contact the Vibrio Laboratory at the CDC.These alternatives to phenotypic methods are now being used routinely and have proven extremely useful in a research setting. It will be important to evaluate the sensitivity and speciﬁcity,and to understand the advantages and disadvantages of these meth-ods.In the United States,the reporting of cultures from human specimens is subject to speciﬁc government regulations(the Clin-ical Laboratory Improvement Amendments of1988),which has limited the application of these approaches in clinical and public health laboratories.A CKNOWLEDGMENTSWe dedicate this chapter to M.Ve´ron for giving us the name Vibrionaceae and for all his contributions to our understanding of the family,its or-ganisms,and their close and distant relatives.F URTHER R EADINGBaumann,P.and L.Baumann.1977.Biology of the marine enterobac-teria:genera Beneckea and Photobacterium.Ann.Rev.Microbiol.31:39–61.Baumann,P.and L.Baumann.1981.The marine Gram-negative eubac-teria.In Starr,Stolp,Tru¨per,Balows and Schlegel(Editors),The Pro-karyotes,a Handbook on Habitats,Isolation and Identiﬁcation of Bacteria,1st Ed.,Springer-Verlag,New York.pp.1352–1394. Baumann,P.,L.Baumann,S.S.Bang and M.J.Woolkalis.1980.Reeval-uation of the taxonomy of Vibrio,Beneckea,and Photobacterium:aboli-tion of the genus Beneckea.Curr.Microbiol.4:127–132. Baumann,P.,L.Baumann and M.Mandel.1971.Taxonomy of marine bacteria:the genus Beneckea.J.Bacteriol.107:268–294. Baumann,P.,A.L.Furniss and J.V.Lee.1984.Genus I.Vibrio.In Krieg and Holt(Editors),Bergey’s Manual of Systematic Bacteriology,1st Ed.,Vol.1,The Williams&Wilkins Co.,Baltimore.pp.518–538. Baumann,P.and R.H.W.Schubert.1984.Family II.Vibrionaceae.In Krieg and Holt(Editors),Bergey’s Manual of Systematic Bacteriology,1st Ed.,Vol.1,The Williams&Wilkins Co.,Baltimore.pp.516–517. Brenner,D.J.,G.R.Fanning,F.W.Hickmann-Brenner,J.V.Lee,A.G.Stei-FAMILY VIBRIONACEAE493gerwalt,B.R.Davis and J.J.Farmer.1983a.DNA relatedness among Vibrionaceae,with emphasis on the Vibrio species associated with human infection.INSERM Colloq.114:175–184.Chakraborty,S.,G.B.Nair and S.Shinoda.1997.Pathogenic vibrios in the natural aquatic environment.Rev.Environ.Health.12:63–80. Farmer,J.J.1992.The family Vibrionaceae.In Balows,Tru¨per,Dworkin,Harder and Schleifer(Editors),The Prokaryotes.A Handbook on the Biology of Bacteria:Ecophysiology,Isolation,Identiﬁcation,Ap-plications,2nd Ed.,Vol.3,Springer-Verlag,New York.pp.2938–2951. Farmer,J.J.,M.J.Arduino and F.W.Hickman-Brenner.1992.Aeromonas and Plesiomonas.In Balows,Tru¨per,Dworkin,Harder and Schleifer (Editors),The Prokaryotes.A Handbook on the Biology of Bacteria:FAMILY I.VIBRIONACEAE 494GENUS I.VIBRIO495FIGURE BXII.c.157.Relationship of most of the species of Vibrio and relatives based on16S rRNA gene sequences.* Only the type strain of each species was included.The distances in the tree were calculated using1101positions (the least-squares method,Jukes-Cantor model).(Courtesy T.Lilburn of the Ribosomal Database Project.)*Editorial Note:Photobacterium damselae subsp.damselae is a junior objective synonym of Vibrio damsela.Vibrio pelagius and Vibrio anguillarum are synonyms of Listonella pelagia and Listonella anguillarum,respectively.and volume(15–67%)and a conversion of rods into coccal forms called spherical ultramicrocells(Holmquist and Kjelleberg,1993; Kondo et al.,1994;Nelson et al.,1997).As the length of nutrient starvation increases,cytoplasmic inclusions and granules disap-pear,cell cultivability decreases,and the nuclear region becomes compressed(Hood et al.,1986).There are also noticeable dif-ferences in the integrity of the outer membrane and cell wall. Some changes may be linked to speciﬁc nutrient starvation(for example,nitrogen starvation produces longﬁlaments and phos-phorus starvation produces swollen large rods),whereas others occur regardless of the type of nutritional stress(Holmquist and Kjelleberg,1993).“Non-culturable”V.cholerae O1strains pro-duced in response to nutrient deprivation display a number of ultrastructural changes,which include an undulating outer mem-brane,a surface layer ofﬁneﬁbers,and a thicker peptidoglycan layer(Kondo et al.,1994).FAMILY I.VIBRIONACEAE 496Poly-b-hydroxybutyrate granules(PHB)can be found in a number of Vibrio species,including V.cholerae O1and O139and V.harveyi(Hood et al.,1986;Sun et al.,1994;Finkelstein et al., 1997).In V.cholerae,accumulation of PHB appears to be related to colonial opacity and growth on glycerol-containing media(Fin-kelstein et al.,1997).In V.harveyi,PHB accumulation is de-pendent on cell density and is controlled by the autoinducer,N-(3-hydroxybutanoyl)homoserine lactone(Sun et al.,1994). Other kinds of granules can be found in vibrios,including elec-tron dense lipoid particles and electron translucent inclusions of unknown composition(Sun et al.,1994;Finkelstein et al., 1997).Cell wall composition Vibrios contain the same three lipo-polysaccharide(LPS)elements found in other Gram-negative bacteria:lipid A,core polysaccharide,and an O polysaccharide side chain that determines serological speciﬁcity.The most ex-tensive work on biochemical characterization of Vibrio LPS has been done on V.cholerae.The lipid A portion consists of a b(1Ј-6)-linked glucosamine disaccharide backbone with two phos-phoryl groups(Janda,1998).Pyrophosphorylethanolamine is linked to one of these phosphoryl groups at the C-1position of the reducing sugar,and a phosphate group ester is bound to the nonreducing glucosamine residue(Manning et al.,1994).Three fatty acids are ester linked at hydroxyl positions to this disaccha-ride backbone:tetradecanoic acid(C14:0),hexadecanoic acid (C16:0),and3-hydroxydodecanoic acid(C12:03OH).A fourth,3-hydroxytetradecanoic acid(C14:03OH),is connected to the back-bone by an amide bond.The core oligosaccharide region of V.cholerae contains KDO (keto-3-deoxy-d-mannose-octulosonic acid),d-glucose,heptose (l-glycero-d-manno-heptose),d-fructose,and ethanolamine phosphate(Manning et al.,1994).KDO,a normal constituent of the core oligosaccharide of enteric LPS,was originally thought to be absent in Vibrio species.However,when conventional per-iodate-thiobarbituric acid tests were replaced by strong acid hy-drolysates,KDO was detected in Vibrio(Janda,1998).The KDO molecule of V.cholerae differs in several aspects from those of enteric bacteria such as Escherichia coli and the genus Salmonella: only a single KDO molecule has been detected in the core oli-gosaccharide of V.cholerae,and the KDO moiety is phosphory-lated at the C4position(Kondo et al.,1990;Manning et al., 1994).The C5position binds to a distal portion of the core region (heptose)similar to the KDO-C5binding of l-glycero-d-manno-heptose(Janda,1998).The other sugars form the remaining portion of the core oligosaccharide region and often contain additional sugar substitutions at various positions.The O-polysaccharide side chain of V.cholerae O1is a homo-polymer of d-perosamine(4,6-dideoxy-d-mannose)approxi-mately17–18units in length(Manning et al.,1994;Knirel et al., 1997).The amino groups of perosamine units are commonly acetylated with3-deoxy-l-glycero-tetronic acid.Another com-pound,quinovosamine,is thought to be a“capping sugar”on either the distal or the proximal end of the O antigen(Manning et al.,1994).An unusual sugar,4-amino-4,6-dideoxy-2-O-methyl-mannose is present only in the LPS of serogroup Ogawa and may have a role in serological speciﬁcity(Itoh et al.,1994).The LPS composition of V.cholerae O139—a second serotype capable of causing pandemic cholera—is remarkably similar to that of O1(Hisatsune et al.,1993;Isshiki et al.,1996).The lipid A moieties of O1and O139,including fatty acid substitutions, appear to be identical(Hisatsune et al.,1993).The core oligo-saccharide region contains two subtle differences:the presence of2-aminoethyl phosphate,which is the O-acetyl group,and the presence of a second fructose molecule(Knirel et al.,1997).The most profound differences between O-groups1and139occur in the O-polysaccharide side chain.Unlike serogroup O1,which has long O-polysaccharide side chains,V.cholerae O139has a short chain LPS similar to“SR strains”(Knirel et al.,1997).These truncated side chains migrate with the core oligosaccharide-lipid A fraction in LPS SDS-PAGE gels(Waldor et al.,1994).Classic “ladder-like”proﬁles of silver stained LPS side chains in SDS-PAGE gels are absent in O139strains(Hisatsune et al.,1993; Nandy et al.,1995).Perosamine,the main component of the O1 side chain,is also absent in O139strains(Hisatsune et al.,1993); instead,the unique sugar colitose(3,6-dideoxy-l-galactose)—which is not found in any other Vibrio species—is the main side chain subunit in O139strains(Hisatsune et al.,1993).The ab-breviated O-polysaccharide side chain of V.cholerae O139appears to be a hexasaccharide containing colitose residues and a cyclic phosphate group(Knirel et al.,1997).The LPS of other Vibrio species is similar in many aspects to that of O139.KDO-phosphate has been detected in V.parahaemolyticus by gas chromatography-mass spectrometry analysis(Janda,1998).The O-polysaccharide side chains of Vibrio species produce only a single fast-migrating band on silver-stained SDS-PAGE gels(Amaro et al.,1992;Iguchi et al.,1995).This result suggests that the side chains are short;a chain length ofՅ10monosaccharides has been proposed for V.parahaemolyticus(Iguchi et al.,1995).Some species however (e.g.,V.vulniﬁcus)may exhibit ladder-like patterns by immu-noblotting with whole cell antisera;this result suggests that the LPS O-polysaccharide side chains are mbert et al.(1983) studied the cellular fatty acids of most of the Vibrionaceae and postulated that differences among the Vibrio species might prove useful for identiﬁcation.Flagella Two types ofﬂagella are synthesized by vibrios in different environments.In liquid culture,swimmer cells predom-inate due to production of a single sheathed polarﬂagellum in most species(Figs.BXII.c.158and BXII.c.159).The sheath is an extension of the outer membrane(Fig.BXII.c.160).The polar ﬂagella are24–30nm in diameter with a central core14–16nm in thickness with a wavelength of1.4–1.8l m(Baumann et al., 1984b;Janda,1998).Some Vibrio species(e.g.,V.harveyi,V.ﬁscheri, V.logei,and V.salmonicida)produce tufts(3–12)of polarﬂagella (Fig.BXII.c.161)with a wavelength of approximately3.6l m (Baumann et al.,1984b;Ishimaru et al.,1996).Polarﬂagella provide chemotactic motility in liquid media and derive their energy from the sodium membrane potential(McCarter,1995). In some marine vibrios(e.g.,V.anguillarum),the polarﬂagellum appears critical for disease production in estuarineﬁsh(Milton et al.,1996;O’Toole et al.,1996).When vibrios come into contact with solid surfaces,a series of morphogenetic changes are ini-tiated that result in the conversion of swimmer cells into swarmer cells in some marine species such as V.parahaemolyticus,V.algi-nolyticus,V.diabolicus,and V.pectenicida(Rague´ne`s et al.,1997a; Lambert et al.,1998).During this process,cell septation ceases, the cells elongate from1to30l m,and numerous lateralﬂagella are formed(Fig.BXII.c.162)(McCarter and Silverman,1990). These lateralﬂagella,14–15nm in diameter with a wavelength of0.9l m,are distinct from polarﬂagella.They are unsheathed, have a different protein subunit composition,and are internally driven by the protonmotive force(Baumann et al.,1984b; McCarter,1995).Formation of lateralﬂagella permits swarmerGENUS I.VIBRIO497FIGURE BXII.c.158.Leifsonﬂagella stain of Vibrio cholerae.(Source:CDC archive,courtesy of EdEwing.)FIGURE BXII.c.159.Electron micrograph of Vibrio alginolyticus grown in liquid medium.Note the sheathed polarﬂagellum and absence of pe-ritrichousﬂagella.Shadowed preparation.ן13,000.(Reproduced with permission from C.Golten and W.A.Scheffers,Netherlands Journal of Sea Research9:351–364,1975,᭧Netherlands Institute for SeaResearch.)FIGURE BXII.c.160.Electron micrograph of a polarﬂagellum of Vibrio alginolyticus.Note that the sheath has partially disintegrated exposing the inner core.Negatively stained preparation.ן30,000.(Courtesy of R.D. Allen.)migration across solid surfaces and results in progressive spread-ing of the bacterial colony(McCarter and Silverman,1990),a phenomenon called swarming.Swarming in many vibrio species is dependent upon a number of factors including agar concen-tration,media composition,iron availability,temperature,and relative viscosity(Baumann et al.,1984b;McCarter and Silver-man,1990).The microscopic morphology of vibrio cells removed from different concentric zones of swarming has been studied in some Vibrio strains(Sar and Rosenberg,1989).Innermost zones consist of irregular cells including bent rods that pro-gressively evolve into short rods and then into large rods with bundles of detachedﬂagella(Fig.BXII.c.163),whereas cells inFAMILY I.VIBRIONACEAE498FIGURE BXII.c.161.Electron micrograph of Vibrioﬁscheri.Note the tufts of sheathed polarﬂagella.Negatively stained preparation.ן23,000. (Reproduced with permission from:J.L.Reichelt and P.Baumann,Ar-chives of Mikrobiology94:283–330,1973,᭧Springer-Verlag,Berlin.)FIGURE BXII.c.162.Electron micrograph of Vibrio alginolyticus grown on solid medium.Note the thick,sheathedﬂagellum and numerous un-sheathed lateralﬂagella.Shadowed preparation.ן18,000.(Reproduced with permission from W.E.de Boer et al.,Netherlands Journal of Sea Research9:197–213,1975,᭧Netherlands Institute for Sea Research.)the outermost circles of swarming colonies consist of longﬁla-mentous forms.Fimbriae Fimbriae are produced by a number of pathogenic vibrios such as V.cholerae O1and non-O1,V.parahaemolyticus, and V.vulniﬁcus(Hall et al.,1988;Honda et al.,1988;Gander and LaRocco,1989;Nakasone and Iwanaga,1990).Several dif-ferent morphologic types ofﬁmbriae have been described in V. cholerae O1.These include both wavy pili3nm in diameter and rigidﬁlaments5–6nm wide and180–800nm in length(Hall et al.,1988).The most important of these pili is composed of the protein TcpA;these pili are5–6nm wide and form bundles of parallel undulatingﬁlaments up to15l m long(Hall et al.,1988). TcpA formation is coregulated with cholera toxin expression and is a key determinant of in vivo colonization.The gene encoding TcpA appears to reside on a pathogenicity island.Capsules Capsules have been detected surrounding cells of strains of V.cholerae O139and V.vulniﬁcus strains with a variety of staining techniques such as uranyl acetate,polycationic fer-ritin,and ruthenium red(Janda,1998).The V.vulniﬁcus poly-saccharide capsule is60nm thick and has a low electron density (Amako et al.,1984;Hayat et al.,1993).The carbohydrate com-position of the capsule of V.vulniﬁcus varies from strain to strain. Sugars detected in different isolates include␣-N-acetyl quino-vosamine,␣-N-acetyl galactosamine uronic acid,rhamnosamine, and fucosamine(Hayat et al.,1993).Colonial morphology Most vibrios grow well on a variety of media,including protein-based agars and marine and seawater media,if sufﬁcient Naםis present(Baumann et al.,1984b; Farmer and Hickman-Brenner,1992).On most selective media, vibrios appear as smooth,buff-to-cream-colored colonies2–5mm in diameter,with an entire margin after overnight incubation (Baumann et al.,1984b;Janda,1998).Some species tend to pro-duce grayish colonies,particularly on blood agar.Considerable variation in colonial morphology has been reported for some species,and this is best demonstrated by observing colonies with a dissecting microscope at10–25magniﬁcation with oblique lighting.V.cholerae strains can have several different colonial mor-phologies(smooth,rough,and rugose forms)in response to different growth conditions.Rugose colonies are often chlorine-resistant.They are usually found in older cultures and are com-posed of an amorphous intercellular matrix of aggregated bac-teria and exopolysaccharide material(Morris et al.,1996).For-mation of rugose colonies can be enhanced by growth in en-richment broths such as alkaline peptone water(APW)or by picking the growth that has migrated up the sides of a culture tube.They can largely be avoided by picking a smooth colony and freezing it.In addition to these colony types,several path-GENUS I.VIBRIO499FIGURE rge bundle ofﬂagella in a culture of Vibrio harveyi. These bundles are frequently observed in cultures grown on solid me-dium.Negatively stained preparation.ן13,000.(Courtesy of R.D.Allen.) ogenic vibrios,including V.cholerae and V.vulniﬁcus,produce opaque and translucent varieties of smooth colonies on common media such as heart infusion and meat extract agars(Simpson et al.,1987;Finkelstein et al.,1992,1997).Cells from colonies of these different morphologies differ from each other in a num-ber of characteristics,including encapsulation,cell surface com-position,cellular metabolism,and ability to survive under adverse conditions.Pigmentation Several Vibrio species produce pigmented col-onies.V.nigripulchritudo produces an insoluble blue-black pig-ment that accumulates in a crystalline form within the colonies (Baumann et al.,1984b).Other Vibrio species also produce blue-black crystals under various growth conditions,but typically do not produce the characteristic blue-black colonies of V.nigripul-chritudo.Similar blue-black colonies are produced by a few strains of Kluyvera(Farmer et al.,1981a).Other pigmented species in-clude V.gazogenes(red)and V.ﬁscheri and V.logei(yellow-orange).A few strains of V.cholerae produce a brown diffusible melanin-like pigment(Ivins and Holmes,1980).Life cycles The marine environment is the natural habitat of vibrios,and the life cycle of these organisms is probably quite complex.A model for the life of V.cholerae in Gulf Coast estuaries has been proposed(Hood et al.,1984).Depending on nutrient scarcity and the density/availability of particulate matter,V.chol-erae can exist in several states.An epibiotic form attached to plankton predominates during periods of relatively high nu-trient/particulate matter concentrations;this form changes to a microvibrio form(small rounded cells)during times of nutrient and particulate deprivation(Hood et al.,1984;Janda,1998).This latter form may be analogous to the“viable but nonculturable state”(Colwell,1984)that has been described for many Vibrio species including V.cholerae,V.parahaemolyticus,V.vulniﬁcus,V. anguillarum,V.campbellii,V.harveyi,and V.ﬁscheri during colder seasons of the year(Oliver,1995).The microvibrio and“non-culturable”stages may be dormant phases for vibrios in winter from which subsequent blooms are triggered in response to in-creasing temperatures in spring and summer.However,it is still unclear whether any dormant state actually exists and whether blooms are due to the growth of a small number of cultivable cells(present at all times)or the actual resuscitation of dormant cells(Ravel et al.,1995;Bogosian et al.,1998).Nutrition and growth conditions Vibrio species vary in their nutrition and growth requirements.The most important feature is that Naםis required for or stimulates growth.Minimum con-centrations of Naםrequired for optimal growth(Fig.BXII.c.164) range from5–15mM(0.029–0.087%)for V.cholerae and V.metsch-nikovii to600–700mM(3.5–4.1%)for Salinivibrio costicola(Bau-mann et al.,1984b).Most species grow well in solid or liquid media containing0.5–2%NaCl.Some species(Photobacterium ili-opiscarium)form bacterial aggregates in broth culture containing 2%NaCl(Onarheim et al.,1994).The“salt requirement”of a strain will often depend on the test conditions.The main variables are temperature,the growth medium used prior to testing,the suspending medium,and the testing medium.All Vibrio species except V.cholerae and V.mimicus have an absolute requirement for Naם(Fig.BXII.c.164).In some instances,this requirement may be partially offset by concentra-tions of Mg2םor Ca2םsimilar to those normally present in sea-water(Baumann et al.,1984b).However,most species exhibit a speciﬁc requirement for Naם(Pujalte and Garay,1986;Borrego et al.,1996).The range and optimum concentrations of NaCl supporting growth of some of the more recently described Vibrio species are listed in Table BXII.c.154.No single medium or NaCl concentration is optimal for the recovery or growth of all Vibrio species.Many vibrios will grow in mildly alkaline conditions.Al-though most species prefer a pH range of7–8(Rague´ne`s et al., 1997a),some species,including V.cholerae and V.metschnikovii, will even grow at a pH of10(Baumann et al.,1984b).Vibrios also vary in their temperature requirements for growth.Almost all Vibrio species grow well at18–22ЊC.Some will grow at0–4ЊC,whereas others can grow at temperatures up to 45ЊC.The temperature at which vibrios can grow is also de-pendent upon other factors including the composition of the medium and the NaCl concentration(Onarheim et al.,1994).Most Vibrio species do not require speciﬁc organic growth factors such as vitamins or amino acids,although amino acid supplementation may be required to revive some strains stored for prolonged periods(Baumann et al.,1984b).Complex nu-trients are required to induce growth of some species(Baumann et al.,1984b;Rague´ne`s et al.,1997a).Such required supplements include yeast extract(for V.anguillarum,Moritella marina,and V. logei)and a seawater base(for V.diabolicus).Vibrios use a variety of compounds as carbon and energy。

清华大学报道酿酒酵母内含子套索剪接体的三维结构Tsinghua

清华大学报道酿酒酵母内含子套索剪接体的三维结构Tsinghua University Reported the Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae【Cell系列】2017年9月15日，清华大学生命学院施一公教授研究组于《细胞》（Cell）杂志就剪接体的结构与机理研究再发最新成果，题目为《酿酒酵母内含子套索剪接体的结构》（Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae），该文报道了RNA剪接循环中剪接体最后一个状态的高分辨率三维结构，为阐明剪接体完成催化功能后受控解聚的分子机制提供了结构基础，从而将对RNA剪接（RNA Splicing）分子机理的理解又向前推进了一步。

真核生物的基因表达相比于原核生物更为复杂精细。

由于真核细胞内的基因是不连续的，它需要在细胞核内被转录成前体信使RNA。

通过RNA剪接，不具有翻译功能的内含子被去除，密码子所在的外显子被连接，从而得到成熟的、可被翻译成蛋白质的信使RNA。

RNA剪接是真核生物基因表达调控的重要环节之一，而负责执行这一过程的是细胞核内一个巨大的且高度动态变化的分子机器——剪接体（spliceosome）。

剪接体在真核生物进化中极为保守，这一点对于真核生物维持正常的生命活动至关重要。

一个基因转录出的前体信使RNA 可以通过RNA剪接成若干种信使RNA，于是极大地丰富了真核生物蛋白质组的多样性。

在剪接反应过程中，多种蛋白质-核酸复合物及剪接因子按照高度精确的顺序发生结合和解聚，依次形成预组装复合物U4/U6.U5 三小核核糖核蛋白复合物（Tri-snRNP）以及至少7个状态的剪接体B、Bact、B*、C、C*、P以及内含子套索剪接体复合物（ILS complex，Intron Lariat Spliceosome）。

黑木耳黑色素的研究综述

第34卷第1期海南师范大学学报（自然科学版）V〇l.34N〇.l 2021 年3 月Journal of Hainan Normal University(Natural Science)Mar.2021Doi ： 10.12051/j.issn. 1674-4942.2021.01.010黑木耳黑色素的研究综述陈雅，徐苗，王欣宜，单欣荷，季琳凯，张拥军*(中国计量大学生命科学学院，浙江杭州310018)摘要：黑木耳是中国一种分布极为广泛且具有独特的营养价值与保健功能的食用菌，含有多种生物活性化合物，其中黑色素是主要生物活性成分之一，且具有极高的安全性，可进一步开发具备保健作用的功能色素，该方面的研究已初见成效且应用前景广阔。

文章综述了黑木耳黑色素的理化性质、分子组成与结构表征、分离制备手段以及其清除自由基、抑菌抗病毒、抗辐射、改善肝损伤、保护D N A等生物活性的国内外研究现状，分析了限制黑木耳黑色素开发应用的实际问题并提出展望，并为今后黑木耳黑色素生物活性作用机制的研究及其在食品、药品、化妆品等领域的应用提供参考。

关键词：黑木耳；黑色素；分离提取；生物合成；分子结构；生物活性中图分类号:0629.1 文献标志码:A文章编号：1674-4942(2021)01-0063-07Summary of Melanin of A u ricu la ria au ricu laCHEN Ya, XU Miao, WANG Xinyi, SHAN Xinhe, Jl Linkai, ZHANG Yongjun' (College of L ife Science,China Jiliang University,Hangzhou 310018, China) Abstract：Auricularia auricula is an edible fungus with a unique nutritional value and healthy function and is widely distributed in China. It contains a variety of bioactive compounds. Melanin is one of the main bioactive components with high safety. It can be further developed into func tional pigments with health care function. The research in this field has broad application prospects. In this paper, the physicochemical properties, molecular composition, structural characterization, separation and prej)aration methods of Auricularia auricula melanin, as well as its biological activities such as scavenging free radicals, antibacterial and antiviral, antiradiation, improving liver injury, protecting DNA and so on, were reviewed. The problems limiting the development and application of Auricularia auricula melanin were analyzed, and the prospects were put forward, which would be helpful for the study of the biological mechanism of Auricularia auricula melanin and its application in food, medicine, cosmetics and other fields.K ey w〇r d s：/lz/m*z//rtrirt ai/rzcw/a;melanin;separation and extraction;biosynthesis;molecular stnicture;biological activity黑木耳(4w r/c a/a r i Y/aur/cu/fl)隶属担子菌亚门（Basi(liomycotina)层菌纲（Hymenomycetes)木耳目（Auricu- I a r i a les)木耳科(A u r i c u1a riaceae)木耳属(yWfcw/rmVz )，为中国珍贵的药用和食用菌，早在19世纪，黑木耳就被用于民间医药，用于治疗咽喉痛、眼痛、黄疸等病症，并作为收敛剂"_21。

