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象鲨(Callorhinchus milii)是澳大利亚南部和新西兰温带水域的一种本地软骨鱼，生活在200米到500米深处，在春季迁移到浅水中进食。
本期Nature发表了这种鱼的基因组序列。与其他脊椎动物基因组所做比较显示，它是所有已知脊椎动物（包括腔棘鱼）中演化最慢的基因组。
基因组分析表明，它有一个不同寻常的适应性免疫系统，缺少CD4受体和一些相关的细胞因子，这说明软骨鱼拥有一个原始的有颌类适应性免疫系统。其基因组中也没有编码分泌的钙结合性磷蛋白的基因，这与软骨鱼没有骨头的事实是一致的。
Nature doi:10.1038/nature12826
Elephant shark genome provides unique insights into gnathostome evolution
Byrappa Venkatesh， Alison P. Lee， Vydianathan Ravi， Ashish K. Maurya， Michelle M. Lian， Jeremy B. Swann， Yuko Ohta， Martin F. Flajnik， Yoichi Sutoh， Masanori Kasahara， Shawn Hoon， Vamshidhar Gangu， Scott W. Roy， Manuel Irimia， Vladimir Korzh， Igor Kondrychyn， Zhi Wei Lim， Boon-Hui Tay， Sumanty Tohari， Kiat Whye Kong， Shufen Ho， Belen Lorente-Galdos， Javier Quilez， Tomas Marques-Bonet， Brian J. Raney
The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations， such as hinged jaws， paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups， the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish， the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates， including the ‘living fossil’ coelacanth， and features extensive synteny conservation with tetrapod genomes， making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore， the adaptive immune system of cartilaginous fishes is unusual： it lacks the canonical CD4 co-receptor and most transcription factors， cytokines and cytokine receptors related to the CD4 lineage， despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.
来自中国科学院动物研究所、深圳华大基因研究院等单位的科研人员成功破译了迄今为止最大的动物基因组–飞蝗全基因组序列图谱，为揭示蝗灾暴发机制，开发可持续性治理策略及新的控制方法提供了宝贵的遗传资源，也为推进飞蝗成为研究人类疾病和行为的生物医学模型奠定了重要基础。最新研究成果于2014年1月14日在《自然o通讯》（Nature Communications）杂志上在线发表。
飞蝗基因组大小约6.5GB，是人类基因组的两倍多，约是果蝇基因组的30倍，是目前科学家成功破译的最大动物基因组。在迄今为止被破译的近百个动物物种中，不乏个体外观较大的动物如小须鲸、藏羚羊、东北虎、白鳍豚，但超乎人们想象的是，个体如此之小的蝗虫竟拥有如此庞大的基因组。动物体形大小与基因组大小并不成比例，这是当今生物学界的一个未解之谜。
在本研究中，科研人员发现飞蝗之所以拥有如此巨大的基因组是由于自身转座因子的扩增以及这些因子相对缓慢的损失速度导致的，一般飞蝗基因组中转座因子损失的速度明显慢于其他昆虫。此外，在飞蝗基因组中，他们发现至少60%的区域存在着大量重复序列，约有2,639个重复序列家族，然而位于前十的重复序列家族总数仅占整个基因组序列的10%，这表明在飞蝗基因组中没有占据主导地位的家族基因。
飞蝗具有聚群和长距离迁徙能力。数以亿计的飞蝗能够突然且不可预计的形成蝗群，它们能够以每天数百千米的速度飞行甚至是穿越海洋。为了探究飞蝗强大的迁徙能力，科研人员发现与脂肪转移和抗氧化剂保护有关的基因发生了显著拷贝数扩增。这些基因包括7个PAT家族蛋白，11个脂肪酸结合蛋白，9个半胱氨酸抗氧化剂蛋白，12个谷胱甘肽巯基转移酶基因。此外，这些家族基因的部分基因在飞蝗飞行前后的脂肪体组织中发生了差异性表达，这说明这些家族基因可能在飞蝗飞行过程中扮演了重要的角色。由于飞行是昆虫消耗能量最大的运动之一，参与脂肪代谢的基因的扩增表明飞蝗已经形成了高效的能量供应系统，从而满足其在长距离飞行中高强度的能量消耗。
型变是一种依赖于密度的非遗传多型性，是蝗虫的一个重要特征，它涉及到一系列的生物和表型特性，包括身体颜色、形态、行为、生理、免疫反应及其它方面的变化。群体密度的增加可引发蝗虫从散居向群居的转变，导致蝗虫大量地聚集到一起，这种变化可快速发生、具有可逆性，并且在不同的亚种中转变的速度迥异。在本次研究中，科研人员首次揭示出两型转变的深层次原因，并表明型变与飞蝗周边及中枢神经系统中参与微管动力调控的多分子进程有关。科研人员表示调控神经可塑性的基因在表达量、DNA甲基化以及可变剪切方面均发生了明显变化。这些基因可能在控制飞蝗形成大的群体过程中发挥重要功能。
此外，科研人员还发现数百个潜在的杀虫剂目标基因，包括半胱氨酸环配体门控离子通道，G蛋白偶联受体以及一些致死基因，对今后控制蝗虫灾害提供了新的思路。飞蝗喜欢禾本科植物作为食物来源，研究人员发现飞蝗身体中的代谢解毒酶类（糖苷键转移酶）的基因数目在所有已测序昆虫中最为丰富，这类酶能够降解禾本科植物中存在的特定次生代谢物，为解释飞蝗以禾本科植物为食的原因提供了重要的科研依据。
