·Nature系列期刊導讀·

重新看待細胞衰老

細胞衰老(cellular senescence)被看作是機體老化的原因。近來研究發(fā)現(xiàn)衰老細胞會抑制自身增殖，吸引免疫細胞將自己清除，最終促進組織再生。在老化的組織或患病組織中，這一系列事件未能正常完成，導致衰老細胞累積。機體啟動細胞衰老程序是對癌基因活化、抗癌基因失效、端?？s短等刺激產(chǎn)生應答以進行自我保護。此外，細胞衰老還參與了發(fā)育過程。因此，研究人員對衰老有了一個完整的認識，即細胞衰老的目的是“清除不再需要的細胞”，以便進行組織再生和替換。

X射線捕捉光合作用中光系統(tǒng)Ⅱ的分子過程

科學家第一次利用X射線自由電子激光器和精確延遲的X射線閃光捕獲了運行中的光合作用的一個關(guān)鍵步驟，并獲得了光系統(tǒng)Ⅱ(photosystemⅡ)將水分解為氫和氧時這一分子復合體的第一批快照。觀察結(jié)果以分子分辨率顯示出在這一過程中光系統(tǒng)Ⅱ顯著地改變了形狀。更深入地了解光合作用有可能有助于開發(fā)出更好的太陽能電池，并有可能推動追求生物化學的圣杯——人工光合作用。

論文鏈接: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 ? 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.

單細胞western blot

研究人員開發(fā)出一種單細胞western blot(scWestern)方法，它采用一塊可擴展的開放式微孔芯片結(jié)構(gòu)，能在4 h內(nèi)同時分析2 000個細胞。scWestern整合了所有關(guān)鍵的western blot步驟，實現(xiàn)了高度平行分析，通過被動重力驅(qū)動的細胞裝置，細胞懸液接種到微孔中，不需單獨獲取每個細胞。應用這種方法研究了體外刺激下的干細胞信號和分化反應。scWestern突破了其他單細胞蛋白分析方法的限制，作為一種多功能工具，能夠以單細胞分辨率研究復雜的細胞群體。

論文鏈接: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.

應高度重視基質(zhì)細胞和免疫細胞的細胞代謝研究

癌細胞一直處于細胞代謝研究的中心。盡管基質(zhì)細胞和免疫細胞能對癌癥、炎癥和代謝疾病產(chǎn)生重要影響，但這些細胞并沒有得到應有的重視。科學家們系統(tǒng)論述了基質(zhì)細胞和免疫細胞在健康/疾病狀態(tài)下的代謝變化，以及代謝對細胞分化和功能的決定機制。呼吁研究者們將更多的精力投入到基質(zhì)細胞和免疫細胞的代謝研究中，理解這些細胞在疾病發(fā)展中起到的作用。這類研究將為人們開辟治療糖尿病、炎癥性疾病和癌癥的新途徑。

論文鏈接: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法生成的干細胞與iPS細胞相比或更適于細胞治療

研究人員比較了通過“體細胞核轉(zhuǎn)移”(SCNT)方法生成的人類多能干細胞與通過“轉(zhuǎn)錄因子介導的重新編程”方法生成的誘導多能干細胞(iPS細胞)的不同遺傳特征、表觀遺傳特征和轉(zhuǎn)錄特征。在iPS細胞中觀察到親本體細胞典型的殘留DNA甲基化，而在通過SCNT方法生成的干細胞中卻未觀察到。表明人類體細胞可以通過SCNT方法被重新編程為具有多能性的細胞，與iPS細胞相比或許更適合用于細胞治療。

論文鏈接: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.

艾滋病免疫療法的雙面效應

SIV是類似于HIV的猴免疫缺陷病毒。研究人員發(fā)現(xiàn)給予感染猴免疫缺陷病毒(SIV)的獼猴Ⅰ型干擾素可產(chǎn)生有益和有害雙重效應，而這種不同影響取決于給藥的時間。在SIV感染初期給予猴子干擾素，可通過抑制炎癥來阻礙感染加重;但在感染已經(jīng)建立后給予它們干擾素，結(jié)果會適得其反。因此，對于HIV患者而言，干擾素有可能是一種有價值的治療，不過仍需要研究來確定干擾素在人體中是否以相同的方式發(fā)揮作用。

論文鏈接: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.

通過免疫接種幫助青蛙抵抗“蛙壺菌”

真菌病原體“蛙壺菌”已在世界范圍內(nèi)造成很多兩棲物種數(shù)量下降。此前幾乎沒有證據(jù)證明兩棲動物能獲得對這種病原體的抵抗力，科研人員對包括古巴樹蛙在內(nèi)的幾種兩棲類所做的實驗顯示，青蛙能學會避開這種病原體，能克服在反復接觸“蛙壺菌”后由其所誘導產(chǎn)生的免疫抑制，并能利用死病原體獲得對它的免疫力。因此可用疫苗誘導青蛙產(chǎn)生抵抗力，幫助它們在已發(fā)生災難性種群數(shù)量下降的區(qū)域重新繁衍。

論文鏈接: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.

人類基因存在程序性核糖體移碼

利用在HIV感染中起至關(guān)重要作用的一種基因展開研究，研究人員發(fā)現(xiàn)某些人類基因具有一套備用操作指令。備用指令可迅速地改變蛋白質(zhì)的成分、功能以及生存的能力。這一稱作為程序性核糖體移碼(programmed ribosomal frameshifting)的現(xiàn)象，是在1985年于病毒中被發(fā)現(xiàn)，而該研究第一次證實了人類基因利用程序性核糖體移碼來改變其組裝蛋白質(zhì)的方式。

論文鏈接: 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種miRNA表達平臺的比較

microRNA(miRNA)作為基因表達的重要調(diào)節(jié)因子，在近年來被廣泛研究。目前，人們利用各種不同的技術(shù)來確定生物樣品中miRNA的相對豐度，包括小RNA測序、RT-qPCR和芯片等。這些技術(shù)究竟孰優(yōu)孰劣，研究者比較了12種miRNA表達分析平臺，分析表明，基于相同技術(shù)的平臺可能有著非常不同的性能。

論文鏈接: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.