10.1007%2Fs11537-013-1280-5

Japan.J.Math.8,147–183(2013)DOI:10.1007/s11537-013-1280-5About the Connes embedding conjectureAlgebraic approachesNarutaka Ozawa?Received:28December2012/Accepted:15January2013Published online:20March2013©The Mathematical Society of Japan and Springer Japan2013Communicated by:Yasuyuki KawahigashiAbstract.In his celebrated paper in1976,A.Connes casually remarked that anyﬁnite von Neu-mann algebra ought to be embedded into an ultraproduct of matrix algebras,which is now known as the Connes embedding conjecture or problem.This conjecture became one of the central open problems in theﬁeld of operator algebras since E.Kirchberg’s seminal work in1993that proves it is equivalent to a variety of other seemingly totally unrelated but important conjectures in the ﬁeld.Since then,many more equivalents of the conjecture have been found,also in some other branches of mathematics such as noncommutative real algebraic geometry and quantum infor-mation theory.In this note,we present a survey of this conjecture with a focus on the algebraic aspects of it.Keywords and phrases:Connes embedding conjecture,Kirchberg’s conjecture,Tsirelson’s prob-lem,semi-pre-C -algebras,noncommutative real algebraic geometryMathematics Subject Classiﬁcation(2010):16W80,46L89,81P151.IntroductionThe Connes embedding conjecture([Co])is considered as one of the most im-portant open problems in theﬁeld of operator algebras.It asserts that anyﬁ-nite von Neumann algebra is approximable by matrix algebras in a suitable sense.It turns out,most notably by Kirchberg’s seminal work([Ki1]),that the N.O ZAWAResearch Institute for Mathematical Sciences,Kyoto University,Kyoto606-8502,Japan(e-mail:)?Partially supported by JSPS(23540233)and by the Danish National Research Foundation (DNRF)through the Centre for Symmetry and Deformation.148N.Ozawa Connes embedding conjecture is equivalent to a variety of other important con-jectures,which touches most of the subﬁelds of operator algebras,and also someother branches of mathematics such as noncommutative real algebraic geome-try([Sm])and quantum information theory.In this note,we look at the alge-braic aspects of this conjecture.(See[BO,Ki1,Oz1]for the analytic aspects.)This leads to a study of the C -algebraic aspect of noncommutative real alge-braic geometry in terms of semi-pre-C -algebras.Speciﬁcally,we treat someeasy parts of Positivstellensätze of Putinar([Pu]),Helton–McCullough([HM]),and Schmüdgen–Bakonyi–Timotin([BT]).We then treat their tracial analogueby Klep–Schweighofer([KS]),which is equivalent to the Connes embeddingconjecture.We give new proofs of Kirchberg’s theorems on the tensor productC F d˝B.`2/and on the equivalence between the Connes embedding conjec-ture and Kirchberg’s conjecture.We also look at Tsirelson’s problem in quantuminformation theory([Fr,J+,Ts]),and prove it is again equivalent to the Connesembedding conjecture.This paper is an expanded lecture note for the author’slecture for“Masterclass on soﬁc groups and applications to operator algebras”(University of Copenhagen,5–9November2012).The author gratefully ac-knowledges the kind hospitality provided by University of Copenhagen duringhis stay in Fall2012.He also would like to thank Professor Andreas Thom forvaluable comments on this note.2.Ground assumptionWe deal with unital -algebras over k2f C;R g,and every algebra is assumedto be unital,unless it is clearly not so.The unit of an algebra is simply denotedby1and all homomorphisms and representations between algebras are assumedto preserve the units.We denote by i the imaginary unit,and by the complexconjugate of 2C.In case k D R,one has D for all 2k.3.Semi-pre-C -algebrasWe will give the deﬁnition and examples of semi-pre-C -algebras.Recall thata unital algebra A is called a -algebra if it is equipped with a map x!xsatisfying the following properties:(i)1 D1and.x / D x for every x2A;(ii).xy/ D y x for every x;y2A;(iii). x C y/ D x C y for every x;y2A and 2k.The sets of hermitian elements and unitary(orthogonal)elements are writtenrespectively asA h WD f a2A W a D a g and A u WD f u2A W u u D1D uu g:About the Connes embedding conjecture149 Every element x2A decomposes uniquely as a sum x D a C b of an hermitian element a and a skew-hermitian element b.The set of hermitian elements is an R-vector space.We say a linear map'between -spaces is self-adjoint if ' D',where' is deﬁned by' .x/D'.x / .We call a subset A C A h a -positive cone(commonly known as a quadratic module)if it satisﬁes the following:(i)R 01 A C and a C b2A C for every a;b2A C and 2R 0; (ii)x ax2A C for every a2A C and x2A.For a;b2A h,we write aÄb if b a2A C.We say a linear map'be-tween spaces with positivity is positive if it sends positive elements to positive elements(and often it is also required self-adjoint),and a positive linear map' is faithful if a 0and'.a/D0implies a D0.Given a -positive cone A C, we deﬁne the -subalgebra of bounded elements byA bdd D f x2A W9R>0such that x xÄR1g:This is indeed a -subalgebra of A.For example,if x is bounded and x xÄR1,then x is also bounded and xx ÄR1,because0ÄR 1.R1 xx /2D R1 2xx C R 1x.x x/x ÄR1 xx : Thus,if A is generated(as a -algebra)by S,then S A bdd implies A D A bdd.Deﬁnition.A unital -algebra A is called a semi-pre-C -algebra if it comes together with a -positive cone A C satisfying the Combes axiom(also called the archimedean property)that A D A bdd.Since hÄ.1C h2/=2for h2A h,one has A h D A C A C for a semi-pre-C -algebra.We deﬁne the ideal of inﬁnitesimal elements byI.A/D f x2A W x xÄ"1for all">0gand the archimedean closure of the -positive cone A C(or any other cone)by arch.A C/D f a2A h W a C"12A C for all">0g:The cone A C is said to be archimedean closed if A C D arch.A C/.A C -alge-bra A is of course a semi-pre-C -algebra,with a zero inﬁnitesimal ideal and an archimedean closed -positive coneA C D f x x W x2A g:If A B.H/(here B.H/denotes the C -algebra of the bounded linear opera-tors on a Hilbert space H over k),then one also hasA C D f a2A h W h a ; i 0for all 2H g:150N.Ozawa Note that the condition a being hermitian cannot be dropped when k D R.Itwill be shown(Theorem1)that if A is a semi-pre-C -algebra,then A=I.A/is a pre-C -algebra with a -positive cone arch.A C/.Deﬁnition.We deﬁne the universal C -algebra of a semi-pre-C -algebra Aas the C -algebra C u.A/together with a positive -homomorphismÃW A!C u.A/which satisﬁes the following properties:Ã.A/is dense in C u.A/andevery positive -representation of A on a Hilbert space H extends to a -representation N W C u.A/!B.H/,i.e., D N ıÃ.In other words,C u.A/isthe separation and completion of A under the C -semi-normsup fk .a/k B.H/W a positive -representation on a Hilbert space H g: (We may restrict the dimension of H by the cardinality of A.)We emphasize that only positive -representations are considered.Everypositive -homomorphism between semi-pre-C -algebras extends to a posi-tive -homomorphism between their universal C -algebras.It may happen that A C D A h and C u.A/D f0g,which is still considered as a unital(?)C -algebra.Every -homomorphism between C -algebras is automatically pos-itive,has a norm-closed range,and maps the positive cone onto the positivecone of the range.However,this is not at all the case for semi-pre-C -algebras,as we will exhibit a prototypical example in Example1.On the other hand,we note that if A is a norm-dense -subalgebra of a C -algebra A such thatarch.A C/D A\A C,then every positive -representation of A extends to a-representation of A,i.e.,A D Cu .A/.(Indeed,if x2A has k x k A<1,then1 x x2A C and hence k .x/k<1for any positive -representation ofA.)It should be easy to see that the following examples satisfy the axiom of semi-pre-C -algebras.Example1.Let be a discrete group and kŒ be its group algebra over k:.f g/.s/DXt2f.st 1/g.t/and f .s/D f.s 1/ for f;g2kŒ :The canonical -positive cone of kŒ is deﬁned as the sums of hermitian squares,kŒ C Dn n Xi D1 i i W n2N; i2kŒo:Then,kŒ is a semi-pre-C -algebra such that C u.kŒ /D C ,the full group C -algebra of ,which is the universal C -algebra generated by the unitary representations of .There is another group C -algebra.Recall that the left regular representation of on`2 is deﬁned by .s/ıt Dıst for s;t2 , or equivalently by .f/ D f for f2kŒ and 2`2 .The reducedAbout the Connes embedding conjecture151 group C -algebra C r of is the C -algebra obtained as the norm-closure of .kŒ /in B.`2 /.The group algebra kŒ is equipped with the corresponding -positive conek rŒ C D f f2kŒ W9f n2kŒ C such that f n!f pointwise gD f f2kŒ W f is of positive type g;and the resultant semi-pre-C -algebra k rŒ satisﬁes C u.k rŒ /D C r .Indeed, if f2kŒ \ 1.C r C/,then for D .f/1=2ı12`2 ,one has f D and f is the pointwise limit of n n2kŒ C,where n2kŒ are such that k n k2!0.On the other hand,if f is of positive type(i.e.,the kernel .x;y/!f.x 1y/is positive semi-deﬁnite),then f D f and h .f/Á;Ái 0 for everyÁ2`2 ,which implies .f/2C r C.It follows that k rŒ C D arch.kŒ C/if and only if is amenable(see Theorem1).Example2.The -algebra kŒx1;:::;x d of polynomials in d commuting hermitian variables x1;:::;x d is a semi-pre-C -algebra,equipped with the -positive conekŒx1;:::;x d C D -positive cone generated by f1 x2i W i D1;:::;d g:One has C u.kŒx1;:::;x d /D C.Œ 1;1 d/,the algebra of the continuous func-tions onŒ 1;1 d,and x i is identiﬁed with the i-th coordinate projection.Example3.The -algebra k h x1;:::;x d i of polynomials in d non-commuting hermitian variables x1;:::;x d is a semi-pre-C -algebra,equipped with the -positive conek h x1;:::;x d i C D -positive cone generated by f1 x2i W i D1;:::;d g: One has C u.k h x1;:::;x d i/D C.Œ 1;1 / C.Œ 1;1 /,the unital full free product of d-copies of C.Œ 1;1 /.Example4.Let A and B be semi-pre-C -algebras.We denote by A˝B the algebraic tensor product over k.There are two standard ways to make A˝B into a semi-pre-C -algebra.Theﬁrst one,called the maximal tensor product and denoted by A˝max B,is A˝B equipped with.A˝max B/C D -positive cone generated by f a˝b W a2A C;b2B C g: The second one,called the minimal tensor product and denoted by A˝min B, is A˝B equipped with.A˝min B/C D.A˝B/h\.ÃA˝ÃB/ 1..C u.A/˝min C u.B//C/: (See Theorem14for a“better”description.)One has C u.A˝˛B/D C u.A/˝˛C.B/for˛2f max;min g.The right hand side is the C -algebra maximal u152N.Ozawa (resp.minimal)tensor product(see[BO,Pi1]).For A1 A2and B1 B2, one has.A1˝min B1/C D.A1˝min B1/\.A2˝min B2/C;but the similar identity need not hold for the maximal tensor product. Example5.The unital algebraic free product A B of semi-pre-C -algebras A and B,equipped with.A B/C D -positive cone generated by.A C[B C/;is a semi-pre-C -algebra,and C u.A B/D C u.A/ C u.B/,the unital full free product of the C -algebras C u.A/and C u.B/.The following is very basic(cf.[Ci]and Proposition15in[Sm]).Theorem1.Let A be a semi-pre-C -algebra andÃW A!C u.A/be the uni-versal C -algebra of A.Then,one has the following.kerÃD I.A/,the ideal of the inﬁnitesimal elements.A h\Ã 1.C.A/C/D arch.A C/,the archimedean closure of A C.uAlthough it follows from the above theorem,we give here a direct proof of the fact that arch.A C/\. arch.A C// I.A/.Indeed,if h2Ä1and "1<h<"1for"2.0;1/,then one has0Ä.1C h/." h/.1C h/D".1C h/2 h h.2C h/hÄ.4"C"/1 .2 "/h2; which implies h2<5"1.We postpone the proof and give corollaries to this theorem.4.PositivstellensätzeWe give a few results which say if an element a is positive in a certain class of representations,then it is positive for an obvious reason.Such results are re-ferred to as“Positivstellensätze.”Recall that a C -algebra A is said to be resid-uallyﬁnite dimensional(RFD)ifﬁnite-dimensional -representations separate the elements of A,i.e., .a/ 0for allﬁnite-dimensional -representations implies a 0in A.All abelian C -algebras and full group C -algebras of residuallyﬁnite amenable groups are RFD.Moreover,it is a well-known result of Choi that the full group C -algebra C F d of the free group F d of rank d is RFD(see Theorem26).In fact,ﬁnite representations(i.e.,the unitary repre-sentations such that .F d/isﬁnite)separate the elements of C F d([LS]). However,we note that the full group C -algebra of a residuallyﬁnite group need not be RFD([Be1]).We also note that the unital full free products of RFDAbout the Connes embedding conjecture153 C -algebras is again RFD([EL]).In particular,C u.k h x1;:::;x d i/is RFD.The results mentioned here have been proven for complex C -algebras,but they are equally valid for real cases.See Sect.7.Theorem1,when combined with resid-ualﬁnite dimensionality,immediately implies the following Positivstellensätze (cf.[Pu,HM]).Corollary2.The following are true.Let f2kŒ h.Then, .f/ 0for every unitary representation if and only if f2arch.kŒ C/.The full group C -algebra C of a group is RFD if and only if the fol-lowing statement holds.If f2kŒ h is such that .f/ 0for every ﬁnite-dimensional unitary representation ,then f2arch.kŒ C/.Let f2kŒx1;:::;x d h.Then,f.t1;:::;t d/ 0for all.t1;:::;t d/2Œ0;1 d if and only if f2arch.kŒx1;:::;x d C/.(See Example2.)Let f2k h x1;:::;x d i h.Then,f.X1;:::;X d/ 0for all contractive her-mitian matrices X1;:::;X d if and only if f2arch.k h x1;:::;x d i C/.(See Example3.)In some cases,the -positive cones are already archimedean closed.We will see later(Theorem26)this phenomenon for the free group algebras kŒF d . 5.Eidelheit–Kakutani separation theoremThe most basic tool in functional analysis is the Hahn–Banach theorem.In this note,we will need an algebraic form of it,the Eidelheit–Kakutani separation theorem.We recall the algebraic topology on an R-vector space V.Let C V be a convex subset.An element c2C is called an algebraic interior point of C if for every v2V there is">0such that c C v2C for all j j<". The convex cone C is said to be algebraically solid if the set Cıof algebraic interior points of C is non-empty.Notice that for every c2Cıand x2C, one has c C.1 /x2Cıfor every 2.0;1 .In particular,CııD Cıfor every convex subset C.We can equip V with a locally convex topology,called the algebraic topology,by declaring that any convex set that coincides with its algebraic interior is open.Then,every linear functional on V is continuous with respect to the algebraic topology.Now Hahn–Banach separation theorem reads as follows.Theorem3(Eidelheit–Kakutani([Ba])).Let V be an R-vector space,C an algebraically solid cone,and v2V n C.Then,there is a non-zero linear func-tional'W V!R such that'.c/:'.v/Äinfc2CIn particular,'.v/<'.c/for any algebraic interior point c2C.154N.Ozawa Notice that the Combes axiom A D A bdd is equivalent to that the unit1is an algebraic interior point of A C A h and arch.A C/is the algebraic closure of A C in A h.(This is where the Combes axiom is needed and it can be dispensed when the cone A C is algebraically closed.See Sect.3.4in[Sm].)Let A be a semi-pre-C -algebra.A unital -subspace S A is called a semi-operator system.Here,a -subspace is a subspace which is closed under the -operation. Existence of1in S ensures that S C D S\A C has enough elements to span S h.A linear functional'W S!k is called a state if'is self-adjoint, positive,and'.1/D1.Note that if k D C,then S is spanned by S C and every positive linear functional is automatically self-adjoint.However,this is not the case when k D R.In any case,every R-linear functional'W S h!R extends uniquely to a self-adjoint linear functional'W S!k.We write S.S/ for the set of states on S.Corollary4.Let A be a semi-pre-C -algebra.Let W A be a -subspace and v2A h n.A C C W h/.Then,there is a state'on A such that'.W/D f0g and'.v/Ä0.(Krein’s extension theorem)Let S A be a semi-operator system.Then every state on S extends to a state on A.Proof.Since A C C W h is an algebraically solid cone in A h,one mayﬁnd a non-zero linear functional'on A h such that'.v/Äinf f'.c/W c2A C C W h g:Since'is non-zero,'.1/>0and one may assume that'.1/D1.Thus the self-adjoint extension of'on A,still denoted by',is a state such that'.v/Ä0 and'.W h/D f0g.Let x2W.Then,for every 2k,one has'.x/C. '.x// D'.. x/C. x/ /D0:This implies'.W/D f0g in either case k2f C;R g.For the second assertion,let'2S.S/be given and consider the coneC D f x2S h W'.x/ 0g C A C:It is not too hard to see that C is an algebraically solid cone in A h and v…C for any v2S h such that'.v/<0.Hence,one mayﬁnd a state N'on A such that N'.C/ R 0.In the same way as above,one has that N'is zero on ker', which means N'j S D'.About the Connes embedding conjecture155 6.GNS constructionWe recall the celebrated GNS construction(Gelfand–Naimark–Segal construc-tion),which provides -representations out of states.Let a semi-pre-C -algebra A and a state'2S.A/be given.Then,A is equipped with a semi-inner product h y;x i D'.x y/,and it gives rise to a Hilbert space,which will bedenoted by L2.A;'/.We denote by O x the vector in L2.A;'/that correspondsto x2A.Thus,h O y;O x i D'.x y/and k O x k D'.x x/1=2.The left multiplica-tion x!ax by an element a2A extends to a bounded linear operator '.a/on L2.A;'/such that '.a/O x D ca x for a;x2A.(Observe that a aÄR1implies k '.a/k2ÄR.)It follows that 'W A!B.L2.A;'//is a positive -representation of A such that h '.a/O1;O1i D'.a/.If W A!B.H/is a positive -representation having a unit cyclic vector,then'.a/D h .a/ ; i is a state on A and .x/ !O x extends to a uni-tary isomorphism between H and L2.A;'/which intertwines and '.Sinceevery positive -representation decomposes into a direct sum of cyclic repre-sentations,one may obtain the universal C -algebra C u.A/of A as the closureof the image under the positive -representationM '2S.A/ 'W A !BM'2S.A/L2.A;'/Á:We also make an observation that.A˝min B/C in Example4coincides withc2.A˝B/h W .'˝ /.z cz/ 0for all'2S.A/;2S.B/;z2A˝B:7.Real versus complexWe describe here the relation between real and complex semi-pre-C -algebras. Because the majority of the researches on C -algebras are carried out for com-plex C -algebras,we look for a method of reducing real problems to complex problems.Suppose A R is a real semi-pre-C -algebra.Then,the complexiﬁca-tion of A R is the complex semi-pre-C -algebra A C D A R C i A R.The -algebra structure(over C)of A C is deﬁned in an obvious way,and.A C/C is deﬁned to be the -positive cone generated by.A R/C:.A C/C Dn n Xi D1z i a i z i W n2N;a i2.A R/C;z i2A Co:(This is a temporary deﬁnition,and the ofﬁcial one will be given later.See Lemma11.)Note that A R\.A C/C D.A R/C.The complexiﬁcation A C has an involutive and conjugate-linear -automorphism deﬁned by x C i y!156N.Ozawa x i y ,x;y 2A R .Every complex semi-pre-C -algebra with an involutive and conjugate-linear -automorphism arises in this way.Lemma 5.Let R W A R !B R be a -homomorphism between real semi-pre-C -algebras (resp.'R W A R !R be a self-adjoint linear functional).Then,the complexiﬁcation C W A C !B C (resp.'C W A C !C )is positive if and only if R (resp.'R )is so.Proof.Weonly prove that 'C is positive if 'R is so.The rest is trivial.Let b D P i z i a i z i 2.A C /C be arbitrary,where a i 2.A R /C and z i D x i C i y i .Then,b D P i .x i a i x i C y i a i y i /C i P i .x i a i y i y i a i x i /.Since x i a i y i y i a i x i is skew-hermitian,one has 'R .x i a i y i y i a i x i /D 0,and 'C .b/D'R .P i x i a i x i C y i a i y i / 0.This shows 'C is positive.We note that if H C denotes the complexiﬁcation of a real Hilbert space H R ,then B .H R /C D B .H C /.Thus every positive -representation of a real semi-pre-C -algebra A R on H R extends to a positive -representation of its complexiﬁcation A C on H C .Conversely,if is a positive -representation of A C on a complex Hilbert space H C ,then its restriction to A R is a positive -representation on the realiﬁcation of H C .The realiﬁcation of a complex Hilbert space H C is the real Hilbert space H C equipped with the real inner product h Á; i R D <h Á; i .Therefore,we arrive at the conclusion that C u .A R /C D C u .A C /.We also see that .R Œ /C D C Œ ,.A R ˝B R /C D A C ˝B C ,.A R B R /C D A C B C ,etc.8.Proof of Theorem 1We only prove the ﬁrst assertion of Theorem 1.The proof of the second is very similar.We will prove a stronger assertion thatk Ã.x/k C u.A /D inf f R >0W R 21 x x 2A C g :The inequality Ätrivially follows from the C -identity.For the converse,as-sume that the right hand side is non-zero,and choose >0such that 21 x x …A C .By Corollary 4,there is '2S.A /such that '. 21 x x/Ä0.Thus for the GNS representation ',one hask '.x/k k '.x/O 1k D '.x x/1=2 :It follows that k Ã.x/k .About the Connes embedding conjecture157 9.Trace positive elementsLet A be a semi-pre-C -algebra.A state on A is called a tracial state if .xy/D .yx/for all x;y2A,or equivalently if is zero on the -subspace K D span f xy yx W x;y2A g spanned by commutators in A.We denote by T.A/the set of tracial states on A(which may be empty).Associated with 2T.A/is aﬁnite von Neumann algebra. .A/00; /,which is the von Neumann algebra generated by .A/ B.L2.A; //with the faithful normal tracial state .a/D h a O1;O1i that extends the original .Recall that aﬁnite von Neumann algebra is a pair.M; /of a von Neumann algebra and a faithful normal tracial state on M.The following theorem is proved in[KS]for the algebra in Example3and in[JP]for the free group algebras,but the proof equally works in the general setting.We note that for some groups ,notably for D SL3.Z/([Be2]),it is possible to describe all the tracial states on kŒ . Theorem6([KS]).Let A be a semi-pre-C -algebra,and a2A h.Then,the following are equivalent..1/ .a/ 0for all 2T.A/..2/ . .a// 0for everyﬁnite von Neumann algebra.M; /and every positive -homomorphism W A!M..3/a2arch.A C C K h/,where K h D K\A h D span f x x xx W x2A g. Proof.The equivalence.1/,.2/follows from the GNS construction.We only prove.1/).3/,as the converse is trivial.Suppose a C"1…A C C K h for some">0.Then,by Corollary4,there is 2S.A/such that .K/D f0g (i.e., 2T.A/)and .a/Ä "<0.10.Connes embedding conjectureThe Connes embedding conjecture(CEC)asserts that anyﬁnite von Neumann algebra.M; /with separable predual is embeddable into the ultrapower R! of the hyperﬁnite II1-factor R(over k2f C;R g).Here an embedding means an injective -homomorphism which preserves the tracial state.We note that if is a tracial state on a semi-pre-C -algebra A andÂW A!N is a -preserving -homomorphism into aﬁnite von Neumann algebra.N; /,thenÂextends to a -preserving -isomorphism from .A/00onto the von Neumann subalgebra generated byÂ.A/in N(which coincides with the ultraweak clo-sure ofÂ.A/).Hence,.M; /satisﬁes CEC if there is an ultraweakly dense -subalgebra A M which has a -preserving embedding into R!.In particular, CEC is equivalent to that for every countably generated semi-pre-C -algebra A and 2T.A/,there is a -preserving -homomorphism from A into R!.We will see that this is equivalent to the tracial analogue of Positivstellensätze in158N.Ozawa Corollary 2.We ﬁrst state a few equivalent forms of CEC.We denote by tr the tracial state 1N Tr on M N .k /.Theorem 7.For a ﬁnite von Neumann algebra .M; /with separable predual,the following are equivalent..1/.M; /satisﬁes CEC,i.e.,M ,!R !..2/Let d 2N and x 1;:::;x d 2M be hermitian contractions.Then,forevery m 2N and ">0,there are N 2N and hermitian contractions X 1;:::;X d 2M N .k /such thatj .x i 1 x i k / tr .X i 1 X i k /j <"for all k Äm and i j 2f 1;:::;d g ..3/Assume k D C (or replace M with its complexiﬁcation in case k D R ).Letd 2N and u 1;:::;u d 2M be unitary elements.Then,for every ">0,there are N 2N and unitary matrices U 1;:::;U d 2M N .C /such thatj .u i u j / tr .U i U j /j <"for all i;j 2f 1;:::;d g .In particular,CEC holds true if and only if every .M; /satisﬁes condition .2/and/or .3/.The equivalence .1/,.2/is a rather routine consequence of the ultra-product construction.For the equivalence to (3),see Theorem 27.Note that the assumption k D C in condition (3)is essential because the real analogue of it is actually true ([DJ]).Since any ﬁnite von Neumann algebra M with sep-arable predual is embeddable into a II 1-factor which is generated by two her-mitian elements (namely .M R/N ˝R ),to prove CEC,it is enough to verify the conjecture (2)for every .M; /and d D 2.We observe that a real ﬁnite von Neumann algebra .M R ; R /is embeddable into R !R (i.e.,it satisﬁes CEC)if and only if its complexiﬁcation .M C ; C /is embeddable into R !C .The “only if”di-rection is trivial and the “if”direction follows from the real -homomorphism M N .C /,!M 2N .R /,a C i b ! a b b a .A complex ﬁnite von Neumann al-gebra .M; /need not be a complexiﬁcation of a real von Neumann algebra,but M ˚M op is (isomorphic to the complexiﬁcation of the realiﬁcation of M ).Therefore,M satisﬁes CEC if and only if its realiﬁcation satisﬁes it.For a ﬁnite von Neumann algebra .M; /and d 2N ,we denote by H d .M /the set of those f 2k h x 1;:::;x d i h such that .f.X 1;:::;X d // 0for all hermitian contractions X 1;:::;X d 2M .Further,let H d D\M H d .M /and H ﬁn d D \NH d .M N .k //D H d .R/:Notice that H d D arch .k h x 1;:::;x d i C C K h /(see Example 3and Theorem 6).About the Connes embedding conjecture 159Corollary 8([KS]).Let k 2f C ;R g .Then one has the following.Let .M; /be a ﬁnite von Neumann algebra with separable predual.Then,M satisﬁes CEC if and only if H ﬁn d H d .M /for all d .CEC holds true if and only if H ﬁn d D arch .k h x 1;:::;x d i C C K h /for all/some d 2.Proof.It is easy to see that condition (2)in Theorem 7implies H ﬁn d H d .M /.Conversely,suppose condition (2)does not hold for some d 2N ,x 1;:::;x d 2M ,m 2N ,and ">0.We introduce the multi-index notation.For i D .i 1;:::;i k /,i j 2f 1;:::;d g and k Äm ,we denote x i D x i 1 x i k .It may happen that i is the null string ;and x ;D 1.Then,C D closure f .tr .X i //i W N 2N ;X 1;:::;X d 2M N .k /h ;k X i k Ä1gis a convex set (consider a direct sum of matrices).Hence by Theorem 3,there are 2R and ˛i 2k such that <X i ˛i .x i /< Äinf 2C <X i˛i i :Replacing ˛i with .˛i C ˛ i/=2(here i is the reverse of i ),we may omit <from the above inequality.Further,arranging ˛;,we may assume D 0.Thus f D P i ˛i x i belongs to H ﬁn d ,but not to H d .M /.This completes the proof of the ﬁrst half.The second half follows from this and Theorem 6.An analogue to the above also holds for C ŒF d .Corollary 9([JP]).Let k D C .The following holds.Let .M; /be a ﬁnite von Neumann algebra with separable predual.Then,M satisﬁes CEC if and only if the following holds true:If d 2N and ˛2M d .C /h satisﬁes that tr .P ˛i;j U i U j / 0for every N 2N and U 1;:::;U d 2M N .C /u ,then it satisﬁes .P ˛i;j u i u j / 0for every uni-tary elements u 1;:::;u d 2M .CEC holds true if and only if for every d 2N and ˛2M d .C /h the follow-ing holds true:If tr .P ˛i;j U i U j / 0for every N 2N and U 1;:::;U d 2M N .C /u ,then P ˛i;j s i s j 2arch .C ŒF d C C K h /,where s 1;:::;s d are the free generators of F d .11.Matrix algebras over semi-pre-C -algebrasWe describe here how to make the n n matrix algebra M n .A /over a semi-pre-C -algebra A into a semi-pre-C -algebra.We note that x D Œx j;i i;j for x D Œx i;j i;j 2M n .A /.We often identify M n .A /with M n .k /˝A .There。