Nature Communications doi:10.1038/ncomms3957
The locust genome provides insight into swarm formation and long-distance flight
Xianhui Wang,Xiaodong Fang, Pengcheng Yang,Xuanting Jiang,Feng Jiang,Dejian Zhao, Bolei Li,Feng Cui,Jianing Wei,Chuan Ma,Yundan Wang,Jing He,Yuan Luo, Zhifeng Wang,Xiaojiao Guo,Wei Guo,Xuesong Wang,Yi Zhang,Meiling Yang,Shuguang Hao et al.
Locusts are one of the world’s most destructive agricultural pests and represent a useful model system in entomology. Here we present a draft 6.5?Gb genome sequence of Locusta migratoria, which is the largest animal genome sequenced so far. Our findings indicate that the large genome size of L. migratoria is likely to be because of transposable element proliferation combined with slow rates of loss for these elements. Methylome and transcriptome analyses reveal complex regulatory mechanisms involved in microtubule dynamic-mediated synapse plasticity during phase change. We find significant expansion of gene families associated with energy consumption and detoxification, consistent with long-distance flight capacity and phytophagy. We report hundreds of potential insecticide target genes, including cys-loop ligand-gated ion channels, G-protein-coupled receptors and lethal genes. The L. migratoria genome sequence offers new insights into the biology and sustainable management of this pest species, and will promote its wide use as a model system.
doi: 10.3732/apps.1300070
Roxana Yockteng, Ana M. R. Almeida, Stephen Yee, Thiago Andre, Colin Hill, and Chelsea D. Specht
Premise of the study: To study gene expression in plants, high-quality RNA must be extracted in quantities sufficient for subsequent cDNA library construction. Field-based collections are often limited in quantity and quality of tissue and are typically preserved in RNAlater. Obtaining sufficient and high-quality yield from variously preserved samples is essential to studies of comparative biology. We present a protocol for the extraction of high-quality RNA from even the most recalcitrant plant tissues.
Methods and Results: Tissues from mosses, cycads, and angiosperm floral organs and leaves were preserved in RNAlater or frozen fresh at −80°C. Extractions were performed and quality was measured for yield and purity.
Conclusions: This protocol results in the extraction of high-quality RNA from a variety of plant tissues representing vascular and nonvascular plants. RNA was used for cDNA synthesis to generate libraries for next-generation sequencing and for expression studies using quantitative PCR (qPCR) and semiquantitative reverse transcription PCR (RT-PCR).
