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Life In Science. 科学生活。
Pub Date : 2015-01-26 eCollection Date: 2015-01-01 DOI: 10.1080/21597081.2014.997143
Robert L Sinsheimer
As a boy in the 1920s and 30s, I was always interested in science. It was an era in which chemists were developing new materials, physicists were developing new instruments and discovering new particles, and engineers were creating new devices (such as radios, airplanes, and refrigerators). As my senior thesis in high school, I researched and wrote a paper on the possibility of transmutation of the elements and of atomic energy. (I did not envision a bomb!) Two high school teachers, one in mathematics and one in chemistry, intrigued and guided my interests, influcening me to enroll at the Massachusetts Institute of Technology (MIT). It was a serious, high-intensity school. During my sophomore year, MIT launched a new program in physical and chemical biology that instantly seized my imagination. After four years’ time out for World War II, (radar research and development) I started work research on nucleic acids in 1946. The nature and mode of action of the gene was mysterious and clearly central to biology. The work of Avery, McLeod, and McCarty (1) with Pneumococcus strongly suggested that the genes were DNA. Uber’s work upon ultraviolet-induced mutation suggested that damage to DNA had genetic effects (2). But the structure of DNA and its biochemistry were essentially unknown. The ultraviolet absorption bands of DNA were broad. We sought to narrow them (so as to be able to produce more specific effects) by taking spectra at low temperatures (liquid nitrogen and liquid hydrogen) (3), but the effects were small. However, upon ultraviolet irradiation of uracil, I discovered a reversible photochemical (4) effect at the same time that Renato Dulbecco discovered a photochemical effect on phage (5). Upon completing my PhD, I obtained a position as Associate Professor of Biophysics at Iowa State (a position earlier held by Uber). To pursue this line of research further it seemed desirable to use deoxynucleotides rather than the purine and pyrimidine bases. At that time the only techniques to isolate deoxynucleotides had yields of about 1%. I developed the technique to obtain 100% yield of nucleotides from DNA (6), and then quantitated and characterized their ultraviolet absorption. I also isolated all of the possible dinucleotides (7). This permitted me to show that (a) the methylcytosine was always adjacent to guanine, and (b) the molar equalities of A and T and of G and C demonstrated by Chargaff (8) could not arise from a sequential order but more likely required two strands of complementary sequence. But I had no proof, only surmise. At this time I realized that if I was to advance further with DNA I needed a biological system in which DNA was active. Bacteriophage, as elucidated by Max Delbruck, was such a system. (Max, at my invitation, had earlier visited at Iowa State to present a series of excellent lectures on bacteriophage.) I was able to take a six month leave of absence from Iowa State, and Max arranged a stipend for me to come to Caltech
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引用次数: 0
Niche-dependent genetic diversity in Antarctic metaviromes. 南极元病毒组中依赖生态位的遗传多样性。
Pub Date : 2014-12-16 eCollection Date: 2014-01-01 DOI: 10.4161/21597081.2014.980125
Olivier Zablocki, Lonnie van Zyl, Evelien M Adriaenssens, Enrico Rubagotti, Marla Tuffin, Stephen C Cary, Don Cowan

The metaviromes from 2 different Antarctic terrestrial soil niches have been analyzed. Both hypoliths (microbial assemblages beneath transluscent rocks) and surrounding open soils showed a high level diversity of tailed phages, viruses of algae and amoeba, and virophage sequences. Comparisons of other global metaviromes with the Antarctic libraries showed a niche-dependent clustering pattern, unrelated to the geographical origin of a given metavirome. Within the Antarctic open soil metavirome, a putative circularly permuted, ∼42kb dsDNA virus genome was annotated, showing features of a temperate phage possessing a variety of conserved protein domains with no significant taxonomic affiliations in current databases.