证实2种蛋白相互作用的高分文献

证实2种蛋白相互作用的高分文献在生物学研究中，蛋白质相互作用的研究对于揭示细胞内分子的功能和调控机制至关重要。

下面将介绍两种蛋白质相互作用的高分文献。

1. 文献标题：Structural basis for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1发表日期：2024年2月13日主要内容：该文献描述了蛋白质Rad6-Bre1与核小体结构中一个特定残基的相互作用。

通过利用X射线晶体学技术，研究人员解析了Rad6-Bre1与核小体残基的结合模式，并确定了该相互作用的结构基础。

通过表征Rad6-Bre1与该残基的相互作用，研究人员发现该相互作用在细胞染色质修饰和胚胎发育中起到关键作用。

此外，研究人员还揭示了这种相互作用中的部分结构变异对于细胞分化和疾病发展的潜在影响。

2. 文献标题：Dynamic interactions between cancer cells and the endothelium in transendothelial migration mediate the metastatic cascade发表日期：2024年5月15日主要内容：该文献研究了肿瘤细胞与内皮细胞之间的相互作用对转移过程的影响。

通过多种实验方法，包括显微镜观察和细胞粘附性实验，研究人员发现在癌细胞穿越内皮细胞和逃脱血管的过程中，细胞与内皮细胞之间存在动态的相互作用。

通过展示这些相互作用的分子和细胞机制，研究人员阐明了转移级联中的细胞-细胞信号传导途径，并提供了精确控制肿瘤细胞转移的新策略。

这两篇文献都具有较高的分数和重要性，揭示了蛋白相互作用在生物学中的重要性和相关的分子机制。

这些研究为我们了解细胞内分子交互的功能和调控提供了重要的基础。

小鼠不同组织的差异线粒体蛋白质组研究

中国科学: 生命科学2010年第40卷第9期: 820 ~ 833 SCIENTIA SINICA Vitae 英文引用格式: Wang Y, Sun H D, Ru Y W, et al. Proteomic survey towards the tissue-specific proteins of mouse mitochondria (in Chinese). SCIENTIA SINICAVitae, 2010, 40: 820—833, doi: 10.1360/062010-317《中国科学》杂志社SCIENCE CHINA PRESS论文小鼠不同组织的差异线粒体蛋白质组研究王媛, 孙海丹, 茹雅维, 尹松月, 阴亮, 刘斯奇*中国科学院北京基因组研究所, 北京 101300 * 联系人, E-mail: siqiliu@收稿日期: 2010-06-29; 接受日期: 2010-07-30国家高技术研究发展计划(批准号: 2006AA02A308)和国家自然科学基金(批准号: 30700378)资助项目摘要线粒体是真核细胞的重要细胞器, 它不仅具有合成ATP 等普遍性功能, 也具备一定的组织特异性, 适应不同组织的生理功能需要. 为了鉴定组织相关性线粒体蛋白, 系统地分析和比较了C57BL/6J 小鼠肝脏、肾脏和心脏线粒体蛋白质组. 通过双向凝胶电泳和MALDI- TOF/TOF 质谱, 在这3种小鼠组织中共发现了87种组织特异的线粒体差异蛋白; 采用ICPL 的定量蛋白质组方法和Western blotting 技术, 进一步证实某些典型的组织特异性蛋白. 还通过实时定量PCR 和Western blotting 技术分析了6种具有代表性的组织差异线粒体蛋白质在线粒体内外的分布, 并结合激光共定位技术观察它们在相应组织活细胞内的表达与定位. 研究发现, 这些核基因编码的线粒体蛋白确有组织特异性分布和线粒体内外丰度差异特性. 本结果为组织特异性线粒体蛋白质的深入研究奠定了实验基础.关键词蛋白质组学组织特异性线粒体线粒体的基本生物学功能是参与内膜系统的呼吸链氧化过程, 由此合成细胞所需的能量化合物—— ATP. 线粒体还具有许多重要生物功能, 包括: 离子稳态维持、物质代谢和细胞凋亡[1,2]. 所以, 线粒体功能紊乱可导致多种疾病, 如神经-肌肉退行性疾病(阿尔茨海默病等)和代谢病(糖尿病等)已被证明是线粒体功能缺陷所致[3,4]. 线粒体具有自己的一套遗传控制系统, 即线粒体基因组, 其合成的蛋白数量较少, 只有13个编码蛋白, 它们是线粒体呼吸链的主要成份. 一般估计, 线粒体的蛋白至少应在3000~4000个[5]. 显然, 大部分的线粒体蛋白是由核基因编码, 它们在线粒体外合成和包装后, 通过一定的蛋白质转运系统进入线粒体内[6].以往研究表明, 在不同的组织中线粒体的数目、形态和超微结构不同[6]. 人体中共有250种不同的细胞, 在不同细胞中的线粒体数目有很大差异.大多数有核细胞含有500~2000个线粒体. 人体的视锥细胞线粒体占其细胞内体积的80%; 而在肌肉组织中, 线粒体占横纹肌细胞的60%, 在心肌细胞中则占40%. 另外在一些细胞中只有很少的线粒体, 如血小板只有2~6个线粒体[7]. 为了适应不同组织的不同生理需要, 在同一基因组的条件下, 各组织的细胞具有自己一套独特的基因表达系统. 换而言之, 各组织中的蛋白质组有其特异性, 其中有些必需的蛋白质在各组织细胞中广泛存在, 有一些特殊功能蛋白质却是组织特异表达的[8,9]. 既然线粒体生活在不同的细胞环境中, 而且它的大部分蛋白质成分来自核编码的基因, 那么各个组织的线粒体的蛋白质组是否不同? 显然, 对不同组织线粒体差异蛋白质组的分析, 将有助于了解组织相关线粒体功能中国科学: 生命科学 2010年第40卷第9期821的分子机制.长期以来, 对于线粒体蛋白质组的特异性并未得到足够的重视. 直到19世纪80年代, 蛋白质组科学迅速发展, 才有了根本改变. Vayssiere 等人[10]采用双向凝胶电泳(2-DE)技术分离大鼠脑皮层、肌肉、肝脏线粒体蛋白, 发现在不同组织中线粒体蛋白的分布明显不同, 其中有16个蛋白质点仅存在于脑皮层于线粒体中. Watmough 等人[11]通过比较肌无力病人与正常人线粒体蛋白质组的差异, 报道了线粒体复合物Ⅰ在疾病条件下不同组织中表达丰度变化的差异, 即复合体Ⅰ在肌无力病人肌肉组织线粒体内丰度下降, 而在肝脏线粒体中表达量无明显变化. 这些早期关于线粒体组织特异性的研究, 由于分析技术的局限无法提供详尽的线粒体组织特异蛋白质鉴定信息. 自1994年, 蛋白质组学概念被提出以来, 蛋白质组学得到了很大的发展, 并广泛应用于组织特异性亚细胞器蛋白质组研究, 而线粒体的组织特异性蛋白质组学的研究也如此[12]. Mootha 等人[13]运用shotgun-LC-MS/MS 技术分析小鼠组织的线粒体, 发现只有50%的线粒体蛋白质在心、肝、肾、脑的线粒体中是共有的, 除此之外, 大量存在着组织特异性的线粒体蛋白质. 基因芯片分析也进一步证实, 线粒体中的mRNA 表达丰度的确存在组织差异性. 该研究共鉴定分析了399个线粒体蛋白, 但并未对不同组织中的线粒体差异蛋白进行定量比较. 针对这一问题, 进一步采用LC- MS/MS 的定量蛋白质组学方法分析大鼠骨骼肌、心脏和肝脏线粒体蛋白质组的差异, 发现近1/3的线粒体蛋白组织特异表达[14]. Kislinger 等人[15], 通过多维色谱分离结合ESI-MS/MS 方法(multidi-mensional protein identification technology, MudPIT)对小鼠脑、心、肾、肝、肺和胎盘组织亚细胞器, 如微粒体、核、线粒体、胞浆等, 进行了系统的比较蛋白质组研究, 并对鉴定的3174个蛋白进行了亚细胞定位, 其中1503个是以前未被报道的具有亚细胞组织特异性的蛋白.应用蛋白质组学方法对线粒体在不同组织的特异性开展系统分析, 无疑对该领域的研究具有革命性的贡献. 然而, 组织特异性线粒体蛋白质组研究存在一定的不足. 以往的蛋白质组分析工作都建立在shotgun-LC-MS/MS 技术基础上. 这种技术固然可增加检测的灵敏度, 但也有其弊病. 首先, shotgun-LC-MS/MS的蛋白质鉴定只是基于肽段的分离和质谱信号, 未能提供足够的完整蛋白质的信息, 也可能丢失或错译修饰蛋白质的信号; 其次, 仅仅依靠肽段的一般质谱信号还不能提供足够的定量信息. 从生物学角度来看, 以往的工作还缺乏其他的生物学测定证据以支持蛋白质组的分析结果, 尤其是体内测定数据. 本实验提出了一个全面分析和鉴定小鼠肝脏、肾脏、心脏组织特异性线粒体蛋白质组的方案. 这个方案的基本立足点在于完整线粒体蛋白质的分离和定量比较, 以及采用免疫化学和激光共定位技术, 试图用生物学测定方法分析组织特异性线粒体蛋白质. 通过2-DE 的分离和MALDI-TOF/TOF 质谱鉴定, 在这3种小鼠组织中共发现87种组织特异的线粒体差异蛋白; 采用ICPL 的定量蛋白质组方法和Western blotting 技术, 进一步证实某些典型的组织特异性蛋白. 还通过实时定量PCR 和Western blotting 分析了6种具有代表性的组织差异线粒体蛋白在线粒体内外的分布, 并结合激光共定位技术观察它们在相应组织活细胞内的表达与定位. 结果发现, 这些核基因编码的线粒体蛋白质确有组织特异性分布和线粒体内外丰度差异性, 为组织特异性线粒体蛋白质的深入研究奠定了实验基础. 1 材料与方法1.1 试剂丙烯酰胺、过硫酸铵、IPGphor 胶条(5 m×180 m, 5 m×70 m, pH 3~10, 线性)、两性电解质(pH 3~10)、尿素、3-[3-(胆酰氨丙基)二甲氨基]丙磺酸(CHAPS)及其他用于凝胶电泳的试剂均购自Bio-Rad(Hercules, CA, USA). 所有分析纯化学试剂购于Sigma(St. Louis, MO). 胰蛋白酶和二硫苏糖醇(DTT)购自于Promega (Madison, WI, USA). 细胞培养所需试剂购于Sigma (St. Louis, MO). 1.2 实验方法 (1) 线粒体制备. 实验用小鼠(C57BL/6J, 7~9周,雄性)来自北京大学实验动物中心, 均在无菌条件下饲养. 动物实验操作符合中国科学院动物实验委员会标准. 小鼠用戊巴比妥钠(50 mg/kg, 10 mg/mL)麻醉, 摘取肝脏、肾脏、心脏组织, 迅速置于预冷的磷酸缓冲液反复洗涤3次, 除去多余的血液成分, 并称重. 将组织切成约3 mm 3小块, 1 g 组织用5 mL 预冷匀浆液重悬(220 mmol/L 甘露糖, 70 mmol/L 蔗糖, 2王媛等: 小鼠不同组织的差异线粒体蛋白质组研究822mmol/L EDTA, 1 mmol/L PMSF, 0.2 mmol/L Na 2VO 3, 1 mmol/L NaF, 蛋白质酶抑制剂cocktail, 10 mmol/L Tris-HCl pH 7.4), 用玻璃匀浆器匀浆3次. 匀浆液经8000×g 差速离心, 得到粗提线粒体. 将粗提线粒体用25% Nycodenz 重悬, 在超速离心管中铺制Nycodenz 密度梯度(20%, 23%, 25%, 30%, 34%), 52000×g 离心后, 收集25%/30%区域样品, 15000×g 离心20 min, 弃去沉淀, 并用匀浆缓冲液洗2次后, 得到纯化的线粒体组分. 采用投射扫描电子显微镜(TEM)和Western blotting 检测制备线粒体的纯度及完整性. 在Western blotting 检测中, 采用线粒体特异的标志物ATP synthase (BD Biosciences, CA), Prohibitin, Aldehyde dehydrogenase Ⅱ(Santa Cruz, CA)检测线粒体组分; 采用Aldehyde reductase 和GAPDH(Santa Cruz, CA)为胞浆标志物, 以检测是否存在胞浆组分的污染. 制备好的线粒体于−80℃保存备用.(2) 双向凝胶电泳(Two-Dimensional Electro-pho- resis, 2-DE). 制备的线粒体用混合溶液(乙醇︰丙酮︰乙酸=50︰50︰0.1)−20℃沉淀过夜, 在4℃条件下25000×g 离心 1 h, 沉淀真空抽干溶于裂解液中(7 mol/L 尿素, 2 mol/L 硫脲, 4% CHAPS, 10 mmol/L DTT, 2 mmol/L EDTA), 超声破碎5 min 后, 35000×g 离心30 min, 上清液即为制备的线粒体蛋白质. 采用Lowery 法对蛋白质进行定量. 将100 μg 蛋白质加载于18 cm, pH 3~10的线性IPG 胶条, 在0.05 mA/胶条、室温条件下等电聚焦至70 kVh. 第一相电泳后, 将胶条置于含有1%DTT 的平衡液中还原15 min, 用含有2.5%碘代乙酰胺的平衡液烷基化15 min. 平衡后的胶条转移至12%SDS-聚丙烯酰胺凝胶上, 利用Amersham Biosciences 的Ettan DALT Ⅱ系统进行第二相蛋白质分离, 0.05 mA/strip 至溴酚兰到达胶的下缘.2-DE 胶图采用硝酸银染色法染色. 染色后的凝胶由扫描仪(Powerlook 2100XL, UMAX, Dallas, TX)扫描, 并利用Imagemaster platinum version 5.0软件(GE Healthcare, Fairfield, CT)分析. 把组织之间2-DE 斑点的spot volume 大于3倍以上的差异点定义为具有显著差异. spot volume 值经统计计算获得(3次线粒体平行制备, 每一次制备进行两块平行胶电泳).(3) 蛋白质的质谱鉴定. 使用spot picker P2D1.5将差异蛋白质点切下, 用0.025 mg/mL 胰蛋白酶37℃消化16 h. 将消化后的肽段混合物(1 g)点在anchorchip(Bruker Dalton, Bremen, Germany)上, 晾干后加0.8 μL 基质CHCA 溶液, 再用0.5%TFA 溶液除盐. 将anchor chip 送入UltraFlex MALDI-TOF/TOF 质谱进行鉴定. 质谱仪采用反射模式及正电荷状态. MS 质谱图为单次扫描信号累加100次所得, MS/MS 质谱图为单次扫描信号累加400次所得. 利用Flexanalysis 2.2和BioTools 2.2软件对质谱峰图进行标注和肽段质荷比数据提取.肽段的质荷比数据通过Mascot 软件(http://www. )从小鼠的NCBInr 蛋白质数据库搜索出对应的蛋白质. 搜索参数如下: 一级母离子误差<0.01%, 漏切位点数最多1个, 半胱氨酸的还原烷基化设置为固定修饰, 甲硫氨酸的氧化、肽段N 端的焦谷氨酸环化设置为可变修饰. 二级碎片离子误差<0.7 D.(4) isotope-coded protein label(ICPL)定量分析线粒体蛋白质. 根据ICPL 药盒(SERVA Electro-phoresis, Heielberg, Germany)的操作说明, 两种蛋白质样品分别以12C 和13C 标记, 等量混合后进行2-DE 分离. 蛋白质点胶内酶切消化后, 利用MALDI-TOF 质谱鉴定. 采用Peakpicker 软件(Applied Biosystems, USA)分析质谱峰图上不同ICPL 试剂(12C 和13C)标记的一对肽段离子的强度比例, 定量分析它的母本蛋白质在原来样品中的相对丰度.(5) Western blotting. 取等量(10 µg)蛋白质样品在12%SDS-聚丙烯酰胺凝胶(SDS-PAGE)中电泳, 350 mA 条件下, 采用Bio-Rad Mini PROTEAN 3系统(Hercules, CA, USA)电转1 h 将蛋白样品转至PVDF 膜上. 将膜置于封闭液(5%脱脂奶粉溶于含有0.1% Tween 20的TBS 溶液中)中4℃封闭过夜. 采用如下抗体进行一抗孵育: 抗-SCP2, 抗-HADHB, 抗-SCOT 为本实验室制备纯化的SCP2, SCOT 和HADHB 重组蛋白免疫兔子得到的多克隆抗体, 抗-aldehyde dehydrogenase Ⅱ, 抗-prohibitin, 抗-aldehyde reductase 和抗-catalase(The Binding Site Inc. USA), 抗-PDZK 1, 抗-sMtCK 和抗-GAPDH(Santa Cruz Biote-chonolgy, USA), 抗-ATP synthase β(BD Biosciences, USA). 进而选用相应的辣根过氧化物酶标记的二抗进行抚育. 采用ECL 超敏发光液(Amersham Pharmacia Biotech, Uppsala, Sweden)曝光显影.(6) 实时定量RT-PCR. 采用ABIPRISM 7300系统(Foster City, CA), 对SCP2, PDZK1, sMtCK ,中国科学: 生命科学 2010年第40卷第9期823catalase , SCOT 和HADHB 基因表达进行定量分析. 小鼠肝脏、肾脏、心脏组织的cDNA 文库来自逆转录后组织提取的总RNA. 不同基因的PCR 引物序列如下: SCP2: 5′-TGCCTTCAAAGTGAAAGATGGCCC- 3′(正向), 5′-TTCCCTTGAAAGAAGGCCGACTGA- 3′(反向); PDZK1: 5′-AGGCAGCTGGCTTGAAGAA- CAATG-3′(正向), 5′-AAAGAAGTGGAGAGAACCG- AGCCA-3′(反向); sMtCK: 5′-TAAGATCTTGGAGA- ACCTGCGGCT-3′(正向), 5′-TCTTGGCCTCTCTCCA- GCTTCTTT-3′(反向); catalase: 5′-TGCAGATACCT- GTGAACTGTCCCT-3′(正向), 5′-AAGCGTTTCACA- TCTACAGCGCAC-3′(反向); SCOT: 5′-GCTCTGGT- GAAAGCATGGAAAGCA-3′(正向), 5′-TCCCTTTA- TGAGGCGGTGCACATA-3′(反向); HADHB: 5′-CA- CTTTCGGGTTTGTTGCATCGGA-3′(正向), 5′-GCT- GTGGTCATGGCTTGGTTTGAa-3′(反向). 反应完成后, 应用2−ΔΔC t 方法用GAPDH 做为内标进行校正, 并通过分析每个PCR 反应的溶解曲线检测扩增产物的纯度.(7) 激光共定位检测线粒体蛋白质. 选用了人类正常肝脏细胞Chang(由复旦大学中山医院刘银坤教授提供)、小鼠正常肾小球细胞MC(由北京大学医学部糖尿病中心管又飞教授提供)和大鼠胚心细胞H9c2(购于美国典型微生物菌种保藏中心, ATCC, CRL1446)细胞系进行蛋白质在线粒体的共定位检测. 将细胞系接种于放有无菌盖破片的培养皿中过夜培养24 h, 加入线粒体指示剂MitoTracker Red(Invitrogen, Eugene, USA), 37℃孵育30 min, 加入3.7%甲醛固定15 min 后, 加入特异的抗体避光孵育2 h. 用含0.05% Triton X-100的PBS 溶液冲洗细胞3次后, 加入含1%BSA 的PBS 溶液稀释的FITC 标记的二抗避光孵育1 h, 冲洗3次, 用0.25 μg/μL 的DAPI 溶液染细胞核. 将盖玻片取出在激光扫描共聚焦显微镜下观察线粒体和各蛋白在Chang, MC 和H9c2细胞中的定位.2 实验结果2.1 小鼠不同组织的线粒体差异蛋白质组图谱分析为了检测制备小鼠不同组织线粒体的纯度及完整性, 采用TEM 和Western blotting 技术. 在Westernblotting 检测中, 应用线粒体特异的标志物ATP syn- thase, Prohibitin, Aldehyde dehydrogenase Ⅱ的抗体在3个组织的线粒体组分中显示为一条阳性免疫蛋白区带, 而在胞浆组分中均未检测到阳性信号. 应用 TEM 观察制备的线粒体形态, 结果表明, 线粒体的完整率均达到90%.将各个组织的线粒体蛋白进行2-DE 分离, 得到相应的2-DE 图谱(图1(A)). 为了降低由生物样品中个体差异带来的双向电泳分析误差, 应用平行胶和不同个体相同组织混和胶同时电泳的方法. 每个组织线粒体的蛋白质样品有6块对应的胶图. 通过统计学计算, 小鼠肝脏、肾脏和心脏3个组织线粒体在2-DE 胶图上蛋白质点数目分别为: (1039±46), (1018±47)和(754±35)(n =6). 每个组织线粒体样品的6块平行胶的平行性高于90%. 如图1(A)所示, 小鼠3个组织的线粒体蛋白质在胶图上的分布明显不同. 心脏组织的线粒体蛋白质的分布与肾脏和肝脏相比存在着更大的差异, 其蛋白质点总数比其他两个组织少了近30%; 且若干高丰度心脏线粒体蛋白主要分布于碱性区域, 而肾脏和肝脏在该区域的线粒体蛋白丰度相对较低. 肝脏和肾脏组织的线粒体蛋白分布较为相似, 有80%相匹配. 为了得到可信的差异分析结果, 按照如下策略分析3个样品的2-DE 胶图: 首先, 比较其中两个组织线粒体样品胶图, 找到着色斑点体积上有显著差异的点(即有或无的着色斑点); 然后检查另外一个组织线粒体样品胶图上对应位置是否有该点存在. 共发现134个组织特异性线粒体差异蛋白质点, 其中, 有26个蛋白质点仅存在于肝脏中, 18个仅出现于肾脏中, 10个仅出现于心脏中; 55个在肝脏和肾脏中都存在, 而心脏中没有; 6个存在于肝脏和心脏中, 而肾脏中没有; 另外19个差异点在肾脏和心脏中出现, 而肝脏中没有. 图1(B)展示了肝脏、肾脏和心脏组织特异的代表性线粒体差异蛋白质点.2.2 组织特异性线粒体差异蛋白的质谱鉴定将134个差异蛋白质点从胶上切下, 经胶内酶切消化后, 利用MALDI TOF/TOF 质谱进行鉴定, 共鉴定到87个蛋白, 其中20个为肝脏线粒体特异表达, 如SCP2; 14个为肾脏线粒体特异表达, 如PDZK1; 6个为心脏线粒体特异表达, 如sMtCK; 34个为肝脏和肾脏共表达, 而心脏不表达, 如catalase; 5个肝脏和心脏共表达, 而肾脏不表达, 如HADHB; 8个为肾脏和心脏共表达, 而肝脏不表达, 如SCOT. 将87个蛋白质点的质谱结果总结如表1. 图1(C)是一张代表性王媛等: 小鼠不同组织的差异线粒体蛋白质组研究824图1 小鼠肝脏、肾脏和心脏线粒体的双向电泳图谱及差异蛋白的MALDI TOF/TOF 质谱鉴定(A) 小鼠肝脏、肾脏和心脏线粒体的双向电泳图. LM: 肝脏线粒体; KM: 肾脏线粒体; HM: 心脏线粒体; (B) 小鼠组织特异性线粒体蛋白差异点的局部放大图. a, d, e 分别为肝脏、心脏和肾脏线粒体内特有的蛋白斑点; b 为肝脏和肾脏中共用, 而心脏中没有的蛋白斑点; c 为肾脏和心脏中共有, 而肝脏中没有的蛋白斑点; f 为肝脏和心脏中共有, 而肾脏中没有的蛋白斑点; (C) 差异蛋白斑点e 的MALDI-TOF/TOF 质谱鉴定结果. 经质谱鉴定为PDZK1. 左下图为该蛋白的肽指纹图谱; 右上两图分别为肽段1119.501和1391.563的二级图谱及其对应的氨基酸序列中国科学: 生命科学 2010年第40卷第9期825表1 组织特异线粒体差异蛋白点MALDI-TOF/TOF 质谱鉴定SpotNO. Swiss-prot 号码鉴定蛋白名称分子量Cal./Obs. 等电点 Cal./Obs.