Materials and Methods
Sampling — Plant material was collected from greenhouses and botanical gardens ( Table 1 ) and either stored in RNA later (Ambion, Carlsbad, California, USA) or frozen immediately in liquid nitrogen. reserved tissue was placed in long-term storage at −80 ° C. For storage in RNA later , approximately 3 × the volume of RNA later : tissue is used. Tissue stored in RNA later and frozen (−80 ° C) was defrosted just enough to remove the tissue from the RNA later prior to extraction.
Basic protocol — The following protocol was modified from the manufacturer’s provided instructions for effective use of the Plant RNA Reagent from Life Technologies. As indicated, all solutions are prepared with sterile RNase-free water, and all supplies and handling materials are cleaned with RNase AWAY (Ambion) prior to dissection and storage. This protocol is optimized to isolate RNA from approximately 0.1 g of plant tissue. If the amount of plant tissue is increased, reagent volumes must be scaled appropriately.
Grinding the tissue— One of the critical points to obtain high yield in the extraction of genetic material is the grinding. It is essential to grind the tissue as finely as possible, maintaining samples as cold as possible during grinding to avoid degradation. Selection of FastPrep or mortars/pestle depends on the hardness of the tissue being processed.
A FastPrep FP120 Homogenizer (Thermo Savant, Carlsbad, California, USA) was used for grinding floral organs and soft leaf tissue. Approximately 0.1 g of frozen floral organs, whole flowers, and leaves or herbaceous stems were added to FastPrep 2-mL tubes (MP Biomedicals, Santa Ana, California, USA) 1/5 filled with bulk Lysing Matrix D (MP Biomedicals). FastPrep tubes containing the frozen tissue plus Lysing Matrix beads were shaken in the homogenizer (FastPrep) for 40 s at speed 6 (6 m/s) without buffer at room temperature.
For grinding hard tissue or ligneous tissue, such as cone scales from gymnosperms, the tissue was ground under liquid nitrogen in a mortar and pestle that was sterilized and baked (minimum 12 h at 150 ° C). The tissue was ground as finely as possible, and the powdered material was placed in a 1.5-mL tube. The manually ground sample can be added to the FastPrep tube with Lysing Matrix beads (see above) for additional pulverization.
RNA extraction— Once the tissue is sufficiently homogenized:
1. Add 0.6 mL of cold (4 °C) Plant RNA Reagent (Life Technologies) to pulverized tissue. Mix by brief vortexing or flicking the bottom of the tube until the sample is thoroughly resuspended. If tissue was ground with a FastPrep, homogenize with the cold buffer for an additional 40 s. If the tissue is not completely ground, repeat 1–2 × until the tissue is pulverized. If the tissue was ground with a mortar and pestle and does not need additional grinding, continue with the next step.
2. Incubate 5 min at room temperature. Placing the samples on a rotator or nutator will help to maximize surface area of the tissue with the extraction buffer.
3. Clarify the solution by centrifuging for 2 min at 12,000 × g in a microcentrifuge at room temperature. Transfer the supernatant to a tube with Phase Lock Gel (5 Prime, Gaithersburg, Maryland, USA). Although the Phase Lock Gel tubes are not required, they greatly facilitate separation of the organic and aqueous phases and help ensure cleanliness of the sample.
4. Add 0.1 mL of 5 M NaCl to the sample, tap tube to mix, and add 0.3 mL of chloroform–isoamyl alcohol (24 : 1). Mix thoroughly by inversion and centrifuge the sample at 4 ° C for 10 min at 12,000 × g to separate the phases. Transfer the aqueous (top) phase to an RNase-free tube.
5. Add to the aqueous phase an equal volume of a mix of LiCl (4 M) (3/4 v) and isopropyl alcohol (1/4 v). Mix and let stand at −20 ° C for 30 min to overnight. If the tissue was stored in RNA later , mix and let stand at −20 ° C for a maximum of 3 h (not overnight). If the precipitation is longer, salts from the RNA later solution could also precipitate. (Note: We have also let the sample stand at −80 ° C for 3 h, and this works as well.)