分析了两个不同的南极陆地土壤生态位的后病毒组。在半透明岩石下的微生物组合和周围的开放土壤中,尾状噬菌体、藻类和变形虫病毒以及噬菌体序列都表现出高度的多样性。其他全球元病毒组与南极文库的比较显示出一种依赖于生态位的聚类模式,与给定元病毒组的地理起源无关。在南极开放土壤中,一个假定的循环排列,约42kb的dsDNA病毒基因组被注释,显示出温带噬菌体的特征,具有各种保守的蛋白质结构域,在当前数据库中没有显著的分类关系。
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引用次数: 10
Metagenomic ventures into outer sequence space. 宏基因组探索外序列空间。
Pub Date : 2014-12-15 eCollection Date: 2014-01-01 DOI: 10.4161/21597081.2014.979664
Bas E Dutilh

Sequencing DNA or RNA directly from the environment often results in many sequencing reads that have no homologs in the database. These are referred to as "unknowns," and reflect the vast unexplored microbial sequence space of our biosphere, also known as "biological dark matter." However, unknowns also exist because metagenomic datasets are not optimally mined. There is a pressure on researchers to publish and move on, and the unknown sequences are often left for what they are, and conclusions drawn based on reads with annotated homologs. This can cause abundant and widespread genomes to be overlooked, such as the recently discovered human gut bacteriophage crAssphage. The unknowns may be enriched for bacteriophage sequences, the most abundant and genetically diverse component of the biosphere and of sequence space. However, it remains an open question, what is the actual size of biological sequence space? The de novo assembly of shotgun metagenomes is the most powerful tool to address this question.

直接从环境中对DNA或RNA进行测序通常会导致许多测序读数在数据库中没有同源物。这些被称为“未知”,反映了我们生物圈中巨大的未被探索的微生物序列空间,也被称为“生物暗物质”。然而,未知因素也存在,因为宏基因组数据集没有得到最佳挖掘。研究人员面临着发表和继续研究的压力,而未知的序列往往被保留下来,结论是基于带有注释的同源物的读数得出的。这可能导致大量和广泛的基因组被忽视,例如最近发现的人类肠道噬菌体。噬菌体序列是生物圈和序列空间中最丰富和遗传多样性最丰富的组成部分。然而,生物序列空间的实际大小是多少,这仍然是一个悬而未决的问题。霰弹枪宏基因组的重新组装是解决这个问题的最有力的工具。
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引用次数: 29
Mycobacteriophage cell binding proteins for the capture of mycobacteria 用于捕获分枝杆菌的噬菌体细胞结合蛋白
Pub Date : 2014-10-31 DOI: 10.4161/21597073.2014.960346
D. Arutyunov, U. Singh, Amr M. El-Hawiet, H. Seckler, Sanaz Nikjah, M. Joe, Yu Bai, T. Lowary, John S. Klassen, S. Evoy, C. Szymanski
Slow growing Mycobacterium avium subsp. paratuberculosis (MAP) causes a deadly condition in cattle known as Johne's disease where asymptomatic carriers are the major source of disease transmission. MAP was also shown to be associated with chronic Crohn's disease in humans. Mycobacterium smegmatis is a model mycobacterium that can cause opportunistic infections in a number of human tissues and, rarely, a respiratory disease. Currently, there are no rapid, culture-independent, reliable and inexpensive tests for the diagnostics of MAP or M. smegmatis infections. Bacteriophages are viruses producing a number of proteins that effectively and specifically recognize the cell envelopes of their bacterial hosts. We demonstrate that the mycobacterial phage L5 minor tail protein Gp6 and lysin Gp10 are useful tools for the rapid capture of mycobacteria. Immobilized Gp10 was able to bind both MAP and M. smegmatis cells whereas Gp6 was M. smegmatis specific. Neither of the 2 proteins was able to capture E. coli, salmonella, campylobacter or Mycobacterium marinum cells. Gp6 was detected previously as a component of the phage particle and shows no homology to proteins with known function. Therefore, electrospray ionization mass spectrometry was used to determine whether recombinant Gp6 could bind to a number of chemically synthesized fragments of mycobacterial surface glycans. These findings demonstrate that mycobacteriophage proteins could be used as a pathogen capturing platform that can potentially improve the effectiveness of existing diagnostic methods.