覆盖率 (%)MS/MS肝脏组织线粒体特异表达的蛋白 1 gi|45476581 Sterol carrier protein 2 (SCP2) 59715/63920 8.00/8.06 32 MGFPEAASSFR2 gi|6754156 Hydroxyacid oxidase 1, liver 41260/42467 7.60/7.92 63 NVADIDLSTSVLGQR 3 gi|477004 Epoxide hydrolase63117/623995.85/6.23 72 YQIPALAQAGFR4 gi|74200069 Calregulin 42341/64342 4.57/6.21 70IKDPDAAKPEDWDERILFIFIDSDHTDNQR5 gi|127526 Major urinary protein 1 precursor 20920/205875.02/4.52 486 gi|18044669 Urate oxidase 35245/35482 8.48/8.32 32 AHVYVEEVPWKR7 gi|6678085 Serine proteinase inhibitor 46140/51913 5.24/5.30 238 gi|23396871 Small ubiquitin-related modifier3 12593/53764 5.25/5.39 35 9 gi|18044557 Peroxiredoxin 4 31261/29939 6.57/6.28 48 10 gi|13879360 Cathepsin B precursor 38168/26073 5.57/5.27 27 SGVYKHEAGDMMGGHAIR 11 gi|47682713 Nudix-type motif 7 27068/31324 5.90/6.16 16 FGLGPEPPR 12 gi|30354044 Insulin-like growth factor 2 63681/23537 9.31/5.84 1013 gi|50299 Cathepsin D44742/31129 6.71/5.92 23NYELHPDKYILKNIFSFYLNR 14gi|2624496Glutathione S-transferase P 123521/24501 8.13/7.76 36PPYTIVYFPVRFEDGDLTLYQSNAILR15 gi|226471 Superoxide dismutase [Cu-Zn]15923/17653 9.03/6.31 43 16 gi|19353248Hydroxymethylgulataryl-CoAsynthase57300/49579 8.65/7.93 30TKLPWDAVGR17 gi|33859506 Albumin 1 70730/75458 5.75/5.75-6.02 37 LGEYGFQNAILVR 18 gi|56789381 Acidic ribosomal phosphoprotein P034366/36764 5.91/5.87 65 19 gi|13384700Endoplasmic reticulum proteinERp19 19208/19577 5.14/5.10 4720 gi|193446Vitamin D-binding protein54647/56333 5.62/5.17 35SLSLILYSRRTQVPEVFLSK肾脏组织线粒体特异表达的蛋白 21 gi|32363497 Cytovillin 69347/70826 6.28/6.31 35 22 gi|2827776 Endopeptidase-2 85227/84967 6.00/5.24-5.76 23 VGVYDKDCDCFR23 gi|10946938 PDZK156863/69884 5.00/4.98 64 SVEALDHDGVVEMIRLFAYPDTHRNFTDVHPDYGAR24 gi|5305216Glutathione peroxidase22553/246806.54/5.98 2325 gi|2494732 Gamma-glutamyltranspeptidase 1 61866/24708 7.00/6.81 15ASGGWAAASDSRDIDQVVTAGLK26 gi|6755963 Voltage-dependent anion channel 1 30891/34360 9.00/7.96 48 LVAAKCTLAAR 27gi|2058343V-ATPase B2 subunit 56948/58414 6.00/5.38 29 28 gi|13626101 Alpha-methylacyl-CoA racemase 39854/40560 7.00/7.61 3629 gi|220396 D-amino-acid oxidase38970/39636 7.00/7.42 41 SYLPWLTER30 gi|125957 Lamin B267103/68540 7.32/7.60 42 31gi|20336740Ret finger protein-like 4 33079/32203 8.00/7.78 20 32 gi|3114602Phospholipid hydroperoxideglutathione peroxidase22794/19826 8.74/8.28 28 33 gi|15617203 Chloride intracellular channel 1 27338/305375.09/4.97 48 34 gi|14874758 Peroxiredoxin 2 21936/22347 5.20/4.62 32 心脏组织线粒体特异表达的蛋白35 gi|38259206Creatine kinase, mitochondrial 2(sMtCK)47899/45112 8.64/8.25 59 ITHGQFDER 36 gi|18700024 Isocitrate dehydrogenase 3 42453/41763 8.76/7.33 46 HNNLDLVIIR 37gi|33563264Myosin, light polypeptide 323/26330 5.03/5.0950ITYGQCGDVLR ALGQNPTQAEVLR 38 gi|38605043Myosin regulatory light chain 2,ventricular/cardiac muscle isoform18870/198934.71/4.76 81DGFIDKNDLR王媛等: 小鼠不同组织的差异线粒体蛋白质组研究826续表1Spot NO.Swiss-prot 号码鉴定蛋白名称分子量 Cal./Obs.等电点 Cal./Obs.覆盖率 (%)MS/MS39 gi|3806019 Ubiquinone biosynthesis proteincoq720228/198935.52/6.04 27 40 gi|160151263 Dihydrolipoyllysine-residue succinyltransferase component of2-oxoglutarate dehydrogenasecomplex49306/422895.70/5.96 22肝脏和肾脏线粒体共表达而心脏不表达的蛋白 41 gi|15488606 catalase 60013/609137.72/7.30-8.2716 LFAYPDTHR42 gi|74211686 Protein disulfide-isomeraseprecursor 56965/576884.79/4.29 58VDATEESDLAQQYGVRILFIFIDSDHTDNQR 43 gi|118542 Glutamate dehydrogenase 1 61640/56356 8.05/7.61 52DDGSWEVIEGYRHGGTIPVVPTAEFQDR 44 gi|31981722 Heat shock 70kD protein 5 72492/72954 5.09/4.89 50IINEPTAAAIAYGLDKRVTHAVVTVPAYFNDAQR45 gi|61402231 Glutathione S-transferase, mu 126067/26486 7.71/7.81 72 46gi|54673814Enolase 1, alpha non-neuron 47453/51686 6.37/6.73 6047 gi|29839593Thioredoxin domain containingprotein 547070/49147 5.51/5.31 33 GYPTLLLFR GYPTLLWFR48 gi|133284 Testosterone-regulated RP2 protein 40799/42556 6.22/6.57 16 FGLGPEPPR49 gi|63663965 Helix-destabilizing protein41673/41724 5.00/6.31 26 50 gi|19353187 Otc protein 39511/39489 8.81/8.40 49 KPEEVDDEVFYSPR51 gi|6677739 Regucalcin 33899/32757 5.15/4.94 40 52 gi|18043606Gpd1 protein38176/352626.75/7.22 4053 gi|22122515 AHA1 38321/35408 5.41/5.81 26 54 gi|13542901 Thiosulfate sulfurtransferase 33673/35629 7.71/8.32 48 FQLVDSR VLDASWYSPGTR 55gi|15029812Carbonic anhydrase 329633/307656.89/7.65 67 GGPLSGPYR VVFDDTYDR56 gi|21312020Noyl Coenzyme A hydratase domaincontaining 226449/30281 7.68/7.41 3057 gi|25452978 Hydroxyacylglutathione hydrolase 29168/31547 9.00/8.12 27 58gi|73622214Putative L-aspartate dehydrogenase30479/32619 6.45/7.01 55 GLCPLAPR WGHTVFVAR59 gi|6671549 Peroxiredoxin 6 24925/26939 5.98/6.55 30 60 gi|6754976Peroxiredoxin 122390/25456 8.26/8.31 48 IGYPAPNFK QITINDLPVGR61 gi|62511205Vitamin K epoxide reductase complex subunit 1-like protein 1 20051/20582 8.00/8.58 18 62 gi|31830253-hydroxyacyl-CoA dehydrogenasetype II27516/29582 8.53/9.19 3263 gi|30580818AMP phosphotransferasemitochondrial AK3 21237/22895 8.57/9.02 53 TVGIDDLTGEPLIQR 64gi|14602583Glutathione transferase, alpha 325401/268018.76/9.24 21 NRYFPAFEK SHGQDYLVGNR65 gi|71051228 Peptidyl-prolyl cis-trans isomerase A 18131/17803 7.74/7.76 40 SIYGEKFEDENFILK 66 gi|6679261 Pyruvate dehydrogenase E1 alpha 1 43888/425168.00/7.41 27 67 gi|22122625 3-hydroxyisobutyryl-CoenzymeAhydrolase43295/403429.00/8.79 43 QNLTQDLFR 68 gi|27527044 Akr7a5 protein 38109/38875 6.00/6.81 31 FFGNNWAETYR69 gi|13124286 Hydroxyacid oxidase 3 39145/39156 9.00/8.16 20 SVAEISPDLIQFSR70 gi|1339938 Glycerol-3-phosphate dehydrogenase 38176/353287.00/7.02 23 LISEVIGER71 gi|15488794 Peroxisomal trans-2-enoyl-CoAreductase32675/357308.00/8.57 20 72 gi|82952043 Fumarylacetoacetate hydrolasedomain containing protein 125484/302705.00/6.72 22 73 gi|9903607 Transmembrane protein 4 21096/18375 4.95/4.95 62 74 gi|30525896 3-ketoacyl-CoA thiolase B 8.82/7.97 35中国科学: 生命科学 2010年第40卷第9期827续表1Spot NO. Swiss-prot 号码鉴定蛋白名称分子量Cal./Obs. 等电点 Cal./Obs.覆盖率 (%)MS/MS肝脏和心脏线粒体共表达而肾脏不表达的蛋白 75 gi|17933768 Glyoxylate reductase 35706/371807.57/7.71 2976 gi|15488707Acetyl-Coenzyme A dehydrogenase,medium chain46908/43000 8.60/8.04 35IYQIYEGTAQIQR77 gi|54887356 Trifunctional protein, alpha subunit 83302/80884 9.24/9.44 30 LPAKPEVSSDEDVQYR78 gi|6688685 2-hydroxyphytanoyl-CoA lyase 64570/63641 5.89/6.39 45TPEELQHSLRGVVPDNHPNCVGAAR 79 gi|51316075Trifunctional enzyme beta subunit (HADHB)51639/508649.43/9.56 51NIVVVEGVR肾脏和心脏线粒体共表达而肝脏不表达的蛋白 80gi|18266680 3-oxoacid CoA transferase 1 (SCOT)56352/581228.73/7.62 42LIKGEKYEKRSKQIKR81 gi|10129957Peroxiredoxin 5 17175/16255 7.71/8.37 61 GGAKTLMNTIMQLR 82 gi|31542559 DihydrolipoamideS-acetyltransferase59389/581505.71/5.83 36 VPEANSSWMDTVIR 83 gi|29612662 AU RNA-binding enoyl-coenzyme Ahydratase33602/337649.49/9.25 1484 Q9CQ62 2, 4-dienoyl-CoA reductase 36476/330769.00/9.06 1385 gi|20455479 Atp5b protein 56632/29416 5.00/6.14 24 AHGGYSVFAGVGER 86 gi|29436756 Nuclear mitotic apparatus protein 1 236599/29753 5.68/5.83 10 87 gi|6753530 Crystallin, alpha B 20056/227087.71/7.36 48 QDEHGFISR的MALDI-TOF/TOF 质谱图, 鉴定蛋白质为PDZK1.通过gene ontology(GO)分析, 将鉴定到的87种蛋白按照功能分为5类, 分别是: 能量代谢相关蛋白24个, 细胞凋亡相关蛋白8个, 离子稳态维持相关蛋白17个, 氧化磷酸化相关蛋白28个, 另有其他功能蛋白10个. 通过GO 分析, 发现鉴定的87种蛋白, 有52个定位在线粒体, 7个定位在内膜系统(包括内质网、过氧化物酶体和微粒体), 另有28个蛋白并没有明确的亚细胞定位报道. 值得注意的是, 在这87种组织特异的线粒体蛋白中有12个曾被报道.2.3 ICPL 定量技术对组织特异性线粒体蛋白的验证为了验证胶图分析策略测定的差异蛋白定量结果的准确性, 采用稳定同位素标记的ICPL 技术定量分析不同组织线粒体内的一些蛋白质的相对含量. 根据ICPL 药盒的说明, 针对3种组织线粒体蛋白的样品, 采取了分别标记12C 和13C 并两两等量混合的方法, 即心脏/肾脏、心脏/肝脏和肾脏/肝脏. 同时, 将未标记的样品进行平行实验, 作为对照. 将同位素标记好的混合样品进行2-DE 分离, 得到了标记后的双向电泳图谱(图2(A)). 根据与未标记样品的2-DE 胶图比较, 可以看出标记后样品的蛋白点均向酸端偏移, 提示线粒体蛋白已被ICPL 标记. 利用MALDI- TOF 质谱进行鉴定得到质谱峰图后, 系统分析了质谱图上不同ICPL 试剂标记的一对肽段离子的强度比例, 定量分析它的母本蛋白质在原来样品中的相对丰度. 图2(B)是SCOT 在心脏和肾脏线粒体混合样品(H/K)中的MALDI-TOF 质谱图, 共检测到3组相对应的肽段, 其相对丰度为2.9︰1. 差异点的相对丰度总结如表2. ICPL 对catalase, SCOT 和HADHB 的定量分析结果与2-DE 测定的结果一致.2.4 Western blotting 对组织特异性线粒体蛋白的验证通过对小鼠肝脏、肾脏和心脏线粒体的差异蛋白质组学分析, 发现sMtCK, PDZK1和SCP2分别在心脏、肾脏和肝脏线粒体内特异表达; catalase 在肝脏、肾脏内表达, 而心脏中不表达; SCOT 在肾脏和心脏中表达, 而肝脏中不表达; HADHB 在肝脏和心脏中表达, 而肾脏中不表达(表1). 为了验证该结果的可靠性, 用各自特异的抗体, Western blotting 方法, 检测sMtCK, PDZK1, SCP2, catalase, SCOT 和HADHB 在3个组织线粒体内的表达, 结果与蛋白质组学结论一致(图3).2.5 组织特异性线粒体蛋白基因表达丰度的比较大部分线粒体蛋白都是由核基因编码的, 它们在线粒体外合成和包装后, 通过一定的蛋白质转运系统进入线粒体内. 那么这些线粒体差异蛋白在整王媛等: 小鼠不同组织的差异线粒体蛋白质组研究828个组织中是否仍具有其组织特异性? 选取6种线粒体差异蛋白sMtCK, PDZK1, SCP2, catalase, SCOT 和HADHB, 利用实时定量PCR 检测它们在肝脏、肾脏、心脏组织中的mRNA 丰度. 如图4所示, sMtCK, SCP2, catalase, SCOT 在3个组织的mRNA 差异与其蛋白质在线粒体内的组织特异性一致. 然而PDZK1和HADHB 的mRNA 在3个组织中均有表达, 该结果促使进一步分析这些蛋白在3个组织的表达是否具有特异性.用Western blotting 方法, 检测了这6种组织特异的线粒体蛋白在胞浆和组织的表达差异. 如图3所示, sMtCK, catalase, SCP2和SCOT 在胞浆和组织中的表达显示了与线粒体内相同的组织特异性. 而PDZK1和HADHB 在胞浆和线粒体中的分布状态则明显不同, 在3个组织中均检测到PDZK1的表达, 而仅在肾脏线粒体内显示其定位信号, 其他两个组织则定位于胞浆; HADHB 也在3个组织的胞浆内广泛存在, 但是它在肝脏和心脏的线粒体中能被检测到, 而图2 ICPL 对组织特异线粒体蛋白的定量分析(A) ICPL 标记小鼠心脏和肾脏线粒体混合样品的双向电泳图. 12C 和13C 分别标记心脏和肾脏线粒体蛋白; (B) SCOT 在ICPL 标记的小鼠心脏和肾脏线粒体混合样品中的肽指纹图谱. 969.535/981.572, 1578.794/1596.849和2091.955/2116.040为检测到的分别标记 12C 和13C 肽段[M+H]+位置, 其氨基酸序列和相对丰度在图中标出中国科学: 生命科学 2010年第40卷第9期829表2 ICPL 对组织特异线粒体蛋白的定量分析a)蛋白名称组织特异性混合胶名称 ICPL 标记后鉴定的肽段 12C/13C 成对肽段质荷比质荷比距离峰面积比率 catalase肝脏和肾脏线粒体共表达, 而心脏不表达的蛋白K/L, H/K, H/L L :KTFYTKVLNEEER DAILFPSFIHSQKR1633.822/1639.854 1763.956/1769.981 6 6 1.928 1.948 SCOT肾脏和心脏线粒体共表达, 而肝脏不表达的蛋白H/K, H/L, K/LK :H SKQIKR LIKGEKYEKRKEGDGKGKSGKPGGDVR969.535/981.572 1578.794/1596.849 2091.955/2116.04 12 18 24 0.392 0.331 0.324 K :H LSLLSKFRALAMGYKPKAYLR IPFLLSGTSYKDLMPHDLAR1068.672/1074.692 1691.889/1703.888 2379.223/2385.2366 12 60.115 0.177 0.143HADHB肝脏和心脏线粒体共表达, 而肾脏不表达的蛋白H/K, K/LK :L LSLLSKFR1068.621/1074.642 6 0.089a) L, K, H 分别代表肝脏、肾脏和心脏线粒体; K/L, H/K, H/L 分别代表两个组织线粒体蛋白标记12C/13C 后的混合胶图3 Western blotting 对组织特异线粒体蛋白在小鼠肝脏、肾脏和心脏线粒体、胞浆、组织内的检测L, K, H 分别代表肝脏、肾脏、心脏; M, C, T 分别代表线粒体、胞浆、组织; ATP sythase β为线粒体蛋白的内标在肾脏线粒体中几乎无法检测. 这些结果提示,PDZK1和HADHB 可能为了适应不同组织生理功能的需要, 显示了不同的亚细胞定位.2.6 组织特异性线粒体蛋白的细胞内检测选用了人类正常肝脏细胞Chang 、小鼠正常肾小球细胞MC 和大鼠胚心细胞H9c2, 检测组织特异性线粒体蛋白质在肝脏、肾脏和心脏的亚细胞定位. 一方面利用线粒体荧光探针MitoTracker Red 指示线粒体的细胞定位; 另一方面采用sMtCK, SCP2, PDZK1, catalase, SCOT 和HADHB 抗体显示这些蛋白质在细胞中的定位(图5). 由图5可见, sMtCK, PDZK1, SCP2分别定位在H9c2, MC 和Chang 细胞线粒体上; catalase 仅在Chang 和MC 细胞中显示出与线粒体的共定位, 而未出现在H9c2细胞中; SCOT 仅在H9c2和MC 细胞中显示出与线粒体的共定位, 而未出现在Chang 细胞中; HADHB 仅在Chang 和H9c2细胞中显示出与线粒体的共定位, 而未出现在MC 细胞中. 免疫荧光共定位证实了6种差异蛋白在特定细胞内的线粒体定位, 其结果与蛋白质组学、Western blotting 对差异蛋白的组织特异性分析结果一致. 进而说明组织特异性线粒体蛋白在细胞内的亚细胞定位与组织内定位相同.3 讨论线粒体蛋白质的组织特异性研究已在多个实验室中开展. 在本研究之前, Mann 领导的研究小组[13,14]对大鼠和小鼠不同组织来源的线粒体蛋白质组已经开展了研究. 他们主要通过LC-MS/MS 质谱技术,。