6. Centrifuge the sample at 4 ° C for 20 min at 12,000 × g .
7. Decant or remove supernatant with a pipette, taking care not to lose the pellet. Add 1 mL of 75% ethanol to the pellet. The pellet may be diffi cult to see. To help to see the pellet, you can add 1 μ L of GlycoBlue (Ambion) in step 5.
8. Centrifuge at 4 ° C for 5 min at 12,000 × g . Decant the liquid carefully, taking care not to lose the pellet. Briefly centrifuge to collect the residual liquid, and remove it with a pipette.
9. Let dry on ice for 15 min at room temperature and elute pellet in 10–30 μL of RNase-free water. Pipette the water up and down over the pellet to dissolve the RNA. If the pellet is difficult to dissolve, add more water or warm to 37 °C to facilitate the dissolution. It is important to resuspend the pellet completely to obtain an accurate measure of the concentration of your RNA. If the sample is not clean, it can be purified by the cleanup step suggested later. Although some protocols have suggested that performing an additional step of chloroform would clean the RNA samples (e.g., Accerbi et al., 2010 ), we found that an additional chloroform step decreases the RNA yield substantially.
A NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA) can be used to verify the concentration and purity of the RNA obtained. To assess the presence and purity of extracted RNA, use the ratio of absorbance at 230 nm, 260 nm, and 280 nm. If the RNA is pure, we expect a 260/280 ratio around 2, although this ratio does not guarantee pure RNA (see below). If the ratio is appreciably lower, it is an indication of the presence of protein, phenol, or other contaminants that absorb strongly at or near 280 nm. The ratio 260/230 is expected to be around 2–2.2. If this value is appreciably lower, it is an indication that contaminants such as carbohydrates, EDTA, guanidine isothiocyanate, and phenol that absorb at 230 nm are present in the sample. Ratios lower than expected could indicate that additional cleaning is necessary and the optional cleanup should be followed. While a more accurate assessment of the quality will be determined with a bioanalyzer prior to sequencing, this initial NanoDrop read will provide an indication of the presence of RNA, enabling the researcher to continue.
Optional cleanup — If the sample is not clean, the following modified cleanup procedure will help to purify the total RNA. This protocol is adapted from that published for DNA cleanup by Rohland and Reich (2012) , using magnetic beads to capture nucleotide material and permit additional washing steps that aid in the removal of undesirable metabolites. All stock solutions and reagents must be prepared with RNase-free water.
Preparing Sera-Mag beads solution— Begin by preparing 50 mL of SeraMag beads. Add 9 g of PEG-8000, 10 mL of 5 M NaCl, 500 μ L of 1 M Tris-HCl (pH 8.0), and 100 μ L of 0.5 M EDTA (pH 8.0) to a 50-mL Falcon tube. Add 1 mL (9 g) of the carboxyl-modified Sera-Mag Magnetic SpeedBeads (Thermo Scientific, Waltham, Massachusetts, USA; cat. no. 09-981-123) to a 1.5-mL tube. Pellet the beads for 5 min with a magnetic stand designed for 1.5–2-mL tubes. Remove the storage buffer, leaving the tubes in the magnetic stand. Wash beads 1 × with 1 mL TE or water. Remove the tube from the magnetic stand and immediately re-suspend the beads in an additional 1 mL TE or water.
Add the bead suspension to the prepared 50-mL Falcon tube and wrap the Falcon tube with aluminium foil. Store at 4 ° C for further use. The final concentration of the bead solution is 0.1% carboxyl-modified Sera-Mag Magnetic SpeedBeads, 18% PEG-8000 (w/v), 1 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), and 0.05% Tween 20.
RNA cleanup— Bring a measured amount of prepared Sera-Mag beads solution to room temperature at least 30 min prior to use. For maximum binding, measure out 3 × the total RNA volume of the beads solution and place into a 1.5-mL tube along with the RNA. Place tubes on a rotator at room temperature for 10 min.