生长缓慢的鸟分枝杆菌亚种。副结核在牛中引起一种称为约翰氏病的致命疾病,其中无症状携带者是疾病传播的主要来源。MAP也被证明与人类慢性克罗恩病有关。耻垢分枝杆菌是一种模式分枝杆菌,可在许多人体组织中引起机会性感染,很少引起呼吸道疾病。目前,没有快速、不依赖培养物、可靠和廉价的检测方法来诊断MAP或耻垢支原体感染。噬菌体是一种产生大量蛋白质的病毒,这些蛋白质可以有效地、特异性地识别其细菌宿主的细胞包膜。我们证明了分枝杆菌噬菌体L5小尾蛋白Gp6和裂解素Gp10是快速捕获分枝杆菌的有用工具。固定化Gp10能够结合MAP和耻毛分枝杆菌细胞,而Gp6对耻毛分枝杆菌具有特异性。这两种蛋白都不能捕获大肠杆菌、沙门氏菌、弯曲杆菌和海洋分枝杆菌细胞。Gp6以前作为噬菌体颗粒的一个组成部分被检测到,并且与已知功能的蛋白质没有同源性。因此,我们使用电喷雾电离质谱法来确定重组Gp6是否可以与一些化学合成的分枝杆菌表面聚糖片段结合。这些发现表明,分枝噬菌体蛋白可以用作病原体捕获平台,可以潜在地提高现有诊断方法的有效性。
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引用次数: 11
Bacteriophage remediation of bacterial pathogens in aquaculture: a review of the technology 水产养殖中病原菌噬菌体修复技术综述
Pub Date : 2014-10-31 DOI: 10.4161/21597081.2014.975540
G. Richards
Bacteriophages have been proposed as an alternative to antibiotic usage and several studies on their application in aquaculture have been reported. This review highlights progress to date on phage therapies for the following fish and shellfish diseases and associated pathogens: hemorrhagic septicemia (Aeromonas hydrophila) in loaches, furunculosis (Aeromonas salmonicida) in trout and salmon, edwardsiellosis (Edwardsiella tarda) in eel, columnaris disease (Flavobacterium columnare) in catfish, rainbow trout fry syndrome or cold water disease (Flavobacterium psychrophilum) in trout and salmon, lactococcosis (Lactococcus spp.) in yellowtail, ulcerative skin lesions (Pseudomonas aeruginosa) in freshwater catfish, bacterial hemorrhagic ascites disease (Pseudomonas plecoglossicida) in ayu fish, streptococcosis (Streptococcus iniae) in flounder, and luminescent vibriosis (Vibrio harveyi) in shrimp. Information is reviewed on phage specificity, host resistance, routes of administration, and dosing of fish and shellfish. Limitations in phage research are described and recommended guidelines are provided for conducting future phage studies involving fish and shellfish.
噬菌体已被提出作为抗生素的替代品,并报道了一些关于其在水产养殖中的应用的研究。本文综述了迄今为止在以下鱼类和贝类疾病及其相关病原体的噬菌体治疗方面的进展:泥鳅的出血性败血症(嗜水气单胞菌)、鳟鱼和鲑鱼的疖病(沙门氏菌气单胞菌)、鳗鱼的爱德华氏菌病(迟发爱德华氏菌)、鲶鱼的柱状菌病(柱状黄杆菌)、鳟鱼和鲑鱼的虹鳟鱼鱼苗综合征或冷水病(嗜冷黄杆菌)、黄尾鱼的乳球菌病(乳球菌)、淡水鲶鱼的溃疡性皮肤病变(铜绿假单胞菌)、阿育鱼的细菌性出血性腹水病(假单胞菌),比目鱼的链球菌病(牛链球菌)和虾的发光弧菌病(哈维弧菌)。综述了有关噬菌体特异性、宿主耐药性、给药途径以及鱼类和贝类给药的信息。本文描述了噬菌体研究的局限性,并为今后开展涉及鱼类和贝类的噬菌体研究提供了建议指南。
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引用次数: 99
Distinguishing direct binding interactions from allosteric effects in the protease–HK97 prohead I δ domain complex by amide H/D exchange mass spectrometry 用酰胺H/D交换质谱法区分蛋白酶- hk97前体I δ结构域复合物的直接结合作用和变构作用
Pub Date : 2014-10-29 DOI: 10.4161/21597073.2014.959816
S. Krishnamurthy, D. Veesler, R. Khayat, J. Snijder, R. Huang, Ajr Heck, Jeremi Johnson, GS Anand
A major question in mapping protein-ligand or protein-protein interactions in solution is to distinguish direct-binding interactions from long-range conformational changes at allosteric sites. We describe here the applicability of amide hydrogen deuterium exchange mass spectrometry (HDXMS) in addressing this important question using the bacteriophage HK97 capsid proteins’ interactions with their processing protease. HK97 is a lambda-like dsDNA bacteriophage that is ideal for studies of particle assembly and maturation. Its capsid precursor protein is composed of two main regions, the scaffolding protein (δ-domain, residues 2-103), and the coat subunit (residues 104-385), which spontaneously forms a mixture of hexamers and pentamers upon association. Activation of the viral protease, which occurs after particle assembly, is initiated by the protease mediated digestion of the scaffolding