·Nature系列期刊导读·

疟原虫青蒿素抗药性的分子标记

青蒿素提取自传统中药青蒿（Artemisia annua），与其他药物联合使用可用于治疗疟疾。为监测具有青蒿素抗性的疟原虫，法国巴斯德研究所的研究人员识别出“镰刀形疟原虫”青蒿素抗药性的一个主要决定因子。该寄生虫的PF3D7＿1343700kelch propeller domain（K-13propeller）中发生的等位基因突变频率增加与最近的抗药性传播有关。该发现除提出一个有用的分子标记外，还有助于加深对抗药性形成的认识，并为在新型抗疟疾药物减少抗药性提供思路。

论文链接： Ariey F，et al..A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.

Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide.To monitor the spread of artemisinin resistance，a molecular marker is urgently needed.Here，using wholegenome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia，we associate mutations in the PF3D7＿1343700kelch propeller domain（‘K13-propeller’）with artemisinin resistance in vitro and in vivo.Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent，and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia.Strong correlations between the presence of a mutant allele，in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance.K13-propeller polymorphism constitutes a useful molecular marker for largescale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

新方法可批量制备用于表观遗传学研究的重组抗体

目前研究中使用的抗体大多经过不同批次从动物体内产生获得，而每批次之间存在着较大的质量变化，对研究工作产生影响。研究人员放弃通过动物进行抗体生产，转而利用遗传手段在细菌体内制造具有高识别度的重组抗体以用于组蛋白的特定修饰。通过与市场上用动物制成的抗体相比较，这种新抗体的表现更佳，质量完全不受批次时间影响。这种抗体制备方法还能开发出经过一种以上化学修饰的抗体，可帮助破解如相邻甲基基团或其他表观遗传修饰的组合是如何影响基因表达等相关问题。

论文链接： Hattori T，et al..Recombinant antibodies to histone post-translational modifications.

Nature Methods，2013，（10），992-995.doi：10.1038／nmeth.2605.

Abstract：Variability in the quality of antibodies to histone post-translational modifications（PTMs）is a widely recognized hindrance in epigenetics research.Here，we produced recombinant antibodies to the trimethylated lysine residues of histone H3with high specificity and affinity and no lot-to-lot variation.These recombinant antibodies performed well in common epigenetics applications，and enabled us to identify positive and negative correlations amonghistone PTMs.

促进肠道固有菌群的互利抗菌机制

人体的上皮细胞通过各种抗菌蛋白的保护，能够与复杂的细菌群落密切共存。C-型外源凝集素RegIII蛋白家族是一类杀菌性蛋白，可限制细菌和肠上皮细胞间的直接接触，从而促进肠道对菌群的耐受性。研究发现，RegIIIα（也称为HIP／PAP）是这一蛋白家族的一员，可结合细胞膜磷脂，并通过形成六聚体低聚物孔隙使膜通透化而杀死革兰氏阳性细菌，对革兰氏阴性菌不起作用。该项研究能够帮助深入了解促进固有菌群的互利抗菌机制。

论文链接： Mukherjee S，et al..Antibacterial membrane attack by apore-forming intestinal C-type lectin.

Nature，doi：10.1038／nature12729.Published online：20November，2013.

Abstract：Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins.C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota.RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate，but the mechanism by which they kill bacteria is unknown.Here we elucidate the mechanistic basis for RegIII bactericidal activity.We show that human RegIIIα （also known as HIP／PAP）binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore.We derive a three-dimensional model of the RegIIIαpore by docking the RegIIIαcrystal structure into a cryo-electron microscopic map of the pore complex，and show that the model accords with experimentally determined properties of the pore.Lipopolysaccharide inhibits RegIIIαpore-forming activity，explaining why RegIIIαis bactericidal for Gram-positive but not Gram-negative bacteria.Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system，and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.

叶酸缺乏导致小鼠精子DNA变化

表观遗传DNA改变（如DNA甲基化）会改变基因活性，但不影响DNA的序列。研究发现，饮食中的叶酸会影响细胞DNA甲基化水平和基因表达，给雄性小鼠终生喂食缺乏叶酸的食物，导致雄性小鼠精子DNA甲基化发生改变且生育能力降低。这些雄性小鼠的雄性和雌性后代产生发育异常的频率都要比采用正常饮食的雄性小鼠的后代高。在一些情况下，这些小鼠会生出有颅面缺陷和脊椎畸形等发育异常的后代。目前这种表观遗传传递的机制还不清楚，造成发育缺陷的分子水平原因尚待研究。

论文链接： R.Lambrot，et al..Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes.

Nature Communications，2013，4（2889）.doi：10.1038／ncomms3889.