Spin the beads solution down before the beads dry permanently onto the wall of the tube. The beads must be spun down very quickly and at low speed to avoid bead precipitation; a mini benchtop centrifuge is recommended, with a spin so quick such that the lid does not need to be closed. Incubate without rotating at room temperature for an additional 10 min to maximize RNA binding. Place the 1.5-mL tubes in the magnetic stand for 5 min, then remove the buffer with a pipette and wash the beads 2 × with 500 μ L of fresh 80% ethanol. After the second wash, remove all ethanol and make sure that no ethanol is left in the samples. Remove the tubes from the magnetic stand and spin beads down from the sides of the wall before they dry. Air-dry the bead pellet for 10 min. Elute with RNase-free water using approximately the same volume as your original sample of RNA.
Vortex beads and water just to mix, and spin down quickly at low speed as before. Place tubes on a rotator at room temperature for 2–5 min for maximum RNA elution, and spin down quickly. Place tubes in the magnetic stand. Let stand for 5 min and recover eluted RNA in a separate tube.
A NanoDrop measure is recommended to check quality and yield of RNA.
by Ruslan Kalendar, David Lee, Alan H. Schulman
Abstract
This chapter introduces the software FastPCR as an integrated tools environment for PCR primer and probe design. It also predicts oligonucleotide properties based on experimental studies of PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, group-specific, unique, and overlap extension PCR for multi-fragment assembly in cloning, as well as bisulphite modification assays. It includes a program to design oligonucleotide sets for long sequence assembly by the ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, and linguistic complexity, and provides a dilution and resuspension calculator. The program includes various bioinformatics tools for analysis of sequences with CG or AT skew, of CG content and purine–pyrimidine skew, and of linguistic sequence complexity. It also permits generation of random DNA sequence and analysis of restriction enzymes of all types. It finds or creates restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It generates consensus sequences and analyzes sequence conservation. It performs efficient and complete detection of various repeat types and displays them. FastPCR allows for sequence file batch processing, which is essential for automation. The FastPCR software is available for download at http://primerdigital.com/fastpcr.html and online version at http://primerdigital.com/tools/pcr.html.
Fig. 1 Principle of the QC cloning strategy.
( a ) DNA fragments containing known and unknown flanking sequences are amplified by PCR using a primer homologous to an adaptor sequence (primer 1 and sequence A) attached to the end of the unknown sequence (U) and a primer homologous to a region of the known sequence (primer 2 and region K2). In addition to specific products, PCR amplification can yield nonspecific products (ns) and primer dimers.
( b ) Fragments are cloned via homology between sequences present in both the insert and the vector: sequences A and sequence K1 (also called the catching sequence, CS). Since primer 2 used for PCR amplification of the insert does not overlap with region K1, nonspecific products and primer dimers cannot be cloned
