·Nature系列期刊导读·

重新看待细胞衰老

细胞衰老(cellular senescence)被看作是机体老化的原因。近来研究发现衰老细胞会抑制自身增殖，吸引免疫细胞将自己清除，最终促进组织再生。在老化的组织或患病组织中，这一系列事件未能正常完成，导致衰老细胞累积。机体启动细胞衰老程序是对癌基因活化、抗癌基因失效、端粒缩短等刺激产生应答以进行自我保护。此外，细胞衰老还参与了发育过程。因此，研究人员对衰老有了一个完整的认识，即细胞衰老的目的是“清除不再需要的细胞”，以便进行组织再生和替换。

X射线捕捉光合作用中光系统Ⅱ的分子过程

科学家第一次利用X射线自由电子激光器和精确延迟的X射线闪光捕获了运行中的光合作用的一个关键步骤，并获得了光系统Ⅱ(photosystemⅡ)将水分解为氢和氧时这一分子复合体的第一批快照。观察结果以分子分辨率显示出在这一过程中光系统Ⅱ显著地改变了形状。更深入地了解光合作用有可能有助于开发出更好的太阳能电池，并有可能推动追求生物化学的圣杯——人工光合作用。

论文链接:Kupitz C，et al..Serial time-resolved crystallography of photosystem Ⅱ using a femtosecond X-ray laser.

Abstract:Photosynthesis，a process catalysed by plants，algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth.Two large membrane protein complexes，photosystem I and II(PSI and PSII)，act in series to catalyse the light-driven reactions in photosynthesis.PSII catalyses the light-driven water splitting process，which maintains the Earth’s oxygenic atmosphere.In this process，the oxygen-evolving complex(OEC)of PSII cycles through five states，S0to S4，in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events.Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography.Structures have been determined from PSII in the dark S1state and after double laser excitation(putative S3state)at 5 and 5.5 Å resolution，respectively.The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5core of the OEC.These include an elongation of the metal cluster，accompanied by changes in the protein environment，which could allow for binding of the second substrate water molecule between the more distant protruding Mn(referred to as the‘dangler’Mn)and the Mn3CaOxcubane in the S2to S3transition，as predicted by spectroscopic and computational studies.This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of cataly tic processes in biomolecules.

单细胞western blot

研究人员开发出一种单细胞western blot(scWestern)方法，它采用一块可扩展的开放式微孔芯片结构，能在4 h内同时分析2 000个细胞。scWestern整合了所有关键的western blot步骤，实现了高度平行分析，通过被动重力驱动的细胞装置，细胞悬液接种到微孔中，不需单独获取每个细胞。应用这种方法研究了体外刺激下的干细胞信号和分化反应。scWestern突破了其他单细胞蛋白分析方法的限制，作为一种多功能工具，能够以单细胞分辨率研究复杂的细胞群体。

论文链接:Hughes A J，et al..Single-cell western blotting.

Nature Methods，2014，11:749-755.doi:10.1038/nmeth.2992.

Abstract:To measure cell-to-cell variation in protein-mediated functions，we developed an approach to conduct ～103concurrent single-cell western blots(scWesterns)in ～4 h.A microscope slide supporting a 30-μm-thick photoactive polyacrylamide gel enables western blotting:settling of single cells into microwells，lysis in situ，gel electrophoresis，photoinitiated blotting to immobilize proteins and antibody probing.We applied this scWestern method to monitor single-cell differentiation of rat neural stem cells and responses to mitogen stimulation.The scWestern quantified target proteins even with off-target antibody binding，multiplexed to 11 protein targets per single cell with detection thresholds of ＜30 000 molecules，and supported analyses of low starting cell numbers(～200)when integrated with FACS.The scWestern overcomes limitations of antibody fidelity and sensitivity in other single-cell protein analysis methods and constitutes a versatile tool for the study of complex cell populations at single-c ell resolution.

应高度重视基质细胞和免疫细胞的细胞代谢研究

癌细胞一直处于细胞代谢研究的中心。尽管基质细胞和免疫细胞能对癌症、炎症和代谢疾病产生重要影响，但这些细胞并没有得到应有的重视。科学家们系统论述了基质细胞和免疫细胞在健康/疾病状态下的代谢变化，以及代谢对细胞分化和功能的决定机制。呼吁研究者们将更多的精力投入到基质细胞和免疫细胞的代谢研究中，理解这些细胞在疾病发展中起到的作用。这类研究将为人们开辟治疗糖尿病、炎症性疾病和癌症的新途径。

论文链接:Ghesquière B，et al..Metabolism of stromal and immune cells in health and disease.