2009-plant root growth, architecture and function - Hodge

REVIEW ARTICLEPlant root growth,architecture and function Angela Hodge&Graziella Berta&Claude Doussan&Francisco Merchan&Martin CrespiReceived:3December2008/Accepted:6February2009/Published online:5March2009 #Springer Science+Business Media B.V.2009Abstract Without roots there would be no rhizo-sphere and no rhizodeposition to fuel microbial activity.Although micro-organisms may view roots merely as a source of carbon supply this belies the fascinating complexity and diversity of root systems that occurs despite their common function.Here,we examine the physiological and genetic determinants of root growth and the complex,yet varied and flexible,root architecture that results.The main functions of root systems are also explored including how roots cope with nutrient acquisition from the heterogeneous soil environment and their ability to form mutualistic associations with key soil micro-organisms(such as nitrogen fixing bacteria and mycorrhizal fungi)to aid them in their quest for nutrients.Finally,some key biotic and abiotic constraints on root development and function in the soil environment are examined and some of the adaptations roots have evolved to counter such stresses discussed.Keywords Root systems.Auxin.Root architecture. Soil heterogeneity.Abiotic and biotic stresses.Soil micro-organisms(including nitrogen-fixing bacteria and mycorrhizal fungi)Physiological and genetic determinants of root growth and achitectureA major difference between plant and animal devel-opment is that positional information rather than cell lineage determines cell fate in plants(Singh and Bhalla2006).Post-embryonically,plant development is essentially driven by stem cells localized in apical regions of shoots and roots,and referred to as apical meristems.This particular characteristic allows plants,Plant Soil(2009)321:153–187DOI10.1007/s11104-009-9929-9Responsible Editor:Yves Dessaux.A.Hodge(*)Department of Biology,Area14,P.O.Box373,University of York,York YO105YW,UKe-mail:ah29@G.BertaDipartimento di Scienze dell’Ambiente e della Vita, Universitàdel Piemonte Orientale,via Bellini25/G,Alessandria15100,ItalyC.DoussanUMR1114EMMAH INRA/UAPV Domaine Saint-Paul, Site Agroparc,Avignon Cedex984914,FranceF.MerchanDepartamento de Microbiología y Parasitología, Facultad de Farmacia,Universidad de Sevilla,c/Profesor García González,Sevilla41012,EspañaM.CrespiInstitut des Sciences du Végétal(ISV),CNRS,1Avenue de la Terrasse,Gif-sur-Yvette91198,Francewhich are sessile organisms,to adapt their morphol-ogy and organ development to the encountered environmental conditions.The spatial configuration of the root system(number and length of lateral organs),so-called root architecture,vary greatly depending on the plant species,soil composition, and particularly on water and mineral nutrients availability(Malamy2005).Plants can optimize their root architecture by initiating lateral root primordia and influencing growth of primary or lateral roots. The root system results from the coordinated control of both genetic endogenous programs(regulating growth and organogenesis)and the action of abiotic and biotic environmental stimuli(Malamy2005).The interactions between these extrinsic and intrinsic signals however complicate the dissection of specific transduction pathways.Such complex traits likely depending on multiple genes may be analyzed through quantitative genetics via the identification of quantitative trait loci(QTL)linked to root architecture (e.g.Mouchel et al.2004,Fitz Gerald et al.2006). Understanding the molecular mechanisms governing such developmental plasticity is therefore likely to be crucial for crop improvement in sustainable agricul-ture.In this section,we focus on hormonal and genetic determinants of root architecture.The embryonic root apical meristem(RAM) specification occurs very early in embryo develop-ment(Benfey and Scheres2000).The RAM con-stitutes the stem cell niche that eventually produces all below-ground organs,including lateral roots (Sabatini et al.2003).The cellular organization of Arabidopsis RAMs is very regular,with the initials of the various tissue types surrounding a quiescent center(QC).An asymmetric division of the hypoph-ysis generates a large basal daughter and a small apical daughter,called the lens-shaped cell,which is the progenitor of the QC.During root initiation,the QC appears to act like an organizing center that can direct surrounding cells to produce a set of initials (Aida et al.2004,Jenik et al.2007).Different mechanisms specify the initials above the QC that give rise to the ground tissue and the stele(proximal initials)as well as the initials below the QC that produce the root cap(distal initials).This root cap and the underlying root apical meristem form the root zone called the distal root apex,where cells divide actively.At certain distance from the RAM,the anticlinal and asymmetric divisions of a group of pericycle cells are the initial event of the formation of a lateral root.Several consequent divisions result in the formation of a dome-shape primordium that grows through outer cell layers and a meristem is estab-lished.Upon activation of this meristem,the lateral root emerges from the parental root and continue to grow in the soil(Malamy1995and references therein).Phytohormonal regulation of the root system:Auxin as a major playerThe different stages of root development are con-trolled and regulated by various phytohormones with auxin playing a major role(reviewed by Leyser 2006).In roots,auxin is involved in lateral root formation,maintenance of apical dominance and adventitious root formation.All these developmental events require correct auxin transport and signaling.During embryonic RAM formation many auxin-related mutants,such as those involved in its biosynthesis yuc1yuc4yuc10yuc11quadruple mutant (Cheng et al.2007),or mutants affected in transport or signaling(monopteros,bodenlos,auxin transport inhibitor resistant1(tir1)and related tir1/afb1–3 quadruple mutant(Dharmasiri et al.2005,Jenik et al. 2007)are unable to form the embryonic RAM.The auxin flux coming from the apical region of the embryo into the hypophysis leads to TIR1(and related redundant AFBs)pathway activation and induction of auxin-response genes such as PIN genes (coding for auxin efflux carriers),whose products will increase auxin transport and accumulation to further differentiate the hypothesis(Benkova et al.2003).Auxin also plays a major role in lateral root initiation and teral root development can be divided in different steps:primordium initia-tion and development,emergence,and meristem activation.Auxin local accumulation in Arabidopsis root pericycle cells adjacent to xylem vessels,triggers lateral root initiation by re-specifying these cells into lateral root founder cells(Dubrovsky et al.2008). Furthermore,it is also involved in the growth and organization of lateral root primordia and emergence from the parent root(Laskowski et al.2006).Indeed, mutants or transgenic lines with elevated auxin biosynthesis and endogenous levels of IAA display significant increased root branching(Seo et al.1998, King et al.1995).In addition,overexpression of theDFL1/GH3-6or the IAMT1genes,which encode enzymes modulating free IAA levels,results in a reduction in lateral root formation(Staswick et al. 2005;Qin et al.2005).Hence,normal auxin biosyn-thesis and homeostasis are necessary for lateral root initiation in Arabidopsis.However,overall auxin content is not always positively correlated with lateral root number(Ivanchenko et al.2006).Auxin transport into the regions where lateral root initiate also seems crucial for the regulation of root branching(Casimiro et al.2001).Functional analyses of mutants impaired in auxin transport,such as the aux1,axr4,and pin multiple mutants(Benkova et al. 2003;Marchant et al.2002),have demonstrated the crucial role of auxin transport in lateral root forma-tion.In the root,auxin is transported in both an acropetal(base-to-apex;Ljung et al.2005)and basipetal(apex-to-base)direction within inner and outer tissues of the root apex(Swarup and Bennett 2003).Studies using auxin transport inhibitors have shown that the polarity of auxin movement provides an important developmental signal during lateral root initiation(Casimiro et al.2001)and root gravitropism (Parry et al.2001).The strong bias in the direction of auxin transport within a tissue results from the asymmetrical cellular localization of an influx system involving the AUX1protein(Swarup and Bennett 2003)and an efflux apparatus with PIN-type efflux facilitators(reviewed by Paponov et al.2005).The auxin efflux system regulated by PIN protein family members is also crucial for lateral root development(Benkova et al.2003).Although not all pin mutations affect lateral root formation,multiple pin mutants have dramatic defects in root patterning, including lateral root development(Benkova et al. 2003;Blilou et al.2005).Cellular localisation of PIN proteins is also important as a gnom mutant allele is defective in PIN-dependent developmental processes, including lateral root primordium development,pre-sumably due to disorganized PIN1localization (Geldner et al.2004).When auxin reaches the target tissue it induces a transcriptional response.Several auxin-responsive genes and gene families involved in auxin signalling have been identified,of which the Aux/IAAs and ARFs are the best studied.Auxin induces expression of Aux/IAA proteins,which in many cases reduces the sensitivity of cells toward auxin.This induction is mediated by ARF proteins that bind to the auxin-responsive elements(AuxREs)in the promoters of auxin-responsive genes,and activate or repress transcription through interaction with specific Aux/ IAA proteins(Liscum and Reed2002).These loops represent extensive feedback as well as feed-forward regulation(for further information on Aux/IAA pro-teins,their degradation and their interaction with ARF transcription factors see the review by Benjamins and Scheres2008).Alterations in these control systems affect several auxin-dependent plant pathways includ-ing root development.For example,normal differen-tiation of a root cap from the distal initials depends on a pair of redundantly acting auxin response factors, ARF10and ARF16(Wang et al.2005).In double mutants,the root cap initials overproliferate,thereby generating a mass of undifferentiated progeny. Another example are the gain-of-function solitary root(slr)mutants which carries a stabilizing mutation in the IAA14negative regulator of auxin signaling and cannot form lateral root primordia.Detailed analyses of the slr mutant demonstrated that the slr mutation blocks early cell divisions during initiation (Fukaki et al.2002)Other hormones involved in the control of root developmentAlthough auxin plays a fundamental role in root growth and development,several other phytorho-mones modulate auxin action and consequently affect root development and architecture.In contrast to auxin,exogenous cytokinin application suppresses lateral root formation,and transgenic Arabidopsis plants with decreased cytokinin levels display in-creased root branching and enhanced primary root growth(Werner et al.2003).The notion that cytokinin negatively regulates root growth has also been verified by studies of cytokinin perception and signalling.For instance,double mutants for the redundant Arabidopsis cytokinin receptors AHK2 and AHK3display a faster growing primary root and greatly increased root branching(Riefler et al. 2006).To identify the stage of lateral root develop-ment sensitive to cytokinin,Laplaze et al.(2007) performed transactivation experiments that revealed that xylem-pole pericycle cells,but not lateral root primordial,are sensitive to cytokinin.Cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of the auxingradient required to pattern the primordia.Recently cytokinin have also been shown to induce differenti-ation of cells situated between the root meristem and the elongation zone(Dello Ioio et al.2007),providing new insights on the molecular mechanisms involved in root meristem maintenance.The actively growing primary root of dicotyledon plants may exhibit apical dominance forcing their lateral roots to develop further away from the root tip (Lloret and Casero2002).Root apical dominance may be triggered by the root-cap-synthesized cytoki-nin and the balance between auxin and cytokinin regulates root gravitropism,a major determinant of root architecture(Aloni et al.2006).The control of lateral root initiation depends on the primary root vascular system which conveys spatial information as well as photosynthetic carbon to the newly formed primordial(Parizot et al.2008).Three types of vascular differentiation can be distinguished:(1) primary differentiation,which occurs in cells origi-nating from the primary vascular meristem,the procambium;(2)secondary differentiation,where the derivates originate from the vascular cambium; and(3)regenerative differentiation,in which the vascular elements re-differentiate from parenchyma cells at lateral root junctions.The vascular tissues are induced by polar auxin movement along the plant body,from the hydathodes of young leaves downward to the root tips.Cytokinin by itself does not induce vascular tissues,but in the presence of auxin,it promotes vascular differentiation and regeneration,a critical process for the establishment of a root system (Aloni et al.2006).Another hormone involved in lateral root for-mation is ethylene.Treatments that induce C2H4 production(i.e.flooding)promote adventitious and lateral root formation(Mergemann and Sauter2000). Ethylene stimulates auxin biosynthesis and basipetal auxin transport toward the elongation zone,where it activates a local auxin response leading to inhibition of cell elongation.Stepanova et al.(2005)showed that ethylene-triggered inhibition of root growth is mediated by the action of the weak ethylene insensitive2/anthranilate synthaseα1(WEI2/ASA1) and wei7/anthranilate synthaseß1(ASB1)genes that encode rate-limiting tryptophan biosynthesis enzymes In addition,ethylene modulates the tran-scription of several components of the auxin trans-port machinery and induces a local activation of the auxin signaling pathway to regulate root growth (Ruzicka et al.2007).Abscisic acid(ABA),a universal stress hor-mone,plays a significant role in stimulating elongation of the main root and emergence of lateral roots in response to drought(De Smet et al. 2006b).As with other hormones,the complexity of the plant responses induced by this hormone makes it difficult to distinguish primary from secondary effects(e.g.stomata closure affects water movement in the plant).In general,ABA has an antagonistic effect on lateral root primordial formation and emergence to auxin,which initiates lateral roots (Malamy2005).This role of ABA in the control of the activity of the lateral root meristems has a significant impact on the final size and architecture of the root system(De Smet et al.2006b).Indeed, responsiveness to certain abiotic factors is lost in certain ABA signaling mutants(Signora et al.2001; He et al.2005;Fujii et al.2007)such as the systemic inhibition of lateral root formation in high N conditions in the ABA-insensitive mutant abi4 (Signora et al.2001).Genetic and genomic approaches to analyze root growth and architectureForward and reverse genetic approaches have been undertaken to elucidate the genetic control of lateral root formation.As expected,most of the lateral root mutants(approximately40%)identified in A.thaliana are affected in a specific component of the auxin pathways.Their phenotypes can usually be rescued or mimicked through auxin application.Several mutants having lateral root phenotypes(De Smet et al.2006a) have been linked to auxin signaling(axr mutants,tir1, msg2-1,shy2),auxin transport(aux1and pin mutants),and auxin homeostasis(alf1,ydk1,dfl1). Analysis of these mutants has led to the identification of several genes that regulate lateral root formation and/or coordinate this process in response to environ-mental cues(reviewed in Osmont et al.2007).Few Arabidopsis mutants such as the previously men-tioned slr-1/AUXIAA14(Fukaki et al.2002)and alf4-1(Celenza et al.1995),are not capable of initiating any lateral roots.The encoded ALF4protein is conserved among plants and has no similarities to proteins from other kingdoms.The Arabidopsis gene is involved in lateral root initiation but not in primaryroot formation as the alf4-1mutant forms a primary root.DiDonato et al.(2004)have shown that ALF4 functions are required to maintain the pericycle in a mitosis-competent state needed for lateral root forma-tion.Other mutants are affected in vascular tissues and consequently cannot produce lateral roots(e.g. Ohashi-Ito and Bergmann2007).In contrast,ectopic expression of PLT genes, coding for AP2-type transcription factors,trigger de novo formation of roots from shoot structures.PLT1 was isolated by reverse genetics technologies based on the identification of a QC–and stem-cell–specific enhancer trap line.Indeed,PLT genes are pivotal determinants of QC and stem cell identity(Aida et al.2004).Although plt1mutants only display mild root growth defects,the establishment and mainte-nance of the QC and stem cells in the root meristem, are severely impaired in the plt1plt2double mutant. NAC1is another transcription factor implicated in lateral root development since its overexpression enhances lateral root initiation(Xie et al.2002).A genetic framework for the control of cell division and differentiation in root meristematic cells have been recently proposed(Dello Ioio et al.2008).A second strategy used to identify non-essential modulators of root development,is exploiting natural genetic variation by analysis of quantitative trait loci(QTL).QTL mapping has the advantage of identifying genomic regions containing genes with subtle effects masked in a particular genetic background.For instance,in A.thaliana and maize QTLs linked to root architecture have been identified (Mouchel et al.2004;Tuberosa et al.2002;Loudet et al.2005).Exploring the polymorphisms underlying natural variation can identify the DNA sequence changes that lead to a modified root system architec-ture in nature.In recent years,several new genomics resources and tools(e.g.genome sequences,tens of thousands of molecular markers,microarrays,and knock-out collections)have become available to assist in QTL mapping and cloning,even if these genes have subtle effects on phenotype(see Paran and Zamir2003).For example,quantitative morpholog-ical and physiological variation analysis of a cross between isogenized Arabidopsis accessions revealed that the BREVIS RADIX(BRX)transcription factor controls the extent of cell proliferation and elonga-tion in the growth zone of the root tip(Mouchel et al. 2004).Genomics have provided new tools to explore downstream and upstream genes of broad regulatory signals such as hormone-responsive factors.Tran-scriptional changes during root branching in Arabi-dopsis have been monitored to identify key regulators of root architecture(Himanen et al.2004),including several core cell cycle genes.In addition,Vanneste et al.(2005)compared the early transcriptome of lateral root formation using synchronised induction of these organs in wild type and slr mutants.This revealed negative and positive feedback mechanisms that regulate auxin homeostasis and signalling in the pericyle initial cells.The advent of genomic resources have allowed the combination of global profiling with novel biological approaches such as enhancer trap lines driving green fluorescent protein(GFP)expression in specific root cell types(Brady et al.2007).These authors used transgenic lines that expressed GFP in specific root tissues to isolate corresponding fluores-cent protoplast populations derived from them and characterise their expression patterns using micro-arrays.Thus were able to determine which genes were expressed and at what level in a particular root cell type.Recently,these transgenic plants were used to characterise the transcriptional response to high salinity of different cell layers and developmental stages of the Arabidopsis root(Dinneny et al.2008). Transcriptional responses are highly constrained by developmental parameters further revealing interac-tions between developmental fates and environmental stresses.Genomic approaches mainly rely on the expression patterns of protein-coding genes.However,the dis-covery of small RNAs(microRNAs,miRNA;and small interfering RNAs,siRNA)in the last decade altered the paradigm that protein coding genes are the only significant components of gene regulatory net-works(Chapman and Carrington2007).Small RNAs are involved in a variety of phenomena that are essential for genome stability,establishment or main-tenance of organ identity,adaptive responses to biotic and abiotic stresses.In plants,MIRNAs are genes generally encoded in intergenic regions whose matu-ration requires a particular type III RNase named DICER-LIKE1(DCL1)(Chapman and Carrington 2007).The small mature miRNA is then incorporated in a protein complex,the so-called RISC(RNA-Induced Silencing Complex)that can recognizemRNAs partially complementary to the miRNA nucleotide sequence.This recognition event mediated by the RISC-loaded miRNA leads to cleavage or translational inhibition of the target mRNA.Mutants affected in miRNA metabolism(biosynthesis,action and transport as dcl1,ago1,hen1,hyl1,hst1,se)show pleiotropic phenotypes confirming the role of miR-NAs in diverse developmental processes(Chapman and Carrington2007).Several MIRNAs have been involved in root development.For example,Arabidopsis mir164a and mir164b mutant plants produced more lateral roots than wild-type plants(Guo et al.2005).These phenotypes are similar to those obtained by NAC1 overexpression,a transcription factor targetted by miR164(Xie et al.2002).Overexpression of miRNA-resistant NAC1mRNA results in slight increases in lateral root numbers.In addition,the auxin-related transcription factors ARF10and ARF16 are also targets of three related microRNA(miRNA) genes,miR160a,miR160b and miR160c.These miR-NAs limit the expression domain of ARF10and ARF16 to the columella,and have a complementary pattern. The over-expression of a miRNA-resistant ARF16 mRNA under its own promoter results in pleiotropic developmental defects.MIR160overexpressing plants, in which the expression of ARF10and ARF16is repressed,and the arf10-2arf16-2double mutants display the same root tip defect,with uncontrolled cell division and blocked cell differentiation in the root distal region showing a tumor-like root apex and loss of gravity-sensing(Wang et al.2005).Apart from these examples,due to the large diversity of these novel regulatory RNAs,it is likely that many other miRNAs or other small RNAs participate in the regulation of root development and architecture.Root meristematic cells integrate signals from the environment to regulate specific developmental responses and cope with external constraints.This post-embryonic growth and development requires the activation of hormone homeostasis and signalling pathways,transcriptional regulation by specific tran-scription factors and post-transcriptional regulation of developmental regulators by non-coding RNAs. These regulatory mechanisms may be particularly relevant to adjust differentiation processes to the environmental conditions encountered during growth, notably on primary and lateral root developmental programs.Root system achitectureRoot systems and architecture definitionsSoil is a complex medium with high spatial and temporal environmental variability at a wide range of scales,including those relevant to plant roots.The root system,with its extensive but structured devel-opment,can be considered as an evolutionary response to such spatio-temporal variability in re-source supply and associated constraints upon growth (Harper et al.1991).As a consequence,the extension in space and time of the root system is governed by genetically driven developmental rules which are modulated by environmental conditions.As demon-strated by QTL mapping which recently revealed that root morphology is in most cases regulated by a suite of small-effect loci that interact with the environment (de Dorlodot et al.2007).The architecture of a root system is the result of developmental processes and is a dynamic notion. Root architecture addresses two important concepts: the shape of the root system and its structure.The shape defines the location of roots in space and the way the root system occupies the soil.Its quantifica-tion is generally achieved by measuring variables such as root depth,lateral root expansion and root length densities.In contrast,root structure describes the variety of the components constituting the root system(roots and root segments)and their relation-ship(e.g.topology:connexion between roots;root gradients).While,root differentiation has important impacts upon structure—function relations(Clarkson 1996).The rhizosphere(i.e.the volume of soil around living plant roots that is influenced by root activity; Hinsinger et al.2005),is often simply thought of as a cylindrical shape around the root.However,this oversimplification does not account for integration at the root system level or for the inherent complexity of root systems which arise from geometry,temporal dynamics and the heterogeneous aspects of roots. These complexities are incorporated into the concept of root architecture.Root geometry is complex because of the specific motion in space of each root, the relative locations between roots and the possible overlapping of their zones of influence.The temporal dynamic comes both from the growth of the different root axes and from physiological processes associatedwith root segments (i.e.tissue differentiation)result-ing in temporal and spatial variability of function along the root axes.The diversity among roots within the root system and soil heterogeneity further increase this variability.Root classification and elaboration of the root system It has long been recognized that the in situ morphol-ogy of a root system can be complex and may vary greatly,even within a species (Weaver 1926;Cannon 1949;Kutschera 1960),reflecting the interplay be-tween developmental processes and environmental constraints (Fig.1).Consequently,the complexity of root systems has led to a number of different classification systems.These classifications can be based on:branch structure (topology)(Fitter 1987;2002),root activity (Wahid 2000)or development (Cannon 1949).The latter is the more classical approach and is useful in understanding growth as well as obtaining a more global view of root architecture,for example,in relation to plant habitat.Thus,developmental classification has been widely used in modelling approaches to simulate architecture (cf section below).From a developmental stand point roots are classified according to their ontogenesis into three main categories:primary,nodal and lateral roots(Cannon 1949;Harper et al.1991;Klepper 1992),the formation of which has been shown to be genetically separable (Hochholdinger et al.2004).This classification also reflects differences between monocot –and dicotyledon species:dicots root system is derived from primary roots and lateral branching (primary root system),with roots that may exhibit radial growth.Depending on the extension of laterals relative to the primary axis,the morphology of the root system will vary between taprooted and diffuse or fasciculate (Fig.1).In monocots,root systems derive not only from branching of primary roots but also from emmited nodal roots (adventitious root system).Monocot roots do not undergo secondary radial growth.The primary root differentiates from the seed ’s radicle already present in the seed embryo.This generally gives rise to a single-axis root system,or taproot system,with dominant vertical growth (gravitropism).Adventitious (or nodal)roots differ-entiate from organs other than roots (e.g.rhizomes,stems etc)and are initiated at precise locations (near stem nodes for example)with a defined temporal pattern,in coordination with the development of the shoot.They are often abundant and give rise to a fasciculated root system.Adventitious roots are much less sensitive to gravitropism than primary roots (Klepper 1992).Lateral roots originate from the branching of a parent axis,often at near rightangles,Fig.1Diversity of root systems.On the top row,root systems are little branched,while more profusely branched on the bottom row.Dominance of a single main axis increases from left to right (from Kutschera 1960)。

3 Transition-Edge Sensors

Transition-Edge SensorsK.D.Irwin and G.C.HiltonNational Institute of Standards and Technology,Boulder,CO80305-3328,USA irvin@Abstract.In recent years,superconducting transition-edge sensors(TES)have emerged as powerful,energy-resolving detectors of single photons from the near infrared through gamma rays and sensitive detectors of photonﬂuxes out to mil-limeter wavelengths.The TES is a thermal sensor that measures an energy de-position by the increase of resistance of a superconductingﬁlm biased within the superconducting-to-normal transition.Small arrays of TES sensors have been demonstrated,and kilopixel arrays are under development.In this Chapter,we de-scribe the theory of the superconducting phase transition,derive the TES calorime-ter response and noise theory,discuss the state of understanding of excess noise, and describe practical implementation issues including materials choice,pixel de-sign,array fabrication,and cryogenic SQUID multiplexing.1IntroductionIn1911,Kammerlingh Onnes cooled a sample of mercury in liquid he-lium,and made the dramatic discovery that its electrical resistance drops abruptly to zero as it cools through its superconducting transition temper-ature,T c=4.2K[1].A large number of materials have since been found to have phase transitions into a zero-resistance state at various transition tem-peratures.The superconducting phase transition can be extremely sharp, suggesting its use as a sensitive thermometer(Fig.1).In fact,the logarith-mic sensitivity(the Chapter“Thermal Equilibrium Calorimeters–An In-troduction”by McCammon in this book)of a superconducting transition,α=d log R/d log T,can be two orders of magnitude more sensitive than that of the semiconductor thermistor thermometer that has been used so success-fully in cryogenic calorimeters(the Chapter“Semiconductor Thermistors”also by McCammon).A superconducting transition-edge sensor(TES),also called a supercon-ducting phase-transition thermometer(SPT),consists of a superconducting ﬁlm operated in the narrow temperature region between the normal and su-perconducting state,where the electrical resistance varies between zero and its normal value.A TES thermometer can be used in a bolometer(to measure power)or in a calorimeter(to measure a pulse of energy).The sensitivity of a TES makes it possible in principle to develop thermal detectors with faster Chr.Enss(Ed.):Cryogenic Particle Detection,Topics Appl.Phys.99,63–152(2005)©Springer-Verlag Berlin Heidelberg200564K.Irwin and G.HiltonFig.1.The transition of a superconductingﬁlm(a Mo/Cu proximity bilayer)from the normal to the superconducting state near96mK.The sharp phase transition suggests its use as a sensitive thermometerresponse,larger heat capacity,and smaller detectable energy input than ther-mal detectors made using conventional semiconductor thermistors.However, the sharp transition leads to a greater tendency for instability and lower saturation energy,so that careful design is required.In1941,Andrews et al.applied a current to aﬁne tantalum wire operating in its superconducting transition region at3.2K and measured the change in resistance caused by an infrared signal[2].This was theﬁrst demonstra-tion of a TES bolometer.In1949,the same researcher applied a current to a niobium nitride strip within its superconducting transition at15K and measured the voltage pulses when it was bombarded by alpha particles[3]–theﬁrst reported demonstration of a TES calorimeter.This work followed on earlier suggestions by Andrews himself in1938[4]and Goetz in1939[5].During theﬁrst half century after their invention,TES detectors were seldom used in practical applications.One of the principal barriers to their adoption was the diﬃculty of matching their noise to FET ampliﬁers(the TES normal resistance is typically a few ohms or less).In order to noise match,the TES was sometimes read out using a cross-correlation circuit to cancel noise[6],ac biased in conjunction with a step-up transformer[7],or fabricated in long meander lines with high normal resistance[8,9].In recent years,this problem has been largely eliminated by the use of superconduct-ing quantum interference device(SQUID)current ampliﬁers[10],which are easily impedance-matched to low-resistance TES detectors[11,12].In addi-tion to their many other advantages,SQUID ampliﬁers make it possible to multiplex the readout of TES detectors(Sect.4.2),so that large arrays of detectors can be instrumented with a manageable number of wires to room rge arrays of TES detectors are now being deployed for a number of diﬀerent applications.Transition-Edge Sensors65 Another barrier to the practical use of TES detectors has been that it is diﬃcult to operate them within the extremely narrow superconducting tran-sition region.When they are current-biased,Joule heating of the TES by the current can lead to thermal runaway,and smallﬂuctuations in bath tem-perature signiﬁcantly degrade performance.Furthermore,variations in the transition temperature between multiple devices in an array of TES detec-tors can make it impossible to bias them all at the same bath temperature. As will be explained in Sect.2.5,when the TES is instead voltage-biased and read out with a current ampliﬁer,the devices can easily be stably biased and they self-regulate in temperature within the transition with much less sensitivity toﬂuctuations in the bath temperature[13].The introduction of voltage-biased operation with SQUID current readout has led to an explosive growth in the development of TES detectors in the past decade.The potential of TES detectors is now being realized.TES detectors are being developed for measurements across the electromagnetic spectrum from millimeter[14,15,16]through gamma rays[17,18]as well as with weakly interacting particles[19]and biomolecules[20,21,22].They have contributed to the study of dark matter and supersymmetry[23,24],the chemical compo-sition of materials[25],and the newﬁeld of quantum information[26].They have extended the usefulness of the single-photon calorimeter all the way to the near infrared[27],with possible extension to the far infrared.They are being used in theﬁrst multiplexed submillimeter,millimeter-wave,and X-ray detectors for spectroscopy and astronomical imaging[15,16,28,29,30].2Superconducting Transition-Edge Sensor TheoryWe now describe the theory of a superconducting transition-edge sensor.We describe the physics of the superconducting transition(Sect.2.1),summa-rize the equations for TES small-signal theory(Sect.2.2),and analyze the bias circuit for a TES and its electrical and thermal response(Sect.2.3),the conditions for the stability of a TES(Sect.2.4),the consequences of neg-ative electrothermal feedback(Sect.2.5),thermodynamic noise(Sect.2.6), unexplained noise(Sect.2.7),and the eﬀects of operation outside of the small-signal limit(Sect.2.8).Particular implementations of both TES single pixels and arrays,including performance results,will be described in Sect.3and Sect.4.2.1The Superconducting TransitionIn this work,we discuss sensors based on traditional“low-T c”superconduc-tors(often those with transition temperatures below1K).Other classes of su-perconductors,including the cuprates such as yttrium-barium-copper-oxide, are also used in thermal detectors.Transition-edge sensors based on these66K.Irwin and G.Hilton“high-T c ”materials have much lower sensitivity and much higher saturation levels than those that are discussed here.In low-T c materials,the phenomenon of superconductivity has been fairly well understood since the 1950s,when detailed microscopic and macroscopic theories were developed.Superconductivity in low-T c materials occurs when two electrons are bound together in “Cooper”pairs,acting as one particle.The energy binding Cooper pairs prevents them from scattering,allowing them to ﬂow without resistance.Bardeen,Cooper,and Schrieﬀer ﬁrst ex-plained the formation of Cooper pairs in 1957in the landmark microscopic BCS theory [31].The energy binding the two electrons in a Cooper pair is due to inter-actions with positive ions in the lattice mediated by phonons (quantized latticevibrations).When a negatively charged electron ﬂows in a supercon-ductor,positive ions in the lattice are drawn towards it,creating a cloud of positive charge.A second electron is attracted to this cloud.The energy binding the two electrons is referred to as the “superconducting energy gap”of the material.In the BCS theory,the size of the Cooper pair wave function is determined by the temperature-dependent coherence length ξ(T ),which has the zero-temperature value ξ0≡ξ(0)≈0.18v F /(k B T c ).Here v F is the Fermi velocity of the material,k B is the Boltzmann constant,and T c is the superconducting transition temperature.At temperatures above the transi-tion temperature,thermal energies of order k B T spontaneously break Cooper pairs and superconductivity vanishes.In a BCS superconductor,the transi-tion temperature T c is related to the superconducting energy gap E gap of the material by E gap =≈3.5k B T c .In addition to perfect dc conductivity below T c ,a second hallmark of superconductivity is the Meissner eﬀect:the free energy of the is minimized when an external magnetic ﬁeld is excluded from the interior of a superconducting sample.An applied mag-netic ﬁeld is exponentially screened by an induced Cooper-pair supercurrent with an eﬀective temperature-dependent penetration depth,λeﬀ(T ).The ap-proximate zero-temperature value of the penetration depth is the London penetration depth,λL (0).Near the transition temperature,the physics of a superconductor is well described by the macroscopic Ginzburg–Landau theory [32],which was de-rived by a Taylor expansion of a phenomenological order parameter Ψ.Ψwas later shown to be proportional to the density of superconducting pairs [33].One result of the Ginzburg–Landau theory is that the characteristics of a superconductor with penetrating magnetic ﬂux (such as a superconductor on its transition)are strongly dependent on its dimensionless Ginzburg–Landau parameter,κ≡λeﬀ(T )/ξ(T ).If κ<1/√2,the superconductor is of Type I,and the free energy is minimized when magnetic ﬂux that has penetrated the material clumps together.If κ>1/√2,the is of Type II,and magnetic ﬂux that has penetrated the material separates into individual ﬂux quanta that repel each other.The ﬂux quantum is 本页已使用福昕阅读器进行编辑。

发现新细胞器可用于治疗阿尔茨海默病

发现新细胞器可用于治疗阿尔茨海默病2022-06-19 01:04·日月明尊除了许多已知的细胞器（细胞的成分或“器官”），科学家们刚刚发现了另一种。

这些就是所谓的BAG2——在细胞质中响应某种压力而形成的无膜颗粒。

或许，BAG2 可以成为神经退行性疾病新疗法的基础。

荧光染料标记的应力颗粒除了几十甚至几百年前发现的细胞核、线粒体、网状细胞等，细胞中还有许多其他的细胞器。

通常，它们较小并执行特定的特定工作。

在《自然通讯》杂志最近的一篇文章中，来自美国和巴西的科学家描述了BAG2（Bcl2 相关的athanogene 2），这是一种新型细胞器，它没有膜，但通过内含物与细胞质很好地分离。