Abstract：Epidemiological studies suggest that a father’s diet can influence offspring health.A proposed mechanism for paternal transmission of environmental information is via the sperm epigenome.The epigenome includes heritable information such as DNA methylation.We hypothesize that the dietary supply of methyl donors will alter epigenetic reprogramming in sperm.Here we feed male mice either a folate-deficient or folate-sufficient diet throughout life.Paternal folate deficiency is associated with increased birth defects in the offspring，which include craniofacial and musculoskeletal malformations.Genome-wide DNA methylation analysis and the subsequent functional analysis identify differential methylation in sperm of genes implicated in development，chronic diseases such as cancer，diabetes，autism and schizophrenia.While＞300genes are differentially expressed in offspring placenta，only two correspond to genes with differential methylation in sperm.This model suggests epigenetic transmission may involve sperm histone H3methylation or DNA methylation and that adequate paternal dietary folate is essential for offspringhealth.

评估荧光蛋白的光激活效率

光控的荧光探针，是对局部进行超高分辨率显微分析的核心。其中，荧光蛋白可以通过基因编码，实现与目的蛋白的1∶1标记。所以，荧光蛋白探针特别适合于单分子计数。ICFO的科学家们对所有已知的“不可逆光控荧光蛋白”，进行了光激活效率的检测，为蛋白的分子定量提供了相当详细的参考框架。研究人员用爪蟾卵母细胞表达的人类甘氨酸受体作为纳米模板，对多种荧光蛋白进行分析，确定了被光激活的蛋白比率。该方法理论上可以对目标蛋白进行分子定量，但仍有限制。通过对该方法参数的优化，可以生成更适合超高分辨率分子计数的探针。

论文链接： Durisic N，et al..Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate.

Nature Methods，doi：10.1038／nmeth.2784.Published online：05January，2014.

Abstract：Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy.Among these probes，fluorescent proteins are appealing because they are genetically encoded.Moreover，the ability to achieve a 1：1labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting.The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form （i.e.，the photoactivation efficiency）plays a crucial part in properly interpreting the quantitative information.It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging.Here，we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy（PALM）to determine the photoactivation efficiency of fluorescent proteins mEos2，mEos3.1，mEos3.2，Dendra2，mClavGR2，mMaple，PA-GFP and PA-mCherry.This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.

大鼠多基因同步敲除技术

实验中敲除一个基因很容易实现，但动物的某个性状往往由几个基因同时控制，因此多基因同步敲除技术更为重要。不过，目前多基因同步敲除还存在技术上的障碍。我国研究人员首次利用CRISPRCas系统诱导大鼠的Tet1／Tet2／Tet3基因敲除，实现了效率高达100%的双等位基因纯合突变的单基因敲除和接近60%高效率的三基因同时敲除大鼠，并且证明CRISPR-Cas系统引入的基因修饰可通过生殖细胞传递到下一代。该技术可推动基因修饰大鼠成为重要的动物模型。

论文链接： Wei Li，et al..Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems.

Nature Biotechnology，2013，31：684-686.doi：10.1038／nbt.2652.

Abstract：CRISPRs are clustered，regularly interspaced，short palindromic repeats present in many bacteria and archaea genomes.Proteins encoded by CRISPR-associated（Cas）genes serve as guardians of the genome，which target foreign DNA at specific sites by means of small CRISPR RNA（crRNA）-guided DNA recognition and degradation.Recently，several groups described how CRISPRCas systems efficiently create site-specific gene modifications in whole organisms such as Streptococcus pneumoniae，Escherichia coli，Danio rerio（zebrafish）and mice，suggesting its potential application in the production of genetically engineered organisms，although germline transmission of the mutations remains to be shown.Here，we report the use of CRISPR-Cas systems to generate multiple gene mutations in rats in a germline-competent manner.

高粱基因组存在遗传变异

中澳两国研究人员合作对重要粮食饲料作物高粱进行了全基因组测序及分析。该研究通过对44株不同来源的高粱样本，包括地方品种、改良品种和野生杂草材料，进行了全基因组重测序及分析，并首次对拟高粱进行了全基因组测序。研究发现，高粱与拟高粱都存在丰富的遗传多样性。通过比较分析，科研人员还发现不同的高粱品种在基因组中存在着强烈的种群结构差异和复杂的驯化历程。为今后高粱及其他粮食作物的育种改良提供了宝贵的遗传资源，也为解决全球日益严峻的粮食问题奠定了重要的科研基础。

论文链接： Emma S.Mace，et al..Whole-genome sequencing reveals untapped genetic potential in Africa’s indigenous cereal crop sorghum.

Nature Communications，2013，4：2320.doi：10.1038／ncomms3320.