Nature，2014，511:167-176.doi:10.1038/nature13312.

Abstract:Cancer cells have been at the centre of cell metabolism research，but the metabolism of stromal and immune cells has received less attention.Nonetheless，these cells influence the progression of malignant，inflammatory and metabolic disorders.Here we discuss the metabolic adaptations of stromal and immune cells in health and disease，and highlight how metabolism determines their differentiation and function.

SCNT法生成的干细胞与iPS细胞相比或更适于细胞治疗

研究人员比较了通过“体细胞核转移”(SCNT)方法生成的人类多能干细胞与通过“转录因子介导的重新编程”方法生成的诱导多能干细胞(iPS细胞)的不同遗传特征、表观遗传特征和转录特征。在iPS细胞中观察到亲本体细胞典型的残留DNA甲基化，而在通过SCNT方法生成的干细胞中却未观察到。表明人类体细胞可以通过SCNT方法被重新编程为具有多能性的细胞，与iPS细胞相比或许更适合用于细胞治疗。

论文链接:Ma H，et al..Abnormalities in human pluripotent cells due to reprogramming mechanisms.

Nature，2014，511:177-183.doi:10.1038/nature13551.

Abstract:Human pluripotent stem cells hold potential for regenerative medicine，but available cell types have significant limitations.Although embryonic stem cells(ES cells)from in vitro fertilized embryos(IVF ES cells)represent the‘gold standard’，they are allogeneic to patients.Autologous induced pluripotent stem cells(iPS cells)are prone to epigenetic and transcriptional aberrations.To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method，genetically matched sets of human IVF ES cells，iPS cells and nuclear transfer ES cells(NT ES cells)derived by somatic cell nuclear transfer(SCNT)were subjected to genome-wide analyses.Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations.In contrast，DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells，whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells.Thus，human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replaceme nt therapies.

艾滋病免疫疗法的双面效应

SIV是类似于HIV的猴免疫缺陷病毒。研究人员发现给予感染猴免疫缺陷病毒(SIV)的猕猴Ⅰ型干扰素可产生有益和有害双重效应，而这种不同影响取决于给药的时间。在SIV感染初期给予猴子干扰素，可通过抑制炎症来阻碍感染加重;但在感染已经建立后给予它们干扰素，结果会适得其反。因此，对于HIV患者而言，干扰素有可能是一种有价值的治疗，不过仍需要研究来确定干扰素在人体中是否以相同的方式发挥作用。

论文链接:Sandler N G，et al..TypeⅠ interferon responses in rhesus macaques prevent SIV infection and slow disease progression.

Nature，doi:10.1038/nature13554.Published online:9 July，2014.

Abstract:Inflammation in HIV infection is predictive of non-AIDS morbidity and deathhigher set point plasma virus load and virus acquisition;thus，therapeutic agents are in development to reduce its causes and consequences.However，inflammation may simultaneously confer both detrimental and beneficial effects.This dichotomy is particularly applicable to typeⅠ interferons(IFN-Ⅰ)which，while contributing to innate control of infection，also provide target cells for the virus during acute infection，impair CD4 T-cell recovery，and are associated with disease progression.Here we manipulated IFN-Ⅰ signalling in rhesus macaques(Macaca mulatta)during simian immunodeficiency virus(SIV)transmission and acute infection with two complementary in vivo interventions.We show that blockade of the IFN-Ⅰ receptor caused reduced antiviral gene expression，increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation.In contrast，IFN-α2a administration initially upregulated expression of antiviral genes and prevented systemic infection.However，continued IFN-α2a treatment induced IFN-Ⅰ desensitization and decreased antiviral gene expression，enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss.Thus，the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation.Yet，the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.

通过免疫接种帮助青蛙抵抗“蛙壶菌”

真菌病原体“蛙壶菌”已在世界范围内造成很多两栖物种数量下降。此前几乎没有证据证明两栖动物能获得对这种病原体的抵抗力，科研人员对包括古巴树蛙在内的几种两栖类所做的实验显示，青蛙能学会避开这种病原体，能克服在反复接触“蛙壶菌”后由其所诱导产生的免疫抑制，并能利用死病原体获得对它的免疫力。因此可用疫苗诱导青蛙产生抵抗力，帮助它们在已发生灾难性种群数量下降的区域重新繁衍。

论文链接:McMahon T A，etal.. Amphibians acquire resistance to live and dead fungus overcoming fungal immunosuppression.