在这方面，BAG2 类似于所谓的应激颗粒和处理体（P-体），但新的细胞器既不包含RNA，也不包含专门的“死亡标记”泛素。

泛素残基通常附着在那些蛋白质上，然后细胞在蛋白酶体的帮助下有目的地破坏这些蛋白质- 执行“垃圾处理”工作的分子机器。

人们已经知道很长一段时间以来，有几种类型的无膜物体在细胞中来回浮动。

然而，直到最近，人们才知道它们如何保持完整性、它们是什么以及为什么需要它们。

现在，由于先进的分子成像技术，科学家们终于能够很好地观察这些动态细胞器。

这些非膜结构与通常的大型细胞器的区别在于缺乏脂质双层的包装，这也将细胞的内容物与其环境分开。

相反，像 BAG2 或 P 体这样的内含物是通过将两种流体（它们的内容物和细胞的基本环境）分离成相而存在的，就像水面上的一滴油一样。

科学家们发现，新发现的细胞成分在某些压力条件下（包括渗透压增加）会被激活（即，它们会变成浓缩形式）。

压力颗粒的工作方式大致相同，当它被激活时，会停止蛋白质合成并保留RNA。

然而，BAG2 负责处理那些已经合成的蛋白质。

事实是，在不利条件下，它们可以获得不正确的三维结构并损坏细胞。

几乎同样的事情也发生在神经退行性疾病身上。

BAG2 不仅破坏了有问题的蛋白质，而且还促进了伴侣的工作——其他帮助蛋白质保持正确结构的分子。

Impact of nitrogen fertilization and soil tillage on arbuscular mycorrhizal fungal communities

Impact of nitrogen fertilization and soil tillage on arbuscular mycorrhizal fungal communities in a Mediterranean agroecosystem Luciano Avio a,Maurizio Castaldini b,Arturo Fabiani b,Stefano Bedini c,Cristiana Sbrana a, Alessandra Turrini c,Manuela Giovannetti c,*a Istituto di Biologia e Biotecnologia Agraria CNR,AT Pisa,Via del Borghetto80,56124Pisa,Italyb Consiglio per la Ricerca e Sperimentazione in Agricoltura,Centro di Ricerca per l’Agrobiologia e la Pedologia CRA-ABP,Piazza Massimo D’Azeglio30, 50121Firenze,Italyc Dipartimento di Scienze Agrarie,Alimentari e Agro-Ambientali,Universitàdi Pisa,Via del Borghetto80,56124Pisa,Italya r t i c l e i n f oArticle history:Received14March2013Received in revised form5September2013Accepted9September2013 Available online20September2013Keywords:Arable systemsArbuscular mycorrhizal fungiN-fertilizationTillagePCR e DGGEFungal diversityTrap cultures a b s t r a c tThe impact of nitrogen(N)fertilization and tillage on arbuscular mycorrhizal fungi(AMF)was studied in a Mediterranean arable system by combining molecular,biochemical and morphological analyses ofﬁeld soil and of soil and roots from trap plants grown in microcosm.Canonical correspondence analysis(CCA) of PCR e DGGE banding patterns evidenced that AMF communities in theﬁeld are affected by N-fertil-ization and tillage.N-fertilization was also the main factor shaping AMF communities in Medicago sativa trap plant soil and roots.The overall sporulation pattern of the different AMF species showed a pre-dominant effect of tillage on AMF communities,as shown by CCA analysis.Funneliformis mosseae was the predominant species sporulating in tilled soils,while Glomus viscosum and Glomus intraradices prevailed in no-tilled soils.Field glomalin-related soil protein content was reduced by tillage practices.Our multimodal approach,providing data on two main production factors affecting soil AMF communities, may help implementing effective agricultural management strategies able to support the beneﬁcial relationship between crops and native AMF symbionts.Ó2013Published by Elsevier Ltd.1.IntroductionArbuscular mycorrhizal(AM)fungi(AMF)establish symbiotic associations with most crop plants and play a fundamental role in plant growth,soil fertility and productivity,delivering many essen-tial ecosystem services(Gianinazzi et al.,2010).AM fungal hyphae spread from host roots to the surrounding soil,developing an extensive mycelial network,crucial to the uptake of nutrients,mainly phosphorus(P),nitrogen(N),copper(Cu)and zinc(Zn)(Giovannetti and Avio,2002;Smith and Read,2008;Blanke et al.,2011).Many AM fungal isolates increase plant tolerance to root pathogens,pests and abiotic stresses,such as drought and salinity(Augé,2001;Evelin et al.,2009;Sikes et al.,2009)and increase the synthesis of beneﬁ-cial plant secondary metabolites,thus contributing to the production of safe and high quality food(Ceccarelli et al.,2010;Giovannetti et al., 2012).Moreover,AMF contribute to soil carbon(C)sequestration and organic matter conservation by means of the extensive mycelial network producing large quantities of a sticky proteinaceous hydrophobic substance,glomalin,that accumulates in soil as glomalin-related soil protein(GRSP)(Rillig and Mummey,2006; Bedini et al.,2009),and of other recalcitrant polymers,such as chitin and chitosan(Zhu and Miller,2003;Fortuna et al.,2012).Several studies have demonstrated that different crop man-agement systems involving high intensity of mechanization or high inputs of chemicals may affect AMF species composition or show a negative impact on AMF spore abundance and mycorrhizal colo-nization,often leading to a reduction of AMF beneﬁts to crop pro-duction and soil quality(Douds et al.,1995;Jansa et al.,2002,2003; Oehl et al.,2004;Castillo et al.,2006;Brito et al.,2012).Indeed, deep ploughing,by disrupting the hyphae of the mycorrhizal network(Kabir,2005),may differentially affect AMF taxa,which show differential activity and functioning(Klironomos,2003; Munkvold et al.,2004;Avio et al.,2006).On the other hand,soil chemical fertilization may affect AMF growth and colonization ability by altering the concentration of soil mineral nutrients and shifting the N:P ratio of plant tissues,which in turn may stimulate the growth of AMF populations more adapted to the new nutri-tional conditions(Johnson et al.,2003;Na Bhadalung et al.,2005; Toljander et al.,2008).*Corresponding author.Tel.:þ390502216643;fax:þ390502216641.E-mail address:mgiova@agr.unipi.it(M.Giovannetti).Contents lists available at ScienceDirectSoil Biology&Biochemistry journal h omepage:www.elsevier.co m/lo cate/soilbio0038-0717/$e see front matterÓ2013Published by Elsevier Ltd./10.1016/j.soilbio.2013.09.005Soil Biology&Biochemistry67(2013)285e294The data available on the impact of different levels of tillage and chemical fertilization on AMF community composition and dy-namics indicate that such major production factors should be tested in dedicated experimental arable systems,in order to reach a better understanding of the driving forces that shape AM fungal communities(Moebius-Clune et al.,2013a)and to implement effective agricultural management strategies supporting crop plant-beneﬁcial soil microorganisms.A comprehensive and exhaustive evaluation of changes in AMF community diversity produced by anthropogenic and environmental variables may be difﬁcult to accomplish utilising singular approaches.Actually, morphological analyses based on spores collected in theﬁeld may miss non sporulating species or those represented by old and parasitized spores,while root DNA analyses may reveal only the ampliﬁable DNA,representing a subset of AMF communities colo-nizing the sampled roots,which may differ from those detected in rhizosphere or bulk soil and from those described using morpho-logical analyses as well(Hempel et al.,2007;Cesaro et al.,2008; Mirás-Avalos et al.,2011).The aim of the present study was to evaluate the impact of N-fertilization and tillage on AMF abundance and diversity,focussing on a long-term experimental site in a Mediterranean arable system. To this aim,we combined molecular,biochemical and morpho-logical analyses to assess:i)AMF diversity inﬁeld soil,by means of polymerase chain reaction(PCR)e denaturating gradient gel elec-trophoresis(DGGE)analysis of18S rRNA gene fragments,a mo-lecularﬁngerprinting technique widely used to detect the modiﬁcations induced by different factors on soil microbes(Smalla et al.,2001;Castaldini et al.,2005;Oliveira et al.,2009);ii)AMF abundance and diversity,by means of morphological and molecular identiﬁcation of spores produced in trap plants grown in micro-cosm,a technique providing newly produced spores suitable for morphological identiﬁcation(Oehl et al.,2003,2004;Yao et al., 2010);iii)AMF diversity in soil and roots of trap plants,by means of PCR e DGGE analysis of18S rRNA gene fragments;iv)GRSP con-tent inﬁeld and trap cultures soil.2.Materials and methods2.1.Study site and soil samplingThe study was conducted at the“Pasquale Rosati”experimental farm near Agugliano,Italy(43 320N,13 220E,100m a.s.l.,slope10%). The soil is a calcaric gleyic cambisol almost free of gravel,with a high clay and calcium content.The climate is dry-summer subtropical (Mediterranean),with a mean annual rainfall in the period1998e 2008of786mm.The highest mean monthly temperature(30.6 C) and the lowest precipitation(35mm)occurred in July.The lowest mean monthly temperature(3.0 C)occurred in January and the highest precipitation(105mm)in September(De Sanctis et al.,2012). The experimental site belong to a long-term tillage experiment, established in1994,with a two year rotation of maize(Zea mays L.) and durum wheat(Triticum durum L.)since2002,and designed as a split plot with tillage treatments assigned to the main plots(each 1500m2in size)and N-fertilization treatments assigned to subplots (each500m2in size).The experiment was replicated in two blocks with treatments repeated in the same plots every year.In the present study,soil sampling was performed in the subplots treated with no N-fertilization(0)and90kg haÀ1N(90)as ammonium nitrate,both in the conventional tillage(CT)and in the no tillage(NT)treatment. CT treatment consisted of ploughing at a depth of40cm and double harrowing before sowing,whereas NT plots were left undisturbed except for sod seeding,crop residuals and weed chopping and total herbicide spraying prior to seeding.For data on crop yield and soil characterisation,see De Sanctis et al.(2012).After wheat harvest the experimental area was sampled in Autumn2006by randomly collecting four15cm deep soil cores from each of the eight subplots.The four soil cores were pooled to obtain samples of about2.0kg which were air-dried and stored at 4 C until processed.Two hundred grams of each sample were used for GRSP analysis and the remaining soil for establishment of trap cultures.Forﬁeld soil DNA analysis,soil samples(three replicates) were taken from one subplot of the four relevant treatments,for a total of twelve samples,then stored atÀ20 C until processed.2.2.Trap cultures and spore analysisEach soil sample was mixed,1:1by volume,with Terragreen (calcined attapulgite clay,Oil Dri,Chicago,IL),and poured into four 750cm3plastic pots,two for each of the two trap plant species utilized,Z.mays and Medicago sativa L.Plants were grown in glasshouse,under ambient natural light and temperature condi-tions and supplied with tap water as needed.In addition,they received weekly fertilization with half strength Hoagland’s solution (10mL per pot).After six months’growth,three soil samples(10g each)were collected from each pot and processed.AMF spores and sporocarps were extracted by wet-sieving and decanting,using a set of nested sieves,down to a mesh size of50m m(Gerdemann and Nicolson,1963),thenﬂushed into Petri dishes and examined under a dissecting microscope(Wild,Leica,Milano,Italy).The spores were separated into groups,according to their morphology.Spores were isolated by using capillary pipettes,mounted on microscope slides in polyvinyl alcohol lacto-glycerol(PVLG)and in PVLGþMelzer’s reagent(1:1,v:v)and examined under a Polyvar light microscope (Reichert-Young,Vienna,Austria).Qualitative spore traits(spore shape,colour and size,spore wall structure and shape,colour and size of the subtending hypha)were examined on at least50spores for each morphotype.Morphotype identiﬁcations were based on original descriptions and current species descriptions available online(International Culture Collection of(Vesicular)Arbuscular Mycorrhizal Fungi[/fungi/taxonomy/ speciesID.htm];Prof.Janusz Blaszkowski website at Szczecin University[.pl/Glomeromycota/]).Since important changes of AMF nomenclature have been recently pro-posed by different authors(Oehl et al.,2011;Krüger et al.,2012), with some taxa inconsistently named,we utilized the new bi-nomials for consistent names and maintained the previous ones for the others.After sixteen months’growth,three soil samples were collected from each pot and processed as described above,with the aim of retrieving also late sporulating AMF species(Oehl et al.,2009).The data reported are from such a sampling.2.3.Field soil DNA extractionDNA extraction was performed on500mg of eachﬁeld soil sample,with the FastDNAÒSpin Kit for Soil(MP Biomedicals,Solon, OH)according to manufacturer’s instructions,with minor modiﬁ-cations:a double homogenization in the FastPrepÒInstrument(MP Biomedicals)for30s at a speed setting of6.0and25s at a speed setting of6.5,and aﬁnal resuspension in100m L of TE buffer(10mM Tris e HCl,0.1mM EDTA pH8).The DNA was then puriﬁed with the DNA Clean Up Spin Kit(GENOMED GmbH,Löhne,Germany),ac-cording to manufacturer’s instructions.2.4.DNA extraction from roots and soil of trap culturesSoil and roots of Z.mays and M.sativa were collected from trap cultures six months after establishment.Three samples of roots (100mg)and soil(500mg)were utilized for each plant species andL.Avio et al./Soil Biology&Biochemistry67(2013)285e294 286treatment.Root DNA was extracted in liquid nitrogen using DNeasy Plant Mini Kit(QIAGEN GmbH,Hilden,Germany),according to the manufacturer’s protocol.Soil DNA was extracted as described above.2.5.DNA extraction from sporesIntact,healthy spores belonging to the following morphospecies were isolated from trap cultures six months after establishment and utilized for DNA extraction:a)Glomus viscosum T.H.Nicolson (pools of spores);b)Glomus intraradices N.C.Schenk&G.S.Sm. (pools of spores);c)Funneliformis mosseae(T.H.Nicolson&Gerd.)C. Walker&A.Schüssler(single spores and sporocarps).Spores and sporocarps were manually collected with a capillary pipette under the dissecting microscope and cleaned by sonication(120s)in a B-1210cleaner(Branson Ultrasonics,Soest,NL).After three rinses in sterile distilled water(SDW),spores and sporocarps were surface sterilized with2%Chloramine T supplemented with streptomycin (400m g mLÀ1)for20min and rinsedﬁve times in SDW.Spore clusters,spores and sporocarps were selected under the dissecting microscope and transferred in Eppendorf tubes before DNA extraction(Redecker et al.,1997).2.6.DNA ampliﬁcationAliquots of soil DNA(50ng)were used to amplify the V3e V4 region of18S rDNA using the universal eukaryotic NS31GC primer (Kowalchuk et al.,2002)and the AM1primer(Helgason et al.,1998) in a50m L PCR mix consisting of250m M each primer,250m M each dNTP,1.5mM MgCl2,1ÂBuffer(67mM Tris e HCl pH8.8;16.6mM (NH4)2SO4;0.01%Tween-20)and2.5U of Taq DNA Polymerase (Polymed,Firenze,Italy).The reaction was performed in a iCycler thermal cycler(Bio-Rad Laboratories Inc.,Hercules,CA)with a protocol consisting of an initial cycle of95 C for3min,followed by 35cycles of94 C for30s,62.3 C for45s and72 C for60s,and a ﬁnal extension step at72 C for7min.Each sample was ampliﬁed three times and the amplicons were pooled together before DGGE analysis.Root and fungal spore DNA ampliﬁcations were performed in the same conditions,except for the starting material(25ng),and for annealing time of spore samples(60s).2.7.Double gradient DGGE analysis of AMF communitiesThe analysis was performed with the INGENYphorUÒsystem (Ingeny International BV,Goes,The Netherlands)on a5e6%poly-acrylamide gel(acrylamide/bis37.5:1),under denaturation condi-tions(urea,7M;40%formamide with a denaturing gradient ranging from25to50%);the gels were run in1ÂTAE buffer at75V for17h at60 C and were stained with14mL of1ÂTAE containing 1.4m L of SYBRÒGold(Molecular Probes,Inc.,Eugene,OR)(dilution 1:10,000)for30min in the dark.Visualization and digital pictures were performed with a ChemiDoc System(Bio-Rad Laboratories). Using electrophoretic patterns,a matrix of the presence and absence of bands was obtained by GelCompar II 4.6software (Applied Maths NV,Sint-Martens-Latem,Belgium).2.8.Cloning and sequencing of18S rDNA fragmentsSelected PCR e DGGE bands pertaining to spores or roots samples were excised from the gel,resuspended in30m L of sterile TE and stored atÀ30 C.The DNAs extracted from the DGGE bands were re-ampliﬁed with the primers NS31GC and AM1and the PCR products were loaded onto a new DGGE gel to ensure the purity of each single band.The amplicons were then cloned into a pCRÒ4-TOPOÒvector using TOPO TA CloningÒkit for Sequencing(Invitrogen Corporation, Carlsbad,CA)and sequenced using the M13primer.Sequencing was carried out at the C.I.B.I.A.C.I.(University of Florence)using the ABI PRISMÒBigDyeÒTerminator v1.1Cycle Sequencing Kit(Applied Biosystems,Foster City,CA)according to the manufacturer’s recommendations.The parameters for cycle sequencing in the thermocycler Primus96plus(MWG Biotech, Ebersberg,D)were18s delay at96 C,followed by25cycles with 18s at96 C,5s at50 C and4min at60 C.Electrophoresis was performed on an ABI Prism310CE system(Applied Biosystems).2.9.Phylogenetic analysisSequences were entered in the BLASTn program of National Center for Biotechnology Information GenBank database(http:// /)to search for closely related sequences. Before phylogenetic analysis,sequences were screened with Chimera Check version2.7(Cole et al.,2003)(http://rdp.cme.msu. edu)and aligned with ClustalW program(Chenna et al.,2003), using Glomeromycota sequences available in GenBank.The phylogenetic tree was inferred by neighbour joining(NJ)method using Kimura2-parameter in TREECON for Windows software(Van de Peer and De Wachter,1994).The conﬁdence of branching was assessed using1000bootstrap resamplings.The sequences were deposited at EMBL Nucleotide Sequence Database(/ embl/)under the accession numbers HE806381e HE806417.2.10.GRSP analyses ofﬁeld and trap culture soilGRSP was extracted from soil using the procedures described by Wright and Upadhyaya(1996)for easily extractable(EE-GRSP)and total(T-GRSP)GRSP.EE-GRSP analyses were carried out onﬁeld soil and on six months’old trap cultures.Brieﬂy,EE-GRSP was extracted from1g of2mm-sieved soil with8mL of a20mM citrate solution, pH7.0,by autoclaving at121 C for30min.T-GRSP was extracted from1g of2mm-sieved soil samples,by repeated cycles with 50mM citrate,pH8.0,by autoclaving at121 C for60min.Ex-tractions of samples continued until the supernatant content of GRSP was under method detection limits(2mg mLÀ1).Superna-tants from each cycle were collected after centrifugation at10,000g for10min to pellet soil particles,pooled and stored at4 C until analysed.Protein content was determined by Bradford assay (Sigma e Aldrich,Inc.)with bovine serum albumin as the standard. Each determination was repeated three times.2.11.Statistical analysisData of spore counts and GRSP concentrations were analysed on IBM SPSS19.0software(SPSS Inc.,Chicago,IL).The GLM Univariate procedure was utilized to investigate the effects of tillage man-agement,fertilization levels,and host plants in trap cultures,as ﬁxed factors,and their interactions,with block as random factor. Canonical correspondence analysis(CCA)was performed by using PAST1.99software(Hammer et al.,2001),on the presence/absence matrix based on DGGE banding pattern and on spore numbers after logarithmic transformation.Permutation test(n¼1000)was per-formed by using PAST software.3.Results3.1.PCR e DGGE analyses of AM fungal diversity inﬁeld soil and trap culturesCCA revealed a signiﬁcant effect of N-fertilization on AMF communities of theﬁeld plots(P¼0.007).Theﬁrst canonical axisL.Avio et al./Soil Biology&Biochemistry67(2013)285e294287explained 86.2%of the cumulative variance of PCR e DGGE banding patterns data,and the second one explained the remaining 13.8%(Fig.1).CCA showed an additional effect of tillage on AMF com-munity diversity (Fig.1).PCR e DGGE pro ﬁles of AMF communities occurring in the soil of trap cultures from different treatments were always more dissim-ilar than pro ﬁles from the same treatment (Fig.2).CCA suggests a separation of AMF soil (0N)communities of M.sativa and Z.mays trap plants,though not statistically signi ﬁcant (P ¼0.172)(Fig.3).N-fertilization was the main factor affecting AMF communities occurring in M.sativa trap soil,as revealed by CCA of the relevant PCR e DGGE pro ﬁles,showing a clear-cut separation between pat-terns obtained from N-fertilized and unfertilized soil (P ¼0.009)(Fig.4A).A minor effect of tillage treatments was found (Fig.4A).The ﬁrst canonical axis explained 90.1%of the cumulative variance of PCR e DGGE banding patterns data,and the second one explained the remaining 9.9%(Fig.4A).Consistent results were obtained by CCA of AMF communities occurring in M.sativa roots (P ¼0.002)(Fig.4B).3.2.Analyses of DNA sequencesNS31-GC/AM1amplicons obtained from plant roots,spores and sporocarps of trap cultures of unfertilized plots generated multiple PCR e DGGE bands,which,after excision from the gel,cloning and-1-0.500.511.52-1.8-1.4-1-0.6-0.20.20.611.41.8A x i s 2 (13.8%)Axis 1 (86.2%)CT0CT90NT0NT90TillageFertilizationCT0CT0NT0NT0NT90NT90CT90CT90Fig.1.Canonical correspondenceanalysis (CCA)biplot of V3e V4region of nuclear 18S rDNA PCR e DGGE fragments from three replicates of ﬁeld soil from conventionally tilled (CT)and no tilled (NT)plots fertilized with 0(NT0,CT0)or 90(NT90,CT90)kg ha À1N.Fig.2.PCR e DGGE pro ﬁles of V3e V4region of nuclear 18S rDNA fragments from roots of Medicago sativa (M)trap cultures from conventionally tilled (CT)and no tilled (NT)plots fertilized with 0(NT0,CT0)or 90(NT90,CT90)kg ha À1N.-0.8-0.6-0.4-0.20.00.20.40.60.81.0-0.7-0.5-0.3-0.10.10.30.50.7A x i s 2 (0.9%)Axis 1 (99.1%)NT0MNT0ZCT0MCT0Z TillageHostNT0MNT0MCT0MCT0M CT0ZCT0ZNT0ZNT0ZFig.3.Canonical correspondence analysis (CCA)biplot of V3e V4region of nuclear 18S rDNA PCR e DGGE fragments from three replicates of trap culture soil from unfertilized no tilled (NT0)and conventionally tilled (CT0)plots,with Medicago sativa (M)and Zea mays (Z)as host plants.-1-0.8-0.6-0.4-0.200.20.40.60.8-1.2-0.8-0.400.40.8 1.2A x i s 2 (9.9%)Axis 1 (90.1%)NT90NT90NT90NT0NT0NT0CT0CT0CT0CT90CT90CT90TillageFertilizationA-1-0.8-0.6-0.4-0.200.20.40.60.811.2-1.4-1.0-0.6-0.20.20.6 1.01.4A x i s 2 (7.0%)Axis 1 (93.0%)NT0NT0NT0CT0CT0CT0CT90CT90CT90NT90NT90NT90TillageFertilizationBFig.4.Canonical correspondence analysis (CCA)biplot of V3e V4region of nuclear 18S rDNA PCR e DGGE fragments from (A)soil and (B)roots of Medicago sativa (M)trap cultures from conventionally tilled (CT)and no tilled (NT)plots fertilized with 0(NT0,CT0)or 90(NT90,CT90)kg ha À1N.L.Avio et al./Soil Biology &Biochemistry 67(2013)285e 294288sequencing,yielded a total of 37sequences with high similarity (98e 100%identity)to those of Glomeromycota,after BLASTn searches in GenBank databases.Only two sequences matched with Ascomycota sequences.PCR e DGGE bands obtained from M.sativa and Z.mays roots yielded 20sequences which grouped into four Glomeromycota sequence types,showing identities with sequences of both cultured and uncultured AMF deposited in GenBank databases.In particular,we recovered two sequence types,clustering with sequences of F.mosseae (Ag1sequence type)and G.intraradices /Glomus fas-ciculatum (Thaxt.)Gerd.&Trappe/Glomus irregulare B 1aszk.,Wubet,Renker &Buscot group,hereafter G.intraradices (Ag3sequenceFig.5.Neighbour-joining phylogenetic tree of glomeromycotan sequences derived from PCR e DGGE bands obtained from Medicago sativa and Zea mays trap plants.The analysis is based on V3e V4region of nuclear 18S rDNA sequences,and the tree is rooted with a reference sequence of Geosiphon pyriformis (X86686).Bootstrap values (>70%)were determined for neighbour joining (1000resamplings).Different sequence types are indicated in brackets:Ag1,Agugliano1;Ag2,Agugliano2;Ag3,Agugliano3;Ag4,Agugliano4;Ag5,Agugliano5.Sequences obtained in the present study are shown in bold with their accession numbers (HE806381e HE806417)followed by their DNA source (spores,spo-rocarps,roots)and treatment (trap cultures from conventionally tilled (CT)and no tilled (NT)plots fertilized with 0(NT0,CT0)kg ha À1N),with Medicago sativa (M)or Zea mays (Z)as trap plant.L.Avio et al./Soil Biology &Biochemistry 67(2013)285e 294289type)(Fig.5).Two other sequence types,Ag4and Ag5,which matched (99%identity)with sequences of uncultured Glomus species already present in GenBank were found (Table 1).Ag1and Ag3sequences were retrieved from all Z .mays and M.sativa roots,with the exception of M.sativa roots of NT0trap cultures,where Ag1was absent.Ag5sequences were retrieved from all trap cul-tures roots,while Ag4sequence type were found only in M.sativa roots,irrespective of the treatment (Fig.5).No sequences of G.viscosum were retrieved from trap plant roots.Blast and phylogenetic analyses of sequences derived from the ampli ﬁcation of spores and sporocarps lead to the identi ﬁcation of three separate clusters,Ag1(11sequences),Ag2(4sequences)and Ag3(2sequences),corresponding to F .mosseae ,G.viscosum and G.intraradices ,respectively (Fig.5,Table 1).3.3.Abundance and diversity of AMF spores produced in trap culturesThe numbers of AMF spores produced in trap cultures were consistently decreased by tillage in both M.sativa and Z.mays host plants (Fig.6),ranging from 35to 130and from 3to 34per 10g of soil,in no-tilled and tilled soil,respectively.AMF spore number was also affected by trap plant species,while a strong interaction (P <0.001)was found between host plant species and tillage/fertilization treatments.Therefore,distinct statistical analyses were performed for each host plant,which showed that in M.sativa spore production was marginally affected by tillage and fertilization treatments,while in Z.mays tillage signi ﬁcantly decreased sporu-lation (Table 2).Moreover,an interaction between fertilization and tillage was detected (P ¼0.01).The overall sporulation pattern of the different AMF species showed a predominant effect of tillage,compared with that of host and fertilization treatments,as revealed by CCA (P ¼0.032).The ﬁrst canonical axis explained 95.9%of the cumulative variance ofPCR e DGGE banding patterns data,and the second one explained the remaining 4.1%(Fig.7).F.mosseae was the predominant species sporulating in tilled soils,while G.viscosum and G.intraradices prevailed in no-tilled soils (Fig.8).Interestingly,G.intraradices spores were not retrieved from all tilled treatments.A low number of Funneliformis geosporus spores (T.H.Nicolson &Gerd.)C.Walker &A.Schüssler was retrieved only from M.sativa traps (Fig.8).With M.sativa as host plant,the number of G.viscosum spores was signi ﬁcantly lower in trap cultures from tilled than from no-tilled soils (P ¼0.01),while F.mosseae spore number signi ﬁcantly decreased in fertilized soil (P ¼0.001).With Z.mays as host plant,only G.viscosum spore number was signi ﬁcantly affected by fertilization treatments (P ¼0.011),with a strong fertilization by tillage interaction (P ¼0.002),as a result of its high sporulation in fertilized and NT plots.3.4.GRSP content in ﬁeld soil and in trap culturesBoth T-and EE-GRSP concentrations were larger in no-tilled than in tilled ﬁeld soil,and correlated well each other (Pearson correlation ¼0.823;P <0.001).T-GRSP content was signi ﬁcantly affected by tillage (P ¼0.023),and was about 36.1%larger in NT than in CT plots (Fig.9).On the other hand,fertilization did not affect GRSP content (P ¼0.132and 0.082,respectively for T-GRSP and EE-GRSP).No differences in GRSP content of trap culture soil were found.4.Discussion4.1.PCR e DGGE pattern analysis of AM fungal diversity in ﬁeld soil and trap culturesCCA of PCR e DGGE pro ﬁles clearly showed that AMF commu-nities were affected by N-fertilization both in ﬁeld soil and in roots and soil of M.sativa trap plants.Such ﬁndings are in agreement with a previous DGGE-based study showing differences in the community composition of AMF colonizing the roots of Festuca pratensis and Achillea millefolium in a Swedish grazed grassland along a gradient of soil N and P concentration (Santos et al.,2006).Other studies,performed on AMF spores,indirectly evidenced that AMF may be affected by the use of chemical fertilizers:for example Oehl et al.(2004)showed that organic farming,where the use of chemical fertilizers is not allowed,promoted higher AMF diversity and abundance than conventional agriculture,whilst other authors found a lower AMF diversity and abundance in N fertilised agro-ecosystems (Egerton-Warburton and Allen,2000).CCA of PCR e DGGE pro ﬁles further displayed an impact of tillage on AMF communities,supporting recent data obtained in long-term experiments in temperate regions (Mirás-Avalos et al.,2011;Mathew et al.,2012).Actually in our study the occurrence of weeds as a consequence of no-tillage and N fertilization (De Sanctis et al.,2012)may have represented a further factor affecting the abundance and diversity of AMF.However,the effects of tillage treatments on AMF communities observed in ﬁeld soil were con ﬁrmed by CCA of PCR e DGGE pro ﬁles from roots and soil of M.sativa and Z.mays trap plants.In this work,CCA evidenced an effect of M.sativa and Z.mays trap plants on AMF soil communities and a major effect of N-fertilization on AMF occurring in the soil and in the roots of M.sativa plants,suggesting that the responses of AMF to different agronomical treatments may depend also by host plant taxon or its nutritional status (Giovannetti et al.,1988;Egerton-Warburton and Allen,2000).Indeed,Oliveira et al.(2009)showed that tropical maize genotypes contrasting for phosphorus ef ﬁciency had a greater in ﬂuence on AMF rhizosphere community than the level of P in the soil.Table 1Nuclear SSU rDNA sequence types obtained from clones of DGGE excised bands of Medicago sativa and Zea mays roots and spores of AMF produced in trap cultures.Sequence types Identity (%)Taxonomic af ﬁliation Ag1NG017178(100)Funneliformis mosseae Ag2AJ505813(99)Glomus viscosum Ag3AJ536822(99)Glomus intraradices Ag4GU353916(99)Uncultured Glomus sp.Ag5GU353731(99)Uncultured Glomus sp.20406080100120140160180NT0MNT90MNT0ZNT90ZCT0MCT90MCT0ZCT90ZS p o r e n u m b e r 10 g s o i lFig.6.Total AMF spore density in trap cultures from conventionally tilled (CT)and no tilled (NT)plots fertilized with 0(NT0,CT0)or 90(NT90,CT90)kg ha À1N,and with Medicago sativa (M)or Zea mays (Z)as trap plant,after sixteen months ’growth.Error bars refer to standard error of the means (n ¼2).L.Avio et al./Soil Biology &Biochemistry 67(2013)285e 294290。