Abstract
Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.
Exeter Medical Schoo大学研究人员日前揭示了新生儿糖尿病的两个新的遗传原因。他们完成的这项研究发表在Cell Metabolism杂志上，进一步揭示了β细胞在胰腺中如何形成。
研究小组发现，对胰腺发育重要的两个特定基因的基因突变后，可引起新生儿糖尿病。这些发现将新生儿糖尿病的遗传原因数量增至20个。论文主要作者Sarah Flanagan表示：我们非常自豪能够给予参与研究的家庭解答，为什么他们的孩子有糖尿病。
新生儿糖尿病在孩子不到半岁时诊断，而其中一些患者增加了并发症，如肌肉无力和学习困难有或无癫痫。我们的新基因发现是关于β细胞在胰腺如何形成，这对研究操纵干细胞治愈疾病产生了重大影响。
研究英国糖尿病协会主任Alasdair Rankin说：除了进一步揭示新生儿糖尿病的遗传原因，新研究还提供答案给父母，为什么孩子会有这种罕见的疾病，这项工作有助于我们了解胰腺发育。
新生儿糖尿病是由影响胰岛素生成的基因发生变化引起的。这意味着，血糖在体内的水平危险性的升高。Exeter团队招募来自80多个国家的1200多名患者。这项研究集中在147名新生儿糖尿病的年轻人上，经过系统的筛选， 110例患者接受了基因诊断。
至于其余的37例患者，研究人员对人类胰腺发育的重要基因突变进行了筛选。突变发现在11名患者中存在，其中四个分别是两个基因NKX2- 2和MNX1（先前不知会导致新生儿糖尿病）中的一个。
对于其余121名（82％）接受基因诊断的患者，知道糖尿病的原因会导致得到更好的治疗，并给所有患者在将来怀孕时提供新生儿患糖尿病风险的重要信息。
doi:10.1016/j.cmet.2013.11.021
Analysis of Transcription Factors Key for Mouse Pancreatic Development Establishes NKX2-2 and MNX1 Mutations as Causes of Neonatal Diabetes in Man
Sarah E. Flanagan, Elisa De Franco, Hana Lango Allen, Michele Zerah, Majedah M. Abdul-Rasoul, Julie A. Edge, Helen Stewart, Elham Alamiri, Khalid Hussain, Sam Wallis, Liat de Vries, Oscar Rubio-Cabezas, Jayne A.L. Houghton, Emma L. Edghill, Ann-Marie Patch, Sian Ellard, Andrew T. Hattersley
Understanding transcriptional regulation of pancreatic development is required to advance current efforts in developing beta cell replacement therapies for patients with diabetes. Current knowledge of key transcriptional regulators has predominantly come from mouse studies, with rare, naturally occurring mutations establishing their relevance in man. This study used a combination of homozygosity analysis and Sanger sequencing in 37 consanguineous patients with permanent neonatal diabetes to search for homozygous mutations in 29 transcription factor genes important for murine pancreatic development. We identified homozygous mutations in 7 different genes in 11 unrelated patients and show that NKX2-2 and MNX1 are etiological genes for neonatal diabetes, thus confirming their key role in development of the human pancreas. The similar phenotype of the patients with recessive mutations and mice with inactivation of a transcription factor gene support there being common steps critical for pancreatic development and validate the use of rodent models for beta cell development.
Source from Bioon
青蒿素抗药性在所分离出的东南亚疟疾病原体“镰刀形疟原虫”中的传播，可能会葬送为在全世界根除这种疾病所作努力。监测抗药性这一重要工作一直受阻于缺少一个分子标记。现在，Frederic Ariey及同事识别出了“镰刀形疟原虫”青蒿素抗药性的一个主要决定因子，它也许能提供这样一个标记。他们发现，该寄生虫的PF3D7_1343700 kelch propeller domain 中发生的突变与抗药性最近的传播有关。与在2001年和2012年间收集的样本所作比较显示，与抗药性的传播相一致的是，该标记的频率也增加了。除了提出一个有用的标记外，这些发现还有可能加深我们对抗药性怎样形成的认识，同时为在寻找新型抗疟疾药物中怎样绕过抗药性提供思路。
Didier Menard & Frederic Ariey
Table 1: Primary and secondary forward (F) and reverse (R) PCR primers
| Primer name | PCR | Sequence (5’– 3’) |
| K13_PCR_F | PCR | CGGAGTGACCAAATCTGGGA |
| K13_PCR_R | GGGAATCTGGTGGTAACAGC | |
| K13_N1_F | Nested | GCCAAGCTGCCATTCATTTG |
| K13_N1_R | GCCTTGTTGAAAGAAGCAGA |
Figure 1: PCR products for Nested PCR for PF3D7_1343700.
*S1: 3D7, S2-S16: tested samples, Neg: PCR negative controls
Figure 2: P. falciparum 3D7 protein coding gene on Pf3D7_13_v3 from 1,724,817 to 1,726,997 (Chromosome: 13)
*2181 bp sequences flanking candidate marker SNPs from 3D7 complete genome are given. Positions of primary primers (yellow) and secondary primers (green) are shown.