Abstract：Sorghum is a food and feed cereal crop adapted to heat and drought and a staple for 500million of the world’s poorest people.Its small diploid genome and phenotypic diversity make it an ideal C4grass model as a complement to C3rice.Here we present high coverage（16-45×）resequenced genomes of 44sorghum lines representing the primary gene pool and spanning dimensions of geographic origin，end-use and taxonomic group.We also report the first resequenced genome of S.propinquum，identifying 8Mhigh-quality SNPs，1.9Mindels and specific gene loss and gain events in S.bicolor.We observe strong racial structure and a complex domestication history involving at least two distinct domestication events.These assembled genomes enable the leveraging of existing cereal functional genomics data against the novel diversity available in sorghum，providing an unmatched resource for the genetic improvement of sorghum and other grass species.

并非所有物种都因衰老而加速死亡

多数动物的死亡率会随着年龄的增长而大幅增加。然而有些动物享有近乎恒定水平的繁殖力和死亡率。研究人员对比了46个物种的标准人口统计学模式，其中包括11种哺乳动物、12种其他脊椎动物、10种无脊椎动物、12种维管植物和一种绿藻，通过平均死亡率将生命周期中每一节点的死亡率区分开来，发现生命长度与衰老程度之间并没有直接的联系。有24个物种的死亡率随着衰老而表现了最为突兀的增长，其中的11个物种具有相对较长的生命周期，而另13个物种则寿命相对较短。生命周期中的类似分裂也发生在那些死亡率并没有明显增加的物种中。这是第一次尝试使跨物种的死亡率及存活比较标准化。

论文链接： Owen R.Jones，et al..Diversity of ageing across the tree of life.

Nature，2013，doi：10.1038／nature12789.

Abstract：Evolution drives，and is driven by，demography.A genotype moulds its phenotype’s age patterns of mortality and fertility in an environment；these two patterns in turn determine the genotype’s fitness in that environment.Hence，to understand the evolution of ageing，age patterns of mortality and reproduction need to be compared for species across the tree of life.However，few studies have done so and only for a limited range of taxa.Here we contrast standardized patterns over age for 11mammals，12other vertebrates，10invertebrates，12vascular plants and a green alga.Although it has been predicted that evolution should inevitably lead to increasing mortality and declining fertility with age after maturity，there is great variation among these species，including increasing，constant，decreasing，humped and bowed trajectories for both long-and short-lived species.This diversity challenges theoreticians to develop broader perspectives on the evolution of ageing and empiricists to study the demography of more species.

研究开发出抗旱水稻新品种

水稻种植需要大量水，所以干旱天气是个大敌。日本研究人员对一种名为IR64的水稻（属于籼稻）进行了基因研究，发现其DRO1基因有部分缺损。通过杂交，研究人员为IR64重新植入DRO1基因。结果发现其扎根深度达到以前的2倍以上。在水稻几乎会绝收的严重干旱环境下，植入DRO1基因后，收获量能够达到通常水平的30%左右。这一抗旱水稻新品种有助于对抗干旱对水稻种植区的影响。此外，玉米等其他作物也有类似基因，所以此次的研究成果也有望促进开发出其他作物的耐旱新品种。

论文链接： Yusaku Uga，et al..Control of root system architecture by DEEPER ROOTING1increases rice yield under drought conditions.

Nature Genetics，2013，45：1097-1102.doi：10.1038／ng.2725.

Abstract：The genetic improvement of drought resistance is essential for stable and adequate crop production in drought-prone areas1.Here we demonstrate that alteration of root system architecture improves drought avoidance through the cloning and characterization of DEEPER ROOTING1（DRO1），a rice quantitative trait locus controlling root growth angle.DRO1is negatively regulated by auxin and is involved in cell elongation in the root tip that causes asymmetric root growth and downward bending of the root in response to gravity.Higher expression of DRO1increases the root growth angle，whereby roots grow in a more downward direction.Introducing DRO1into a shallow-rooting rice cultivar by backcrossing enabled the resulting line to avoid drought by increasing deep rooting，which maintained high yield performance under drought conditions relative to the recipient cultivar.Our experiments suggest that control of root system architecture will contribute to drought avoidance in crops.

参与调控植物分枝（蘖）的生长发育基因发现

我国科学家首次在遗传和生化层面证实D53蛋白可作为独脚金内酯信号途径的抑制子，参与调控植物分枝（蘖）的生长发育。研究人员通过精细定位和图位克隆，获得了位于水稻第11号染色体短臂末端的DWARF53（D53）基因，结果表明，d53是一个独脚金内酯不敏感突变体。在独脚金内酯存在的条件下D53蛋白可与两个已知的独脚金内酯信号分子D14、D3互作形成蛋白复合体，进而使得泛素化的D53蛋白特异地被蛋白酶体系统降解，从而诱导下游目标基因的表达以及独脚金内酯信号的响应。该研究为植物，特别是农作物的株型改良提供了重要的理论基础。

论文链接： Feng Zhou，et al..D14-SCFD3-dependent degradation of D53regulates strigolactone signalling.