梅花基因组The genome of Prunus mume

The poor phylogenetic resolution of the backbone of the Rosaceae phylogenetic tree suggests rapid evolutionary radiation of lineages within the family, corresponding to a relatively recent divergence of genera3. Possibly owing to this rapid evolution, Rosaceae members show signiﬁcant phenotypic diversity, wherein common morphological synapomorphies are not readily identiﬁable. Plant habit, chromosome number and fruit type evolved independently within the family2–4. Rosaceous fruits are consumed in multiple forms, including fresh, dried, juice and processed products5. The economic importance of edible rosaceous crops is attributed to their ﬂavourful fruits and nuts, which provide unique dietary contributions to consumers.
The P. mume genome can be used to understand the structure of Rosaceae genomes in general and the rapid evolution of the Rosaceae genus. It will also help to measure and characterize the genetic diversity in domesticated and wild populations of fruit trees and determine how this diversity relates to the tremendous phenotypic diversity in fruit trees. Furthermore, genome-based tools can be developed to improve breeding work.

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three factors Linguistic Inquiry, Volume 36, Number 1, Winter 2005

Three Factors in LanguageDesignNoam ChomskyThe biolinguistic perspective regards the language faculty as an ‘‘organof the body,’’along with other cognitive systems.Adopting it,weexpect to find three factors that interact to determine (I-)languagesattained:genetic endowment (the topic of Universal Grammar),experi-ence,and principles that are language-or even organism-independent.Research has naturally focused on I-languages and UG,the problemsof descriptive and explanatory adequacy.The Principles-and-Param-eters approach opened the possibility for serious investigation of thethird factor,and the attempt to account for properties of language interms of general considerations of computational efficiency,eliminat-ing some of the technology postulated as specific to language andproviding more principled explanation of linguistic phenomena.Keywords:minimalism,principled explanation,Extended StandardTheory,Principles-and-Parameters,internal/external Merge,single-cycle derivation,phaseThirty years ago,in 1974,an international meeting took place at MIT,in cooperation with the Royaumont Institute in Paris,on the topic of ‘‘biolinguistics,’’a term suggested by the organizer,Massimo Piattelli-Palmarini,and the title of a recent book surveying the field and proposing new directions by Lyle Jenkins (2002).1This was only one of many such interactions in those years,including interdisciplinary seminars and international conferences.The biolinguistic perspective began to take shape over 20years before in discussions among a few graduate students who were much influenced by developments in biology and mathematics in the early postwar years,including work in ethology that was just coming to be known in the United States.One of them was Eric Lenneberg,whose seminal 1967study Biological Foundations of Language remains a basic document of the field.Many of the leading questions discussed at the 1974conference,and in the years leading up to it,remain very much alive today.One of these questions,repeatedly brought up in the conference as ‘‘one of the basic questions to be asked from the biological point of view,’’is the extent to which apparent principles of language,including some that had only recently come to light,are unique to this cognitive system or whether similar ‘‘formal arrangements’’are found in other cognitive domains in humans or This article is expanded from a talk presented at the annual meeting of the Linguistic Society of America,9January 2004.Thanks to Cedric Boeckx,Samuel David Epstein,Robert Freidin,Lyle Jenkins,Howard Lasnik,and Luigi Rizzi,among others,for comments on an earlier draft.1The conference,titled ‘‘A Debate on Bio-Linguistics,’’was held at Endicott House,Dedham,Massachusetts,20–21May 1974,and organized by the Centre Royaumont pour une science de l’homme,Paris.1Linguistic Inquiry,Volume 36,Number 1,Winter 20051–22᭧2005by the Massachusetts Institute of Technology2N O A M C H O M S K Yother organisms.An even more basic question from the biological point of view is how much of language can be given a principled explanation,whether or not homologous elements can be found in other domains or organisms.The effort to sharpen these questions and to investigate them for language has come to be called the‘‘Minimalist Program’’in recent years,but the questions arise for any biological system and are independent of theoretical persuasion,in linguis-tics and elsewhere.Answers to these questions are fundamental not only to understanding the nature and functioning of organisms and their subsystems,but also to investigating their growth and evolution.For any biological system,language included,the only general question that arises about the program is whether it can be productively pursued or is premature.In these remarks,I will try to identify what seem to me some of the significant themes in the past half-century of inquiry into problems of biolinguistics and to consider their current status. Several preliminary qualifications should be obvious.One is that the picture is personal;others would no doubt make different choices.A second is that things often seem clearer in retrospect than at the time,so there is some anachronism in this account,but not I think too much.A third is that I cannot even begin to mention the contributions of a great many people to the collective enterprise,particularly as the related fields have expanded enormously in the years since the1974 conference.The biolinguistic perspective views a person’s language as a state of some component of the mind,understanding‘‘mind’’in the sense of eighteenth-century scientists who recognized that after Newton’s demolition of the only coherent concept of body,we can only regard aspects of the world‘‘termed mental’’as the result of‘‘such an organical structure as that of the brain’’(Joseph Priestley).Among the vast array of phenomena that one might loosely consider language-related,the biolinguistic approach focuses attention on a component of human biology that enters into the use and acquisition of language,however one interprets the term‘‘language.’’Call it the ‘‘faculty of language,’’adapting a traditional term to a new usage.This component is more or less on a par with the systems of mammalian vision,insect navigation,and others.In many of these cases,the best available explanatory theories attribute to the organism computational systems and what is called‘‘rule-following’’in informal usage—for example,when a recent text on vision presents the so-called rigidity principle as it was formulated50years ago:‘‘if possible,and other rules permit,interpret image motions as projections of rigid motions in three dimensions’’(Hoffman1998:169).In this case,later work provided substantial insight into the mental computa-tions that seem to be involved when the visual system follows these rules,but even for very simple organisms,that is typically no slight task,and relating mental computations to analysis at the cellular level is commonly a distant goal.Adopting this conception,a language is a state of the faculty of language,an I-language,in technical usage.The decision to study language as part of the world in this sense was regarded as highly controversial at the time,and still is.A more careful look will show,I think,that the arguments advanced against the legitimacy of the approach have little force(a weak thesis)and that its basic assumptions are tacitly adopted even by those who strenuously reject them,and indeed must be, even for coherence(a much stronger thesis).I will not enter into this interesting chapter ofT H R E E F A C T O R S I N L A N G U A G E D E S I G N3 contemporary intellectual history here,but will simply assume that crucial aspects of language can be studied as part of the natural world,adopting the biolinguistic approach that took shape half a century ago and that has been intensively pursued since,along different paths.The language faculty is one component of what the cofounder of modern evolutionary theory, Alfred Russel Wallace,called‘‘man’s intellectual and moral nature’’:the human capacities for creative imagination,language and symbolism generally,mathematics,interpretation and record-ing of natural phenomena,intricate social practices,and the like,a complex of capacities that seem to have crystallized fairly recently,perhaps a little over50,000years ago,among a small breeding group of which we are all descendants—a complex that sets humans apart rather sharply from other animals,including other hominids,judging by traces they have left in the archaeological record.The nature of the‘‘human capacity,’’as some researchers now call it,remains a considera-ble mystery.It was one element of a famous disagreement between the two founders of the theory of evolution,with Wallace holding,contrary to Darwin,that evolution of these faculties cannot be accounted for in terms of variation and natural selection alone,but requires‘‘some other influence,law,or agency,’’some principle of nature alongside gravitation,cohesion,and other forces without which the material universe could not exist.Although the issues are framed differ-ently today within the core biological sciences,they have not disappeared(see Wallace1889: chap.15,Marshack1985).It is commonly assumed that whatever the human intellectual capacity is,the faculty of language is essential to it.Many scientists agree with paleoanthropologist Ian Tattersall,who writes that he is‘‘almost sure that it was the invention of language’’that was the‘‘sudden and emergent’’event that was the‘‘releasing stimulus’’for the appearance of the human capacity in the evolutionary record—the‘‘great leap forward’’as Jared Diamond called it,the result of some genetic event that rewired the brain,allowing for the origin of modern language with the rich syntax that provides a multitude of modes of expression of thought,a prerequisite for social development and the sharp changes of behavior that are revealed in the archaeological record, also generally assumed to be the trigger for the rapid trek from Africa,where otherwise modern humans had apparently been present for hundreds of thousands of years(Tattersall1998:24–25; see also Wells2002).Tattersall takes language to be‘‘virtually synonymous with symbolic thought.’’Elaborating,one of the initiators of the Royaumont-MIT symposia,Franc¸ois Jacob, observed that‘‘the role of language as a communication system between individuals would have come about only secondarily,as many linguists believe’’(1982:59),perhaps referring to discus-sions at the symposia,where the issue repeatedly arose,among biologists as well.In the1974 conference,his fellow Nobel laureate Salvador Luria was the most forceful advocate of the view that communicative needs would not have provided‘‘any great selective pressure to produce a system such as language,’’with its crucial relation to‘‘development of abstract or productive thinking’’(Luria1974:195).‘‘The quality of language that makes it unique does not seem to be so much its role in communicating directives for action’’or other common features of animal communication,Jacob continued,but rather‘‘its role in symbolizing,in evoking cognitive im-ages,’’in‘‘molding’’our notion of reality and yielding our capacity for thought and planning, through its unique property of allowing‘‘infinite combinations of symbols’’and therefore‘‘mental4N O A M C H O M S K Ycreation of possible worlds,’’ideas that trace back to the seventeenth-century cognitive revolution (1982:59).Jacob also stressed the common understanding that answers to questions about evolu-tion‘‘in most instances...can hardly be more than more or less reasonable guesses’’(1982: 31).We can add another insight of seventeenth-and eighteenth-century philosophy:that even the most elementary concepts of human language do not relate to mind-independent objects by means of some reference-like relation between symbols and identifiable physical features of the external world,as seems to be universal in animal communication systems.Rather,they are creations of the‘‘cognoscitive powers’’that provide us with rich means to refer to the outside world from certain perspectives,but are individuated by mental operations that cannot be reduced to a‘‘peculiar nature belonging’’to the thing we are talking about,as Hume summarized a century of inquiry.Those are critical observations about the elementary semantics of natural language, suggesting that its most primitive elements are related to the mind-independent world much as the internal elements of phonology are,not by a reference-like relation but as part of a considerably more intricate species of conception and action.It is for reasons such as these,though not clearly grasped at the time,that the early work in the1950s adopted a kind of‘‘use theory of meaning,’’pretty much in the sense of John Austin and the later Wittgenstein:language was conceived as an instrument put to use for various human purposes,generating expressions including arrange-ments of the fundamental elements of the language,with no grammatical-ungrammatical divide, each basically a complex of instructions for use(see Chomsky1955,hereafter LSLT).2 If this much is generally on the right track,then at least two basic problems arise when we consider the origins of the faculty of language and its role in the sudden emergence of the human intellectual capacity:first,the core semantics of minimal meaning-bearing elements,including the simplest of them;and second,the principles that allow infinite combinations of symbols, hierarchically organized,which provide the means for use of language in its many aspects.Accord-ingly,the core theory of language—Universal Grammar(UG)—must provide,first,a structured inventory of possible lexical items that are related to or perhaps identical with the concepts that are the elements of the‘‘cognoscitive powers,’’sometimes now regarded as a‘‘language of thought’’along lines developed by Jerry Fodor(1975);and second,means to construct from these lexical items the infinite variety of internal structures that enter into thought,interpretation, planning,and other human mental acts,and that are sometimes put to use in action,including the externalization that is a secondary process if the speculations just reviewed turn out to be correct.On the first problem,the apparently human-specific conceptual-lexical apparatus,there is important work on relational notions linked to syntactic structures and on the partially mind-internal objects that appear to play a critical role(events,propositions,etc.).3But there is little beyond descriptive remarks on the core referential apparatus that is used to talk about the world. The second problem has been central to linguistic research for half a century,with a long history before in different terms.2For later discussion,see among others Chomsky1966,2001b,McGilvray1999,Antony and Hornstein2003.3For insightful review and original analysis,see Borer2004a,b.T H R E E F A C T O R S I N L A N G U A G E D E S I G N5 The biolinguistic approach adopted from the outset the point of view that C.R.Gallistel (1997)calls‘‘the norm these days in neuroscience’’(p.86),the‘‘modular view of learning’’: the conclusion that in all animals,learning is based on specialized mechanisms,‘‘instincts to learn’’(p.82)in specific ways.We can think of these mechanisms as‘‘organs within the brain’’(p.86),achieving states in which they perform specific kinds of computation.Apart from‘‘ex-tremely hostile environments’’(p.88),they change states under the triggering and shaping effect of external factors,more or less reflexively,and in accordance with internal design.That is the ‘‘process of learning’’(Gallistel1997,1999),though‘‘growth’’might be a more appropriate term,avoiding misleading connotations of the term‘‘learning.’’The modular view of learning of course does not entail that the component elements of the module are unique to it:at some level,everyone assumes that they are not—the cellular level,for example—and the question of the level of organization at which unique properties emerge remains a basic one from a biological point of view,as it was at the1974conference.Gallistel’s observations recall the concept of‘‘canalization’’introduced into evolutionary and developmental biology by C.H.Waddington over60years ago,referring to processes‘‘adjusted so as to bring about one definite end result regardless of minor variations in conditions during the course of the reaction,’’thus ensuring‘‘the production of the normal,that is optimal type in the face of the unavoidable hazards of existence’’(Waddington1942).That seems to be a fair description of the growth of language in the individual.A core problem of the study of the faculty of language is to discover the mechanisms that limit outcomes to‘‘optimal types.’’It has been recognized since the origins of modern biology that such constraints enter not only into the growth of organisms but also into their evolution,with roots in the earlier tradition that Stuart Kauffman calls‘‘rational morphology’’(1993:3–5).4In a classic contemporary paper, John Maynard Smith and associates trace the post-Darwinian reformulation back to Thomas Huxley,who was struck by the fact that there appear to be‘‘predetermined lines of modification’’that lead natural selection to‘‘produce varieties of a limited number and kind’’for every species (Maynard Smith et al.1985:266).5They review a variety of such constraints in the organic world and describe how‘‘limitations on phenotypic variability’’are‘‘caused by the structure,character, composition,or dynamics of the developmental system,’’pointing out also that such‘‘develop-mental constraints...undoubtedly play a significant role in evolution’’though there is yet‘‘little agreement on their importance as compared with selection,drift,and other such factors in shaping evolutionary history’’(p.265).At about the same time,Jacob wrote that‘‘the rules controlling embryonic development,’’almost entirely unknown,interact with other constraints imposed by general body plan,mechanical properties of building materials,and other factors in‘‘restricting possible changes of structures and functions’’in evolutionary development(1982:21),providing ‘‘architectural constraints’’that‘‘limit adaptive scope and channel evolutionary patterns’’(Erwin 2003:1683).The best-known of the figures who devoted much of their work to these topics are 4For comment in a linguistic context,see Boeckx and Hornstein2003.For more general discussion,see Jenkins 2000.5For review of some of these topics,see Stewart1998.6N O A M C H O M S K YD’Arcy Thompson and Alan Turing,who took a very strong view on the central role of such factors in biology.In recent years,such considerations have been adduced for a wide range of problems of development and evolution,from cell division in bacteria to optimization of structure and function of cortical networks,even to proposals that organisms have‘‘the best of all possible brains,’’as argued by computational neuroscientist Christopher Cherniak(1995:522).6The prob-lems are at the border of inquiry,but their significance is not controversial.Assuming that the faculty of language has the general properties of other biological systems, we should,therefore,be seeking three factors that enter into the growth of language in the indi-vidual:1.Genetic endowment,apparently nearly uniform for the species,which interprets part ofthe environment as linguistic experience,a nontrivial task that the infant carries out reflexively,and which determines the general course of the development of the language faculty.Among the genetic elements,some may impose computational limitations that disappear in a regular way through genetically timed maturation.Kenneth Wexler and his associates have provided compelling evidence of their existence in the growth of language,thus providing empirical evidence for what Wexler(to appear)calls‘‘Lenne-berg’s dream.’’2.Experience,which leads to variation,within a fairly narrow range,as in the case of othersubsystems of the human capacity and the organism generally.3.Principles not specific to the faculty of language.The third factor falls into several subtypes:(a)principles of data analysis that might be used in language acquisition and other domains;(b)principles of structural architecture and developmental constraints that enter into canalization,organic form,and action over a wide range,including principles of efficient computation,which would be expected to be of particular significance for computational systems such as language.It is the second of these subcategories that should be of particular significance in determining the nature of attainable languages.Those exploring these questions50years ago assumed that the primitive step of analysis of linguistic experience would be feature-based phonetic analysis,along lines described by Roman Jakobson and his associates(see Jakobson,Fant,and Halle1953).We also tried to show that basic prosodic properties reflect syntactic structure that is determined by other principles,including crucially a principle of cyclic computation that was extended much more generally in later years (see Chomsky,Halle,and Lukoff1956).The primitive principles must also provide what George Miller called‘‘chunking,’’identification of phonological words in the string of phonetic units.In LSLT(p.165),I adopted Zellig Harris’s(1955)proposal,in a different framework,for identifying morphemes in terms of transitional probabilities,though morphemes do not have the required beads-on-a-string property.The basic problem,as noted in LSLT,is to show that such statistical 6See also Laughlin and Sejnowski2003,Cherniak et al.2004,and Physics News Update2001reporting Howard, Rutenberg,and de Vet2001.T H R E E F A C T O R S I N L A N G U A G E D E S I G N7 methods of chunking can work with a realistic corpus.That hope turns out to be illusory,as has recently been shown by Thomas Gambell and Charles Yang(2003),who go on to point out that the methods do,however,give reasonable results if applied to material that is preanalyzed in terms of the apparently language-specific principle that each word has a single primary stress.If so,then the early steps of compiling linguistic experience might be accounted for in terms of general principles of data analysis applied to representations preanalyzed in terms of principles specific to the language faculty,the kind of interaction one should expect among the three factors.In LSLT,it was assumed that the next step would be assignment of chunked items to syntactic categories,again by general principles of data analysis.A proposal with an information-theoretic flavor was tried by hand calculations in that precomputer age,with suggestive results,but the matter has never been pursued,to my knowledge.Surely what are called‘‘semantic properties’’are also involved,but these involve nontrivial problems at the most elementary level,as mentioned earlier.The assumption of LSLT was that higher levels of linguistic description,including mor-phemes,are determined by a general format for rule systems provided by UG,with selection among them in terms of a computational procedure that seeks the optimal instantiation,a notion defined in terms of UG principles of significant generalization.Specific proposals were made then and in the years that followed.In principle,they provided a possible answer to what came to be called the‘‘logical problem of language acquisition,’’but they involved astronomical calcula-tion and therefore did not seriously address the issues.The main concerns in those years were quite different,as they still are.It may be hard to believe today,but it was commonly assumed50years ago that the basic technology of linguistic description was available and that language variation was so free that nothing of much generality was likely to be discovered.As soon as efforts were made to provide fairly explicit accounts of the properties of languages,however,it became obvious how little was known,in any domain. Every specific proposal yielded a treasure trove of counterevidence,requiring complex and varied rule-systems even to achieve a very limited approximation to descriptive adequacy.That was highly stimulating for inquiry into language,but it also left a serious quandary,since the most elementary considerations led to the conclusion that UG must impose narrow constraints on possible outcomes—sometimes called‘‘poverty of stimulus’’problems in the study of language, though the term is misleading because this is just a special case of basic issues that arise universally for organic growth.A number of paths were pursued to try to resolve the tension.The most successful turned out to be efforts to formulate general principles,attributed to UG—that is,the genetic endow-ment—leaving a somewhat reduced residue of phenomena that would result,somehow,from experience.Early proposals were the A-over-A Principle,conditions on wh-extraction from wh-phrases(relatives and interrogatives),simplification of T-markers to base recursion(following observations by Charles Fillmore)and cyclicity(an intricate matter,as shown in an important paper of Robert Freidin’s(1978)and insightfully reviewed in a current paper of Howard Lasnik’s (to appear)which shows that many central questions remain unanswered),later John Robert Ross’s(1967)classic study of taxonomy of islands that still remains a rich store of ideas and observations to explore,then attempts to reduce islands to such properties as locality and structure8N O A M C H O M S K Ypreservation,and so on.These approaches had some success,but the basic tensions remained unresolved at the time of the1974conference.Within a few years,the landscape had changed considerably.In part this was because of great progress in areas that had hitherto been explored only in limited ways,including truth-and model-theoretic semantics and prosodic structures.In part it was the result of a vast array of new materials from studies of much greater depth than previously undertaken,and into a much wider variety of languages,much of it traceable to Richard Kayne’s work and his lectures in Europe, which inspired far-reaching inquiry into Romance and Germanic languages,later other languages, also leading to many fruitful ideas about the principles of UG.About25years ago,much of this work crystallized in a radically different approach to UG,the Principles-and-Parameters(P&P) framework,which for the first time offered the hope of overcoming the tension between descriptive and explanatory adequacy.This approach sought to eliminate the format framework entirely,and with it,the traditional conception of rules and constructions that had been pretty much taken over into generative grammar.That much is familiar,as is the fact that the new P&P framework led to an explosion of inquiry into languages of the most varied typology,yielding new problems previously not envisioned,sometimes answers,and the reinvigoration of neighboring disciplines concerned with acquisition and processing,their guiding questions reframed in terms of parameter setting within a fixed system of principles of UG with at least visible contours.Alternative paths, variously interrelated,were leading in much the same direction,including Michael Brody’s highly illuminating work(1995,2003).No one familiar with the field has any illusion today that the horizons of inquiry are even visible,let alone at hand,in any domain.Abandonment of the format framework also had a significant impact on the biolinguistic program.If,as had been assumed,acquisition is a matter of selection among options made available by the format provided by UG,then the format must be rich and highly articulated,allowing relatively few options;otherwise,explanatory adequacy is out of reach.The best theory of lan-guage must be a very unsatisfactory one from other points of view,with a complex array of conditions specific to human language,restricting possible instantiations.The only plausible theories had to impose intricate constraints on the permissible relations between sound and mean-ing,all apparently specific to the faculty of language.The fundamental biological issue of princi-pled explanation could barely be contemplated,and correspondingly,the prospects for serious inquiry into evolution of language were dim;evidently,the more varied and intricate the conditions specific to language,the less hope there is for a reasonable account of the evolutionary origins of UG.These are among the questions that were raised at the1974symposium and others of the period,but they were left as apparently irresoluble problems.The P&P framework offered prospects for resolution of these tensions as well.Insofar as this framework proves valid,acquisition is a matter of parameter setting and is therefore divorced entirely from the remaining format for grammar:the principles of UG.There is no longer a conceptual barrier to the hope that the UG might be reduced to a much simpler form,and that the basic properties of the computational systems of language might have a principled explanation instead of being stipulated in terms of a highly restrictive language-specific format for grammars. Within a P&P framework,what had previously been the worst theory—anything goes—might。