domains to yield Prohead-2. This irreversible step is obligatory for activation of the virus maturation pathway. Here we provide an “addendum” to our previous study of Prohead I and Prohead I+pro (a transient complex of Prohead I and the protease) where we investigated the interactions between the δ domain and the packaged protease using HDXMS. Our results revealed two sites on the δ domain: one set of contiguous peptides that showed decreased exchange at the protease binding site at early time points of deuterium labeling and another separate set of continuous peptides that showed decreased exchange at later time points. Even though this cannot reveal the time scales of molecular processes governing binding and allostery, we believe this differential pattern of exchange across deuteration times can allow spatial distinction between binding sites and long range conformational changes with allosteric implications. This partitioning can be discerned from the lag between noncontiguous regions on a protein showing maximal changes in deuterium exchange and highlights a powerful application of HDXMS in distinguishing direct binding in transient protein-protein interactions from allosteric changes.
在溶液中绘制蛋白质-配体或蛋白质-蛋白质相互作用的一个主要问题是区分直接结合相互作用和变构位点的远程构象变化。我们在这里描述了酰胺氢氘交换质谱(HDXMS)在利用噬菌体HK97衣壳蛋白与其加工蛋白酶相互作用来解决这一重要问题方面的适用性。HK97是一种λ样的dsDNA噬菌体,是研究粒子组装和成熟的理想材料。它的衣壳前体蛋白由两个主要区域组成,即支架蛋白(δ-结构域,残基2-103)和外壳亚基(残基104-385),它们在结合时自发形成六聚体和五聚体的混合物。病毒蛋白酶的激活发生在颗粒组装之后,由蛋白酶介导的支架结构域的消化启动,产生Prohead-2。这个不可逆的步骤是激活病毒成熟途径的必要步骤。在这里,我们提供了一个“附录”,以我们之前的研究Prohead I和Prohead I+pro (Prohead I和蛋白酶的瞬时复合体),我们研究了δ结构域和包装蛋白酶之间的相互作用,使用HDXMS。我们的结果揭示了δ结构域上的两个位点:一组连续肽在氘标记的早期时间点显示蛋白酶结合位点的交换减少,另一组独立的连续肽在后期时间点显示交换减少。尽管这不能揭示控制结合和变构的分子过程的时间尺度,但我们相信这种跨氘化时间的差异交换模式可以允许结合位点之间的空间区分和具有变构含义的长期构象变化。这种分配可以从蛋白质上显示最大氘交换变化的不连续区域之间的滞后中辨别出来,并突出了HDXMS在区分瞬时蛋白质相互作用和变弹性变化中的直接结合方面的强大应用。
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引用次数: 6
Evidence for horizontal gene transfer between Chlamydophila pneumoniae and Chlamydia phage 肺炎衣原体和噬菌体衣原体之间水平基因转移的证据
Pub Date : 2014-10-29 DOI: 10.4161/21597073.2014.965076
A. Rosenwald, B. Murray, Theodore Toth, R. Madupu, A. Kyrillos, Gaurav S. Arora
Chlamydia-infecting bacteriophages, members of the Microviridae family, specifically the Gokushovirinae subfamily, are small (4.5–5 kb) single-stranded circles with 8–10 open-reading frames similar to E. coli phage φX174. Using sequence information found in GenBank, we examined related genes in Chlamydophila pneumoniae and Chlamydia-infecting bacteriophages. The 5 completely sequenced C. pneumoniae strains contain a gene orthologous to a phage gene annotated as the putative replication initiation protein (PRIP, also called VP4), which is not found in any other members of the Chlamydiaceae family sequenced to date. The C. pneumoniae strain infecting koalas, LPCoLN, in addition contains another region orthologous to phage sequences derived from the minor capsid protein gene, VP3. Phylogenetically, the phage PRIP sequences are more diverse than the bacterial PRIP sequences; nevertheless, the bacterial sequences and the phage sequences each cluster together in their own clade. Finally, we found evidence for another Microviridae phage-related gene, the major capsid protein gene, VP1 in a number of other bacterial species and 2 eukaryotes, the woodland strawberry and a nematode. Thus, we find considerable evidence for DNA sequences related to genes found in bacteriophages of the Microviridae family not only in a variety of prokaryotic but also eukaryotic species.