Nature，2014，511:224-227.doi:10.1038/nature13491.

Abstract:Emerging fungal pathogens pose a greater threat to biodiversity than any other parasitic group，causing declines of many taxa，including bats，corals，bees，snakes and amphibians.Currently，there is little evidence that wild animals can acquire resistance to these pathogens.Batrachochytrium dendrobatidis is a pathogenic fungus implicated in the recent global decline of amphibians.Here we demonstrate that three species of amphibians can acquire behavioural or immunological resistance to B.dendrobatidis.Frogs learned to avoid the fungus after just one B.dendrobatidis exposure and temperature-induced clearance.In subsequent experiments in which B.dendrobatidis avoidance was prevented，the number of previous exposures was a negative predictor of B.dendrobatidis burden on frogs and B.dendrobatidis-induced mortality，and was a positive predictor of lymphocyte abundance and proliferation.These results suggest that amphibians can acquire immunity to B.dendrobatidis that overcomes pathogen-induced immunosuppression and increases their survival.Importantly，exposure to dead fungus induced a similar magnitude of acquired resistance as exposure to live fungus.Exposure of frogs to B.dendrobatidis antigens might offer a practical way to protect pathogen-naive amphibians and facilitate the reintroduction of amphibians to locations in the wild where B.dendrobatidis persists.Moreover，given the conserved nature of vertebrate immune responses to fungi and the fact that many animals are capable of learning to avoid natural enemies，these results offer hope that other wild animal taxa threatened by invasive fungi might be rescued by management approaches based on herd immunity.

人类基因存在程序性核糖体移码

利用在HIV感染中起至关重要作用的一种基因展开研究，研究人员发现某些人类基因具有一套备用操作指令。备用指令可迅速地改变蛋白质的成分、功能以及生存的能力。这一称作为程序性核糖体移码(programmed ribosomal frameshifting)的现象，是在1985年于病毒中被发现，而该研究第一次证实了人类基因利用程序性核糖体移码来改变其组装蛋白质的方式。

论文链接: Belew A T，et al..Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway.

Nature，doi:10.1038/nature13429.Published online:9 July，2014.

Abstract:Programmed-1 ribosomal frameshift(-1 PRF)signals redirect translating ribosomes to slip back one base on messenger RNAs.Although well characterized in viruses，how these elements may regulate cellular gene expression is not understood.Here we describe a-1 PRF signal in the human mRNA encoding CCR5，the HIV-1 co-receptor.CCR5 mRNA-mediated-1 PRF is directed by an mRNA pseudoknot，and is stimulated by at least two microRNAs.Mapping the mRNA-miRNA interaction suggests that formation of a triplex RNA structure stimulates-1 PRF.A -1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon，destabilizing it through the nonsense-mediated mRNA decay pathway.At least one additional mRNA decay pathway is also involved.Functional-1 PRF signals that seem to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors，suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells.

12种miRNA表达平台的比较

microRNA(miRNA)作为基因表达的重要调节因子，在近年来被广泛研究。目前，人们利用各种不同的技术来确定生物样品中miRNA的相对丰度，包括小RNA测序、RT-qPCR和芯片等。这些技术究竟孰优孰劣，研究者比较了12种miRNA表达分析平台，分析表明，基于相同技术的平台可能有着非常不同的性能。

论文链接:Mestdagh P，et al..Evaluation of quantitative miRNA expression platforms in the microRNA quality control(miRQC)study.

Nature Methods，doi:10.1038/nmeth.3014.Published online:29 June，2014.

Abstract:MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years.Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing，reverse transcription-quantitative PCR(RT-qPCR)and(microarray)hybridization.In this study，we systematically compared 12 commercially available platforms for analysis of microRNA expression.We measured an identical set of 20 standardized positive and negative control samples，including human universal reference RNA，human brain RNA and titrations thereof，human serum samples and synthetic spikes from microRNA family members with varying homology.We developed robust quality metrics to objectively assess platform performance in terms of reproducibility，sensitivity，accuracy，specificity and concordance of differential expression.The results indicate that each method has its strengths and weaknesses，which help to guide informed selection of a quantitative microRNA gene expression platform for particular stud y goals.