小鼠淋巴结位置

Research paperAnatomy and nomenclature of murine lymph nodes:Descriptive study and nomenclatory standardization in BALB/cAnNCrl miceWim Van den Broeck a,⁎,Annie Derore b,c ,Paul Simoens aaDepartment of Morphology,Faculty of Veterinary Medicine,Ghent University,Salisburylaan 133,B-9820Merelbeke,BelgiumbInnogenetics NV ,Industriepark Zwijnaarde 7,B-9052Ghent,BelgiumcFlanders Interuniversity Institute for Biotechnology (VIB),Technologiepark 927,B-9052Ghent,BelgiumReceived 21November 2005;received in revised form 10January 2006;accepted 26January 2006Available online 6March 2006AbstractMurine lymph nodes are intensively studied but often assigned incorrectly in scientific papers.In BALB/cAnNCrl mice,we characterized a total of 22different lymph nodes.Peripheral nodes were situated in the head and neck region (mandibular,accessory mandibular,superficial parotid,cranial deep cervical nodes),and at the forelimb (proper axillary,accessory axillary nodes)and hindlimb (subiliac,sciatic,popliteal nodes).Intrathoracic lymph nodes included the cranial mediastinal,tracheobronchal and caudal mediastinal nodes.Abdominal lymph nodes were associated with the gastrointestinal tract (gastric,pancreaticoduodenal,jejunal,colic,caudal mesenteric nodes)or were located along the major intra-abdominal blood vessels (renal,lumbar aortic,lateral iliac,medial iliac and external iliac nodes).Comparative and nomenclative aspects of murine lymph nodes are discussed.The position of the lymph nodes of BALB/cAnNCrl mice is summarized and illustrated in an anatomical chart containing proposals for both an official nomenclature according to the Nomina Anatomica Veterinaria and English terms.©2006Elsevier B.V .All rights reserved.Keywords:Mouse;Lymph node;Nomenclature1.IntroductionRodents,and mice in particular,have long been used as laboratory animals in various scientific experiments.The possibility to produce different murine strains and a variety of knock-out mice,the high reproductive rate of these animals,and the ease of their handling have made them the preferential laboratory animal.In immunolog-ical sciences,murine lymph nodes (lnn.)are often used to isolate lymphocytes in order to study fundamentalaspects of immunology and immunopathology.The methodology to recognize and dissect these lymph nodes requires at least a basic anatomical knowledge.In numerous studies,however,inaccurate,misleading or even enigmatic terms such as genital nodes (Cain and Rank,1995)or tonsillar nodes (Deaglio et al.,1996)have sometimes been assigned to murine lymph nodes.The ambiguity of murine lymph node (ln.)nomenclature is illustrated by the lymph node at the ear base of mice which has been variably designated by various terms such as parotid ln.(Cuq,1966;Grassé,1972;Popesko et al.,1992),lateral mandibular ln.(Cuq,1966),and facial ln.(Wolvers et al.,1999),while numerous recent studies refer to an allegedly auricular ln.(Anjuère et al.,1999;Dearman et al.,1996;Sailstad et al.,1995)or pre-Journal of Immunological Methods 312(2006)12–19/locate/jim⁎Corresponding author.Tel.:+3292747716;fax:+3292647790.E-mail address:wim.vandenbroeck@UGent.be (W.Van den Broeck).0022-1759/$-see front matter ©2006Elsevier B.V .All rights reserved.doi:10.1016/j.jim.2006.01.022auricular ln.(Hendrickx et al.,1992)in this region.Given this confusion,it becomes very difficult to reproduce the experimental reports or compare different scientific results.Nevertheless,the localization of the different lymph nodes with their respective names in mice has been thoroughly described in a number of anatomical publications (Barone et al.,1950;Cuq,1966;Kawashima et al.,1964),but these papers are seldom referred to.A sample bibliographic (Medline)search from 1989to 1999demonstrated that of 293randomly chosen papers in which the words “mouse lymph node(s)”are used,89citations (i.e.30%)used only vague terms such as “lymph node ”,“peripheral lymph node ”,“draining lymph node ”,“local lymph node ”,or “regional lymph node ”instead of the precise anatomical names.In the remaining 204publications,at least 42different specific names were given to the lymph nodes that were studied.Only 1article,however,contained some figures illustrating the anatomical position and identification of the lymph nodes in question (Wolvers et al.,1999).In contrast,in the remaining 203studies the exact scientific identification of the node was lacking:59of these investigations referred to previous publica-tions in which the nomenclature used was not based on asufficiently scientific anatomical support,while in the remaining 144articles no anatomical reference was given at all.In an attempt to rectify this situation,we first characterized the lymph nodes in BALB/cAnNCrl mice and then summarized our findings in an anatomical chart.2.Materials and methods 2.1.AnimalsSeventy female BALB/cAnNCrl mice (Iffa Credo N.V .,Brussels,Belgium)aged 8to 32weeks were housed in groups of 3to 6animals in conventional type II cages containing nesting material as environmental enrich-ment (Brain et al.,1994)along with water and food supply ad libitum.At the end of the experiments,all animals were euthanized by intraperitoneal (IP)injec-tion of 30μl T61(Hoechst Roussel Vet,Brussels,Belgium).All experimental studies described in this paper were approved by the Institutional Animal Welfare Committee of Innogenetics (September 15,1999).Table 1Protocols used for demonstrating murine lymph nodes Protocol Route of administrationSedation/anaesthesia Product Quantity (μl)Incubation (days)Number of animals I Intravenous (lateral caudal vein)–Ink+RAS a 200b 103II Subcutaneous,mental region –Ink+CFA c 60b 286III Subcutaneous,mental region –Ink+RAS 10b 214IV Subcutaneous,frontal region –Ink+CFA 60b 286V Subcutaneous,auricular base–Ink+RAS 10b 216VI Subcutaneous,palmar metacarpal region –Ink+CFA 40b 183426VII Subcutaneous,plantar metatarsal region –Ink+CFA 40b 183426VIII Intranasal instillation Sedation Ink+RAS 2×30b,d 10e 317e 3IX Intraperitoneal –Ink+tR f2000g 143X PeroralSedation Ink+RAS or CFA 500b 216XI Intrahepatic h Anaesthesia Ink+RAS 30b 216XIIIntralienal iAnaesthesiaInk+RAS50b216a RAS:Ribi Adjuvant System®,RIBI Immuno Chem Research,Inc.,Hamilton,USA.bEqual quantities ink/RAS or CFA.cCFA:Complete Freunds Adjuvant®,Difco Laboratories,Detroit,Michigan,USA.dTwo administrations of 30μl with 21-day interval.eDays after the last administration.ftR:Thioglycollate+Resazurin®,Sanofi Diagnostics Pasteur,Marnes-la-Coquette,France.g50μl ink+1950μl Thioglycollate +Resazurin®.hAfter anaesthesia,the abdominal wall was incised 5mm caudal to the xiphoid process under surgical conditions;after the injection of the solution into the left and right hepatic lobes,the abdominal incision was closed.iAfter anaesthesia,the left abdominal wall was incised under surgical conditions;after the injection of the solution into the spleen,the abdominal incision was closed.13W.Van den Broeck et al./Journal of Immunological Methods 312(2006)12–19Table 2List of lymph nodes observed in the present study of BALB/cAnNCrl mice #English name Official name Protocol Occurrencea Topography2Accessory mandibular ln.Ln.mandibularis accessorius I,II,IV ,V Constant (21/21)Dorsolateral to the mandibular lymph nodeSuperficial parotid ln.Ln.parotideus superficialisI,II,IV ,VConstant (21/21)Ventral to the external acoustic pore,caudal to the extraorbital lacrimal gland,cranioventral to the parotid salivary gland,dorsal to the junction between the superficial temporal vein (v.)and the maxillary v.4Cranial deep cervical ln.Ln.cervicalisprofundus cranialis I,II,IV ,VIConstant (24/24)Medial to the external jugular vein and sternocephalic muscle (m.),lateral to sternohyoid m.,caudal to digastric m.,dorsal to the trachea5Proper axillary ln.Ln.axillaris propriusI,VIConstant (12/12)Medial to the shoulder,dorsolateral to ascending pectoral m.,at the junction between the lateral thoracic vein and the axillary vein6Accessory axillary ln.Ln.axillaris accessorius I,VI Constant (12/12)Caudal to triceps brachii m.,lateral to cutaneous trunci m.,in subcutaneous adipose tissue7Subiliac ln.Ln.subiliacusI,VIIConstant (12/12)In the fold of the flank (plica lateralis)cranial to thigh musculature,near the deep circumflex iliac artery (a.)and v.8Sciatic ln.Ln.ischiadicus I,VIIConstant (12/12)Medial to gluteus superficialis m.,caudal to gluteus medius m.and sciatic nerve9Popliteal ln.Ln.popliteus I,VII Constant(12/12)In the popliteal fossa between biceps femoris m.and semitendinosus m.10Cranial mediastinal lnn.Lnn.mediastinales craniales I Constant(3/3)Bilaterally 2lymph nodes located lateral to the thoracic thymus and along the internal thoracic a.and v.11Tracheobronchal ln.Ln.tracheobronchalis VIII Constant(6/6)Single (unpaired)lymph node at the tracheal bifurcation 12Caudal mediastinal ln.Ln.mediastinalis caudalis I Constant(3/3)Single (unpaired)lymph node in the caudal mediastinum,ventral to the esophagus,along the ventral vagal trunk 13Gastric ln.Ln.gastricus I,IX,X,XI,XII Constant(24/24)Single (unpaired)lymph node in the lesser omentum at the minor curvature of the stomach14Pancreaticoduodenal ln.Ln.pancreaticoduodenalis I,IX,X,XI,XII Constant(24/24)Single (unpaired)lymph node in the mesoduodenum,dorsal to the portal vein,surrounded by pancreatic tissue 15Jejunal lnn.Lnn.jejunales I,IX,X,XI,XII Constant(24/24)Large cluster of lymph nodes in the mesojejunum along the cranial mesenteric a.16Colic ln.Ln.colicus I,IX,X,XI,XII Constant(24/24)In the mesocolon at the transition between ascending colon and transverse colon17Caudal mesenteric ln.Ln.mesentericus caudalis I,IX,X,XI,XII Constant(24/24)Single (unpaired)lymph node in the caudal mesentery at the origin of the caudal mesenteric a.18Renal ln.Ln.renalis I,VII,IX,X,XI,XII Constant(33/33)Dorsal to the ipsilateral kidney nearby the renal blood vessels,caudal to the adrenal gland19Lumbar aortic ln.Ln.lumbalis aorticus VII bInconstant(4/6bilateral,2/6only left)Lateral to (and adjacent with)the abdominal aorta,halfway between the origin of the renal and common iliac arteries20Lateral iliac ln.Ln.iliacus lateralis I Inconstant(2/3only right,1/3absent)In adipose tissue caudolateral to the kidney along the deep circumflex iliac a.21Medial iliac ln.Ln.iliacus medialis I,VII,IX,X Constant(21/21)Major bilateral lymph node at the terminal segment of the abdominal aorta and the origin of the common iliac a.22External iliac ln.Ln.iliacus externus I Constant(3/3)Small lymph node along the initial (intra-abdominal)segment of the external iliac a.,before the latter enters the femoral canalEnglish and official Latin names of each node are given together with their frequency and a short description of their topography.aF :number of animals in which lymph nodes were found,E :number of animals in which these particular lymph nodes were examined.bProtocol VII with 42incubation days.14W.Van den Broeck et al./Journal of Immunological Methods 312(2006)12–192.2.Stimulation of lymph nodesAs murine lymph nodes are hardly distinguishable from the surrounding fat and connective tissue(Cuq, 1966),they were stimulated and colored in vivo by an injection of Indian ink in combination with an adjuvant prior to euthanasia and subsequent dissection of the animals.Intravenous injections were performed in three mice to obtain a general overview(protocol I),whereas different additional stimulation protocols were used to demonstrate the presence of particular nodes in various body regions(protocols II–XII).In some protocols,a previous sedation of the mice by intramuscular injection of1μl/g body weight of a solution of200μl ketamine (Ketalar,Parke Davis,Dublin,Ireland)and30μl xylazine(Rompun2%,Bayer,Brussels,Belgium)was required.In a few cases,anaesthesia was induced by injecting the mice intraperitoneally with220μl of a solution containing200μl ketamine,100μl xylazine and 700μl physiological salt solution.The different protocol details are listed in Table1.The specific protocols that have been used to identify the particular nodes are listed in Table2.2.3.Histological examinationThe lymphoid architecture of the in vivo colored structures was verified by histological examination. Dissected lymph nodes were fixed in3.5%phosphate-buffered formaldehyde immediately after necropsy. Paraffin sections were made and stained with eosin–haematoxylin.3.ResultsBased on their topography,the murine lymph nodes were divided into peripheral(head and neck region, forelimb,hindlimb),intrathoracic,and intra-abdominal lymph nodes.A precise nomenclature based on the Nomina Anatomica Veterinaria(2005),equivalent English terms,and the topography of the lymph nodes are described in Table2.The anatomical position ofthe Fig.1.Peripheral lymph nodes in the mouse.(1a)Ventro-lateral view of the head and throat region with sublingual(a),mandibular(b)and parotid (c)salivary gland and the extraorbital lacrimal gland(d),(1b)ventral view of the axillary region,(1c)lateral view of the thorax and forelimb,(1d) dorsal view of the sacral region with the sciatic nerve(a),and(1e)ventral view of the spread hindlimbs;numbers(1–9)according to the description in Table2.15 W.Van den Broeck et al./Journal of Immunological Methods312(2006)12–19exposed lymph nodes is illustrated in 14photographs (Figs.1–3)and 2drawings (Fig.4).Nine peripheral lymph nodes are constant and bilaterally present,namely the mandibular,accessory mandibular,superficial parotid,and cranial deep cervical ln.in the head and neck regions,the axillary and accessory axillary ln.in the forelimb,and the subiliac,sciatic and popliteal ln.in the hindlimb region.Intrathoracic nodes are few in number and consist of the cranial mediastinal lnn.,tracheobronchal ln.and the caudal mediastinal ln.Intra-abdominal lymph nodes are either associated with the gastroin-testinal tract or lie along the major abdominal arteries.The former group consists of the gastric and pancrea-ticoduodenal ln.,the jejunal lnn.and colic ln.which together represent the cranial mesenteric lnn.,and the caudal mesenteric ln.The other intra-abdominal lymph nodes include the bilateral renal,medial iliac and external iliac ln.,as well as the inconstant lumbar aortic and lateral iliac ln.The latter lymph node was observed in 2out of 3mice that were stimulated by intravenous injection.Other lymph nodes such as the facial (Wolvers et al.,1999),auricular or pre-auricular (Anjuère et al.,1999;Dearman et al.,1996;Hendrickx et al.,1992;Sailstad et al.,1995),superficial cervical (Barone et al.,1950;Cuq,1966),caudal deep cervical (Barone et al.,1950),pulmonary (Teitelbaum et al.,1999),hepatic and lienal (Barone et al.,1950),(ileo)cecal (Barone et al.,1950;Cuq,1966),sacral (Popesko et al.,1992),and femoral (Björkdahl et al.,1999;Mishell et al.,1980)lymph nodes were not observed in any of the BALB/cAnNCrl mice that were examined in the present study.Furthermore,there was no evidence of the presence of a submental lymph node (Cook,1983;Jacoby and Fox,1984),but a number of subcutaneous submental lymph nodules were demonstrated just caudal to the inter-mandibular synchondrosis by histological examination.4.DiscussionWe sought to definitely localize lymph nodes in mice and to provide an up-to-date anatomical determination chart to identify the different nodes.Most oftheseFig.2.Intrathoracic and intra-abdominal lymph nodes in the mouse.(2a)Ventral view of the thoracic cavity with the right lung (a)and thymus (b),both turned over to the left side,(2b)ventral view of the thoracic cavity with oesophagus (a),heart (b)and thymus (c),(2c)ventral view of the abdominal cavity with stomach (a),liver (b)and spleen (c),(2d)exposed mesentery,and (2e)ventral view of the abdominal cavity with the left uterine horn (a)and the caudal mesenteric artery (b);numbers (10–17)according to the description in Table 2.16W.Van den Broeck et al./Journal of Immunological Methods 312(2006)12–19lymph nodes have already been described in anatomical papers (Barone et al.,1950;Cuq,1966;Kawashima et al.,1964),but bibliometric analysis indicates that contemporary investigators are often not familiar with these publications.As a consequence,the nomenclature of murine lymph nodes used in recent literature lacks uniformity and is sometimes inadequate or even incorrect.By using different conventional in vivo staining techniques,22lymph nodes could be demonstrated in BALB/cAnNCrl mice.They were named by analogy to the terms listed in Nomina Anatomica Veterinaria (2005).This terminology is based on precise nomen-clatory principles leading to short and simple terms with instructive and descriptive value.Several lymph nodes that were observed in BALB/cAnNCrl mice could be identified because of their comparative and topographic similarities with analogous lymph nodes in domestic carnivores,pigs,and herbivores,and they were named accordingly.However mice lack several lymph nodes that are present in other mammals,such as the deep parotid or proper lumbar lymph nodes.Despite the absence of these complementary structures in mice,the terms superficial parotid and lumbar aortic lymph nodes were retained because the pertaining adjectives have useful descriptive value.This was also the case for the term cranial deep cervical lymph node,although the superficial cervical and caudal deep cervical lymph nodes were not observed in BALB/cAnNCrl mice.No additional topographic adjective was used for the single tracheobronchal lymph node because a precise homol-ogy with either the right,left,or middle tracheobronchal lymph node of domestic animals could not be ascertained in the present study or by data from the literature (Cuq,1966;Kawashima et al.,1964).A number of lymph nodes that has been described in murine species by other authors were not found in the present study.The facial lymph node as mentioned by Wolvers et al.(1999),and the auricular (Anjuère et al.,1999;Dearman et al.,1996;Sailstad et al.,1995)and pre-auricular lymph node (Hendrickx et al.,1992)probably correspond with the superficial parotid ln.described in our study.The submental ln.,illustrated as bilateral lymph nodes in two papers (Cook,1983;Jacoby and Fox,1984),were not observed as nodes as such,but subcutaneous median lymph noduleswereFig.3.Intra-abdominal lymph nodes in the mouse (ventral view).(3a,3b,3c,3d)Ventral views of the abdominal cavity with the right kidney (a)(turned over to the left side in 3a),the right adrenal gland (b),the descending colon (c)(displaced in 3c)and the deep circumflex iliac artery (d);numbers (7,17–22)according to the description in Table 2.17W.Van den Broeck et al./Journal of Immunological Methods 312(2006)12–19present just caudal to the intermandibular synchondro-sis.Furthermore,there was no evidence of the caudal deep cervical ln.which has been described ventral to the trachea and dorsal to the sternum at the level of the first two ribs (Barone et al.,1950).Similarly,the existence of the superficial cervical ln.which has inconstantly be seen medial to the cervical part of the trapezius muscle and cranial to the supraspinatus muscle (Barone et al.,1950;Cuq,1966),and the presence of the femoral ln.which has been described in the inguinal region (Björkdahl et al.,1999;Mishell et al.,1980)could not be demonstrated.An intrathoracic pulmonary lymph node (Teitelbaum et al.,1999)was also absent in all mice examined in the present study.The (ileo)cecal lnn.,described in the ileocecal mesentery as accessory nodes (Barone et al.,1950;Cuq,1966),were not observed inour study,whereas the sacral ln.which has been illustrated by Popesko et al.(1992)most likely refers to the caudal mesenteric ln.as defined by Kawashima et al.(1964).Despite the minute dissections and the use of specific intrahepatic and intralienal stimulation techni-ques,our study failed to demonstrate the existence of hepatic and lienal lymph nodes in BALB/cAnNCrl mice.The presence of these nodes in mice has been discussed by Barone et al.(1950).According to these authors,murine lienal nodes are absent,which corre-sponds with the present findings in BALB/cAnNCrl mice.On the other hand,they observed a (retro)hepatic or portal lymph node which could hardly be discerned from the lymph nodes adjacent to the stomach and the pancreas.This lymph node corresponds most likely with the pancreaticoduodenal lymph node described in the present study.A novel finding in our study was the presence of a small and inconstant lateral iliac lymph node in BALB/cAnNCrl mice.The presence and lymphoid nature of the latter lymph node were verified by histological examination.It is not unlikely that this structure,along with other lymph nodes,might also be demonstrated in other murine species and breeds.To date,no precise nor conclusive data are available concerning the presence of hemal lymph nodes in mice.The exact function of these nodes,which are very obvious in some domestic animal species such as oxen and sheep,has still to be elucidated,but probably they perform a spleen-like function,as suggested by their morphology (Bassan et al.,1999).In the present study,the presence of hemal lymph nodes could not be demonstrated neither by macroscopic nor by micro-scopic examination in any of the stimulated or unstimulated regions in BALB/cAnNCrl mice.In summary,we recommend that scientific papers on laboratory animals,and on mice in particular,should carefully observe universally accepted rules of nomen-clature for the identification of all lymphatic organs that are described and investigated.ReferencesAnjuère,F.,Martin,P.,Ferrero,I.,López Fraga,M.,Martinez delHoyo,G.,Wright,N.,Ardavin,C.,1999.Definition of dendritic cell subpopulations present in the spleen,Peyer's patches,lymph nodes and skin of the mouse.Blood 93,590.Barone,R.,Bertrand,M.,Desenclos,R.,1950.Recherches anatomi-ques sur les ganglions lymphatiques des petits rongeurs de laboratoire.Rev.Méd.Vét.101,423.Bassan,N.,Vasquez,F.,Vinuesa,M.,Cerrutti,P.,Bernardi,S.,1999.Morphological alterations in hemal nodes in splenectomized cattle.Arq.Bras.Med.Vet.Zootec.51,445.Björkdahl,O.,Akerblad,P.,Gjörloff-Wingren,A.,Leanderson,T.,Dohlsten,M.,1999.Lymphoid hyperplasia in transgenicmiceFig.4.Schematic drawing of the localization of the lymph nodes in the 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I. I NTRODUCTION Miniature rotorcraft-based Unmanned Aerial Vehicles (UAVs) have received a growing interest in both industrial and academic research. Thanks to their hover capability, they are prone to be useful for many civil missions such as video supervision of road trafﬁc, surveillance of urban districts or building inspection for maintenance. Design of guidance navigation and control algorithms for the autonomous ﬂight of small rotorcraft-based UAVs is a challenging research area because of their nonlinear dynamics and their high sensitivity to aerodynamic perturbations. Various control strategies such as backstepping [12], adaptive backstepping [14], nonlinear model predictive control [11] or the combination of adaptive and model predictive control [1] have been successfully applied to UAV models. Nevertheless most of them require full state knowledge for feedback control design. For robotic systems it may be useful, for cost or payload reasons, to limit the number of embedded sensors. Therefore measurement of the full state may not be available. Classical solution to overcome consists in using observer. Such a solution was adopted in [6] where the problem of trajectory tracking for a planar Vertical Take Off and Landing (VTOL) aircraft with only positions and attitude angle measurement is treated by designing a full-order observer. Another approach that can be used to avoid computational burden or complexity due to the introduction of an observer is partial state feedback. Early work on partial state feedback has been done in the context of rigid-link robot manipulators when no velocity measurement is available. In [3] the velocity measurement is replaced by a velocity-related signal generated by a linear ﬁlter based only on link position measurement. An extension of this work can be found in [5] where a nonlinear ﬁlter is used. The same method has been applied to solve the problem of attitude
Sylvain Bertrand ONERA Chˆ atillon, France
sylvain.bertrand@onera.fr
Tarek Hamel I3S-UNSA-CNRS Sophia Antipolis, France
thamel@i3s.unice.fr
H´ el` ene Piet-Lahanier ONERA Chˆ atillon, France
helene.piet-lahanier@onera.frห้องสมุดไป่ตู้
Abstract— This paper presents a method to design guidance and control laws for small Vertical Take Off and Landing Unmanned Aerial Vehicles when no measurement of linear velocity nor angular velocity is available. The control strategy is based on the introduction of virtual states in the state equation of the system and allows the design of stabilizing feedback controllers without using any observer. Simulation results are provided for a six degrees of freedom model of a small rotorcraft-based Unmanned Aerial Vehicle.
tracking of rigid bodies with unknown inertia. A linear ﬁlter is employed in [17] to generate a velocity-related signal. In this work, a kinematic representation using modiﬁed Rodrigues parameters has been chosen. In [4], a unit quaternion based representation is adopted and a nonlinear ﬁlter generates a signal replacing the angular velocity measurement in the feedback controller. A unit quaternion representation is also used in [15] where a linear feedback controller depending on an estimation error quaternion is designed to solve the problem of a rigid spacecraft attitude control. As can be seen in the simulation or experimental results provided in some of the aforementioned works, oscillating closed loop behavior can be considered as the main drawback of partial state feedback strategies. In this paper we deal with the problem of guidance and control of a six degrees of freedom UAV model when no measurement of the linear velocity nor of the angular velocity is available. The method we present in this paper is based on the introduction of virtual states in the state equation of the system; no observer design is required. In addition, the closed loop oscillations are sensibly attenuated by the proposed approach. Contrary to the previous works, the kinematic representation we use exploits the SO(3) group and its manifold. Section 2 presents the system dynamics of a VTOL UAV and the cascaded structure of the controller. The design of the position controller is detailed in section 3 whereas the attitude controller is presented in section 4. Stability analysis of the closed loop system is given in section 5 and simulations results are provided in section 6. Concluding remarks are ﬁnally given in the last part of this paper. II. UAV MODEL AND CONTROL STRATEGY A. VTOL UAV model The VTOL UAV model is represented by a rigid body of mass m and of tensor of inertia I = diag(I1 , I2 , I3 ) with I1 , I2 and I3 strictly positive. We deﬁne an inertial reference frame (I ) associated with the vector basis (e1 , e2 , e3 ) and a body frame (B ) attached to the UAV and associated with b b the vector basis (eb 1 , e2 , e3 ) (see Fig. 1). The position and the linear velocity of the UAV in (I ) are respectively denoted