感染衣原体的噬菌体是微病毒科的成员,特别是Gokushovirinae亚科,是小的(4.5-5 kb)单链圆环,具有8-10个开放阅读框,类似于大肠杆菌噬菌体φX174。利用GenBank中发现的序列信息,我们检测了肺炎衣原体和感染衣原体的噬菌体的相关基因。这5个完全测序的肺炎球菌菌株含有一个与噬菌体基因同源的基因,该基因被注释为假定的复制起始蛋白(PRIP,也称为VP4),这是迄今为止在衣原体科的任何其他测序成员中未发现的。感染考拉的肺炎球菌菌株LPCoLN还含有另一个与次要衣壳蛋白基因VP3衍生的噬菌体序列同源的区域。在系统发育上,噬菌体的PRIP序列比细菌的更多样化;然而,细菌序列和噬菌体序列在各自的分支中都聚集在一起。最后,我们发现了另一个微病毒科噬菌体相关基因,主要的衣壳蛋白基因VP1在许多其他细菌物种和2种真核生物,林地草莓和线虫中存在的证据。因此,我们不仅在各种原核生物中而且在真核生物物种中发现了大量与微病毒科噬菌体中发现的基因相关的DNA序列的证据。
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引用次数: 14
Bacteriophage HK022 Nun protein arrests transcription by blocking lateral mobility of RNA polymerase during transcription elongation. 噬菌体HK022 Nun蛋白在转录延伸过程中通过阻断RNA聚合酶的横向迁移来阻止转录。
Pub Date : 2014-07-30 eCollection Date: 2014-01-01 DOI: 10.4161/bact.32187
Christal L Vitiello, Max E Gottesman

Coliphage HK022 excludes phage λ by subverting the λ antitermination system and arresting transcription on the λ chromosome. The 12 kDa HK022 Nun protein binds to λ nascent transcript through its N-terminal Arginine Rich Motif (ARM), blocking access by λ N and arresting transcription via a C-terminal interaction with RNA polymerase. In a purified in vitro system, we recently demonstrated that Nun arrests transcription by restricting lateral movement of transcription elongation complex (TEC) along the DNA register, thereby freezing the translocation state. We will discuss some of the key experiments that led to this conclusion, as well as present additional results that further support it.

噬菌体HK022通过破坏λ抗终止系统和阻止λ染色体上的转录而排斥噬菌体λ。12 kDa的HK022 Nun蛋白通过其N端Arginine Rich Motif (ARM)结合到λ新生转录物上,阻断λ N的进入,并通过与RNA聚合酶的c端相互作用阻止转录。在纯化的体外系统中,我们最近证明了Nun通过限制转录延伸复合体(TEC)沿DNA寄存器的横向运动来阻止转录,从而冻结转运状态。我们将讨论导致这一结论的一些关键实验,并提出进一步支持这一结论的其他结果。
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引用次数: 3
Inactivation of Streptomyces phage ɸC31 by 405 nm light: Requirement for exogenous photosensitizers? 405 nm光对链霉菌噬菌体的灭活作用:对外源光敏剂的要求?