Figure 3: Polymorphisms observed in the K13-propeller domain
8. Appendix B. SNPs in PF3D7_1343700 Kelch protein propeller domain already observed
Colour code: dark grey Plasmodium-specific (positions 1-225) then Apicomplexa-specific (225-345), light grey: BTB/POZ domain The individual kelch domains are colour coded as in Figure 5 (see Ariey et al, Nature, 2013)
近日，刊登在国际杂志The Journal of General Physiology的两项研究成果中，来自国外的研究人员通过研究揭示了尼古丁如何利用人类机体的细胞机器促进人类成瘾，这将帮助研究者们开发新型的疗法来帮助个体实现戒烟。
据CDC信息显示，相比任何一种介质，烟草占据着世界上最大数量的可预防个体的死亡量，尼古丁是烟草中的有效成分，其可以激活名为nAChRs的受体；并不像其它滥用药物一样，尼古丁可以扮演分子伴侣的角色将受体固定于内质网中，从而增加受体在细胞表面的含量，而受体nAChRs在尼古丁成瘾过程中也扮演着重要角色，其可以降低吸烟患者患帕金森疾病的易感性。
包含∝6亚单位的nAChRs受体在许多特殊的大脑区域中含量丰富，研究者使用表达∝6（使用荧光蛋白标记的∝6）的小鼠进行研究揭示了，暴露于一定水平尼古丁中可以上调大脑区域中的∝6 nAChRs受体含量。
另外研究者也发现尼古丁上调∝6 nAChRs受体的能力依赖于COPI囊泡介导的∝6 nAChRs受体从高尔基体到内质网的逆向运输过程；研究者认为，高尔基体-内质网间的循环或许是尼古丁上调其它nAChRs受体的常见机制，通过对该机制进行控制或许可以帮助开发新型的戒烟手段以及帮助患者抵御帕金森疾病的神经保护策略。
COPI polices nicotine-mediated up-regulation of nicotinic receptors
Rene Anand
Mayans, Aztecs, and indigenous Americans cultivated tobacco for medicinal and religious purposes well over 2,000 years ago. The subsequent trade and industrial-scale production of tobacco have led to its global recreational use with devastating health consequences. It is currently responsible for the greatest number of preventable deaths worldwide by any single agent, estimated to be 5 million per year by the Center for Disease Control and Prevention. The active ingredient of tobacco, nicotine, efficiently permeates the blood brain barrier and activates neuronal nicotinic acetylcholine receptors (nAChRs) in the brain. In addition, nicotine exposure of the brain during childhood and adolescence is likely to increase susceptibility to neuropsychiatric and addiction disorders.
Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors
Brandon J. Henderson1, Rahul Srinivasan1, Weston A. Nichols1, Crystal N. Dilworth1, Diana F. Gutierrez1, Elisha D.W. Mackey1, Sheri McKinney1, Ryan M. Drenan2, Christopher I. Richards3, and Henry A. Lester1
Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson’s disease. nAChRs containing the α6 subunit (α6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates α6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of α6* or α4* nAChRs but does not participate in basal trafficking. We show that α6β2β3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the β3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of α4β2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Förster resonance energy transfer between α6β2β3 nAChRs and εCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.
Source from 生物谷
一般情况下我们一年会患2-3次感冒，但是普通感冒病毒感染机体的分子机制并不是完全清楚。近日，来自维也纳大学等处的研究者就清楚地解析了普通感冒病毒感染机体的分子机制，相关研究刊登于国际杂志PNAS上。
感冒病毒（鼻病毒）是一种较小的球形颗粒病毒，病毒包裹的遗传物质缠绕在蛋白质外壳（病毒衣壳）上，研究者在文章中揭示了病毒RNA释放衣壳以及有效感染人类机体的分子机制。
研究者Dieter Blaas说道，我们在文章中揭示了感冒病毒感染宿主细胞的分子结构，以及病毒RNA释放和复制的细节。研究者发现病毒RNA的构象以及其与病毒内在的衣壳间的相互作用不断发生着变化。
鼻病毒也会引发脊髓灰质炎和甲型肝炎的发生，该病毒属于微小核醣核酸病毒科，这项研究也为揭示其它疾病，比如甲肝等疾病的发病机理提供了一定思路。然而关于鼻病毒感染的很多机制目前仍不清楚，研究者表示，后期还需要进行大量研究工作来揭示病毒引发疾病的细节，这对于未来开发抵御病毒的药物或者新型疗法非常重要，本文也为后期的研究提供了一定的研究基础。
Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle
Angela Pickl-Herka,1,2, Daniel Luqueb,c,1, Laia Vives-Adriánd, Jordi Querol-Audíd, Damià Garrigae,3, Benes L. Trusf, Nuria Verdaguerd,4, Dieter Blaasa,4, and José R. Castónb,4
During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNA–protein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp38 of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.