Nature，2013，504，406-410.doi：10.1038／nature12878.

Abstract：Strigolactones（SLs），a newly discovered class of carotenoid-derived phytohormones，are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi.Despite the rapid progress in elucidating the SL biosynthetic pathway，the perception and signalling mechanisms of SL remain poorly understood.Here we show that DWARF 53（D53）acts as a repressor of SL signalling and that SLs induce its degradation.We find that the rice（Oryza sativa）d53mutant，which produces an exaggerated number of tillers compared to wild-type plants，is caused by again-of-function mutation and is insensitive to exogenous SL treatment.The D53gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with theα／βhydrolase protein DWARF 14（D14）and the F-box protein DWARF 3（D3），two previously identified signalling components potentially responsible for SL perception.We demonstrate that，in a D14-and D3-dependent manner，SLs induce D53degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth.Our combined genetic and biochemical data reveal that D53acts as a repressor of the SL signalling pathway，whose hormone-induced degradation represents a key molecular link between SL perception and responses.

一种DNA修复酶可修复特定基因组不稳定性

去乙酰化酶1（SIRT1）是一种用来修复受损DNA的酶，由于去乙酰化酶具有防止大脑细胞凋亡的保护作用，研究发现，没有SIRT1，神经元无法修复由有毒化学物质造成的DNA损伤，且SIRT1受到另一种酶的控制调节。该研究认为SIRT1可以修复患有神经退行性疾病诸如阿尔茨海默氏症和肌萎缩侧索硬化症（又名“渐冻人”症，ALS）的小鼠体内的基因组不稳定性。

论文链接： Matthew M Dobbin，et al..SIRT1collaborates with ATM and HDAC1to maintain genomic stability in neurons.

Nature Neuroscience，2013，16，1008-1015.doi：10.1038／nn.3460.

Abstract：Defects in DNA repair have been linked to cognitive decline with age and neurodegenerative disease，yet the mechanisms that protect neurons from genotoxic stress remain largely obscure.We sought to characterize the roles of the NAD＋-dependent deacetylase SIRT1in the neuronal response to DNA double-strand breaks（DSBs）.We found that SIRT1was rapidly recruited to DSBs in postmitotic neurons，where it showed a synergistic relationship with ataxia telangiectasia mutated （ATM）.SIRT1 recruitment to breaks was ATM dependent；however，SIRT1also stimulated ATM autophosphorylation and activity and stabilized ATM at DSB sites.After DSB induction，SIRT1also bound the neuroprotective class I histone deacetylase HDAC1.We found that SIRT1deacetylated HDAC1and stimulated its enzymatic activity，which was necessary for DSB repair through the nonhomologous end-joining pathway.HDAC1mutations that mimic a constitutively acetylated state rendered neurons more susceptible to DNA damage，whereas pharmacological SIRT1activators that promoted HDAC1deacetylation also reduced DNA damage in two mouse models of neurodegeneration.We propose that SIRT1is an apical transducer of the DSB response and that SIRT1activation offers an important therapeutic avenue in neurodegeneration.

构建小鼠基因组CRISPR导向RNAs文库

CRISPR技术是指利用一段与靶序列相同的导向RNA来引导Cas9核酸酶对特异性靶向DNA进行识别和切割，造成DNA的双链或单链断裂，细胞随后会对断裂的DNA进行修复。利用该技术，研究人员构建出了一个可诱导小鼠基因组全基因靶向突变的CRISPR导向RNA（gRNAs）综合文库。利用这些导向RNAs可以靶向和改变小鼠基因组中所有的基因，并已构建小鼠突变胚胎干细胞，进而针对腐败梭菌α毒素进行了一次遗传筛查。研究小组靶向了26个已知与这种细菌毒素受体合成相关的基因，揭示出其中17个基因导致了抗性发生。同时，还发现了一些未知基因，这些基因发生突变赋予了对这种有毒物质的抗性。

论文链接： Koike-Yusa H，et al..Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.

Nature Biotechnology，doi：10.1038／nbt.2800.Published online：23December，2013.

Abstract：Identification of genes influencing aphenotype of interest is frequently achieved through genetic screening by RNA interference（RNAi）or knockouts.However，RNAi may only achieve partial depletion of gene activity，and knockout-based screens are difficult in diploid mammalian cells.Here we took advantage of the efficiency and high throughput of genome editing based on type II，clustered，regularly interspaced，short palindromic repeats（CRISPR）-CRISPR-associated （Cas）systems to introduce genome-wide targeted mutations in mouse embryonic stem cells（ESCs）.We designed 87，897guide RNAs（gRNAs）targeting 19，150mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9.Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4previously unknown genes implicated in these phenotypes.Our results demonstrate the potential for efficient loss-offunction screening using the CRISPR-Cas9system.