Pub Date : 2014-07-28 eCollection Date: 2014-01-01 DOI: 10.4161/bact.32129
Rachael M Tomb, Michelle Maclean, Paul R Herron, Paul A Hoskisson, Scott J MacGregor, John G Anderson

Exposure to narrowband violet-blue light around 405 nm wavelength can induce lethal oxidative damage to bacteria and fungi, however effects on viruses are unknown. As photosensitive porphyrin molecules are involved in the microbicidal inactivation mechanism, and since porphyrins are absent in viruses, then any damaging effects of 405 nm light on viruses might appear unlikely. This study used the bacteriophage ɸC31, as a surrogate for non-enveloped double-stranded DNA viruses, to establish whether 405 nm light can induce virucidal effects. Exposure of ɸC31 suspended in minimal media, nutrient-rich media, and porphyrin solution, demonstrated differing sensitivity of the phage. Significant reductions in phage titer occurred when exposed in nutrient-rich media, with ~3-, 5- and 7-log10 reductions achieved after exposure to doses of 0.3, 0.5 and 1.4 kJ/cm2, respectively. When suspended in minimal media a 0.3-log10 reduction (P = 0.012) occurred after exposure to 306 J/cm2: much lower than the 2.7- and > 2.5-log10 reductions achieved with the same dose in nutrient-rich, and porphyrin-supplemented media, suggesting inactivation is accelerated by the photo-activation of light-sensitive components in the media. This study provides the first evidence of the interaction of narrowband 405 nm light with viruses, and demonstrates that viral susceptibility to 405 nm light can be significantly enhanced by involvement of exogenous photosensitive components. The reduced susceptibility of viruses in minimal media, compared with that of other microorganisms, provides further evidence that the antimicrobial action of 405 nm light is predominantly due to the photo-excitation of endogenous photosensitive molecules such as porphyrins within susceptible microorganisms.

暴露在405nm波长左右的窄波段紫蓝光下可以对细菌和真菌造成致命的氧化损伤,但对病毒的影响尚不清楚。由于光敏卟啉分子参与杀微生物失活机制,且病毒中不存在卟啉,因此405 nm光对病毒的破坏作用似乎不太可能发生。本研究以噬菌体h C31作为非包膜双链DNA病毒的替代物,验证405 nm光是否能诱导灭病毒作用。暴露于微量培养基、富营养培养基和卟啉溶液中,显示出噬菌体的不同敏感性。当暴露在富含营养的培养基中时,噬菌体滴度显著降低,暴露于0.3、0.5和1.4 kJ/cm2剂量后,噬菌体滴度分别降低了~3、5和7 log10。当悬浮在最小的培养基中时,暴露于306 J/cm2后,减少了0.3 log10 (P = 0.012),远低于相同剂量的富营养化和卟啉补充培养基中的2.7和> 2.5 log10减少,这表明介质中光敏成分的光活化加速了失活。本研究首次提供了窄带405 nm光与病毒相互作用的证据,并证明外源光敏成分的参与可以显著增强病毒对405 nm光的敏感性。与其他微生物相比,病毒在最小介质中的敏感性降低,进一步证明405 nm光的抗菌作用主要是由于敏感微生物内的内源性光敏分子(如卟啉)的光激发。
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引用次数: 30
Life in Science: Abraham Eisenstark. 《科学人生》:亚伯拉罕·艾森斯塔克。
Pub Date : 2014-07-28 eCollection Date: 2014-01-01 DOI: 10.4161/bact.29009
Abraham Eisenstark
The Influence of Books and Movies addiction to microbiology came early in my boyhood through books and film. First there was the novel Arrowsmith by sinclair Lewis. in the movie adaptation, actor ronald coleman dealt with bubonic plague and quickly became my scientific hero. throughout the years, my paperback copy of this novel has been loaned to many students. next, i became fascinated by paul muni in The Story of Louis Pasteur. then there was Microbe Hunters by paul DeKruif, and Rats, Lice and History by hans Zinsser. Later came a 1940 biographical film, Dr. Ehrlich’s Magic Bullet starring edward g robinson. in addition to these items from popular culture, i was inspired by books from the Life in Science
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引用次数: 0
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Bacteriophage
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