Source from 生物谷
发表在2014年1月的FASEB Journal杂志上的一项最新研究中，科学家证实随着年龄的增长，棕色脂肪的产热活动减少。
棕色脂肪是“好”脂肪，坐落在我们的脖子的背部，帮助燃烧“坏的”白色脂肪。此外，研究人员还发现了一个可能的代谢开关，可以重新激活棕色脂肪。
Junko Sugatanii博士表示：研究是关于PAF / PAFR如何通过调控动物以及人类的棕色脂肪中β3 -AR生成信号控制UCP1的水平，研究可发现新的治疗靶点来治疗与肥胖症相关的代谢紊乱。
为了获得这一发现，科学家分析了两组小鼠。第一组血小板活化因子受体（PAFR）基因敲除小鼠。第二组是正常小鼠。与野生型同窝小鼠相比，PAFR缺陷的小鼠随着年龄增长发展了更严重的肥胖状态，具有更高的身体和附睾脂肪量。
从PAFR基因敲除小鼠模型结果显示，PAFR缺乏会导致褐色脂肪组织（BAT）功能障碍，从而诱发肥胖的发展，由于BAT产热作用受损。该研究阐明了PAF/PAF受体介导抗肥胖相关的分子机制，将有助发现肥胖症和相关病症如糖尿病，高血压，心脏疾病，癌症，不育和溃疡等治疗的新靶标。
Antiobese function of platelet-activating factor: increased adiposity in platelet-activating factor receptor-deficient mice with age
doi:10.1096/fj.13-233262
Junko Sugatani*,†,1, Satoshi Sadamitsu*, Masahiko Yamaguchi*, Yasuhiro Yamazaki*, Ryoko Higa*, Yoshiki Hattori*, Takahiro Uchida*, Akira Ikari*, Wataru Sugiyama‡, Tatsuo Watanabe†‡, Satoshi Ishii§, Masao Miwa* and Takao Shimizu‖
Platelet-activating factor receptor (PAFR)-deficient mice developed a more severe obese state characterized by higher body mass (～25%) and epididymal fat mass (～55%) with age than that of wild-type (WT) littermates. PAFR-deficient mice did not show changes in the expression of critical genes involved in anabolic and catabolic metabolism in adipose, liver, and muscle tissues between 6 and 36 wk. However, a 38-81% reduction in β3/β1-adrenergic receptor (AR) and uncoupling protein 1 (UCP1) mRNA and protein levels was observed in the interscapular brown adipose tissue (BAT) of PAFR-deficient mice. Whereas a single injection of the β3-adrenergic agonist, CL-316,243 (25 μg/kg) increased temperatures in the brown fat and rectums of WT mice, this increase in temperature was markedly suppressed in PAFR-deficient mice. Acetyl-CoA:lyso-platelet-activating factor (PAF) acetyltransferase, which is involved in PAF biosynthesis, and the PAF receptor were predominantly localized in BAT macrophages, whereas brown adipocytes possessed the enzyme and functional PAF receptors. The stimulation of brown adipocytes by PAF induced the expression of β3-AR mRNA and protein (1.5- and 1.9-fold, respectively), but not that of UCP1. These results indicate that obesity in PAFR-deficient mice resulted from impaired BAT activity and suggest that the antiobese function of PAF occurs through β3-AR/UCP1 expression in BAT.—Sugatani, J., Sadamitsu, S., Yamaguchi, M., Yamazaki, Y., Higa, R., Hattori, Y., Uchida, T., Ikari, A., Sugiyama, W., Watanabe, T., Ishii, S., Miwa, M., Shimizu, T. Antiobese function of platelet-activating factor: increased adiposity in platelet-activating factor receptor-deficient mice with age.
Source from 生物谷
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