Approaches to semi-synthetic minimal cells: a review

Following is a synthetic review on the minimal living cell, defined as an artificial or a semi-artificial cell having the minimal and sufficient number of components to be considered alive. We describe concepts and experiments based on these constructions, and we point out that an operational definition of minimal cell does not define a single species, but rather a broad family of interrelated cell-like structures. The relevance of these researches, considering that the minimal cell should also correspond to the early simple cell in the origin of life and early evolution, is also explained. In addition, we present detailed data in relation to minimal genome, with observations cited by several authors who agree on setting the theoretical full-fledged minimal genome to a figure between 200 and 300 genes. However, further theoretical assumptions may significantly reduce this number (i.e. by eliminating ribosomal proteins and by limiting DNA and RNA polymerases to only a few, less specific molecular species). Generally, the experimental approach to minimal cells consists in utilizing liposomes as cell models and in filling them with genes/enzymes corresponding to minimal cellular functions. To date, a few research groups have successfully induced the expression of single proteins, such as the green fluorescence protein, inside liposomes. Here, different approaches are described and compared. Present constructs are still rather far from the minimal cell, and experimental as well as theoretical difficulties opposing further reduction of complexity are discussed. While most of these minimal cell constructions may represent relatively poor imitations of a modern full-fledged cell, further studies will begin precisely from these constructs. In conclusion, we give a brief outline of the next possible steps on the road map to the minimal cell.

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基于电生理信息的两种桑科植物叶片细胞代谢能比较

植物的细胞代谢能可以表征植物的生理活性和源库状况,而植物的细胞代谢能可由细胞的生理电容、生理电阻和生理阻抗来表征.本研究以桑科植物构树(Broussonetia papyrifera)和桑树(Morus alba)为例研究了不同夹持力下生理电阻、生理阻抗和生理电容的特征.先依据吉布斯自由能方程和Nernst方程构建模型...     

基于mcrA基因的厌氧颗粒污泥产甲烷菌群分析

基于mcrA基因对阿维菌素废水处理工业化UASB厌氧颗粒污泥中产甲烷菌群进行分析,并与基于16S rRNA基因的产甲烷菌群分析结果进行比较.结果表明,基于2种目标基因PCR产物的DGGE图谱存在差异,但根据图谱计算所得产甲烷菌群Shannon多样性指数、Margalef丰富度指数和Berger-Parker优势度指数没有差异,表明基于2种目标基因的产甲烷菌群多样性分析基本一致.基于不同目标基因的优势产甲烷菌群系统发育种属的分析结果大体相似,产甲烷杆菌目和产甲烷八叠球菌目是厌氧颗粒污泥样品中的优势产甲烷种群;同时,分析结果的差异表明2种目标基因的检测特异性不完全相同.基于2种目标基因的产甲烷菌群FISH杂交区域具有很高的一致性,但杂交区域面积有所差异.基于mcrA基因FISH检测的产甲烷菌群平均相对丰度为24.25%±6.47%,低于基于16S rRNA基因FISH检测结果(33.42%±2.34%).以上结果表明,基于mcrA基因与基于16S rRNA基因的的产甲污泥菌群分析结果具有较高的相似度,mcrA基因可以作为16S rRNA基因的替代目标基因.     

Does the Spatial Distribution of the Parasitic Mite Varroa jacobsoni Oud. (Mesostigmata: Varroidae) in Worker Brood of Honey Bee Apis Mellifera L. (Hymenoptera: Apidae) Rely on an Aggregative Process?

  Varroa jacobsoni is an ectoparasite of honey bees which reproduces in capped brood cells. Multi-infestation is frequently observed in worker brood and can be interpreted as an aggregative phenomenon. The aim of this study was to determine whether the distribution of V. jacobsoni in worker brood cells relies on a random or an aggregative process. We studied the distribution of Varroa females in capped worker brood at similar age by comparing, by a Monte Carlo test, the observed frequency distribution of mites per cell to simulated distributions based on a random process. A complementary approach, using the "nearest neighbor distances" (NND) with Monte Carlo tests, was investigated to study the spatial distribution (a) between mites in different cells and (b) between infested cells in brood. The observed distributions did not differ significantly from that expected by a random process, and we conclude that there is no aggregation during invasion of V. jacobsoni in worker brood. Received: 29 April 1999 / Accepted in revised form: 26 August 1999     

玉米根际微生物氮磷转化的功能基因组学分析

化肥减量增效是保障农业生态环境安全的重要基础.微生物是调控土壤氮磷循环的关键驱动力,研究根际微生物氮磷转化功能可以为进一步提高土壤氮磷利用率提供微生物学调控途径.基于3种典型农田土壤（黑土、潮土和红壤）的田间微区试验,利用宏基因组测序技术研究玉米根际微生物在土壤氮磷转化过程中功能基因的差异及调控因子.结果表明,根际微生物功能多样性受土壤类型影响,黑土和潮土的根际微生物功能多样性主要受含水量和养分含量的影响,红壤受全磷（TP）和速效磷（AP）影响.在土壤氮转化方面,编码氮转化过程通路中相关酶的基因丰度以脲酶基因（ureC）和葡萄糖脱氢酶基因（gdh）丰度最高,分别为7.25×10^-5~12.88×10^-5和4.47×10^-5~7.49×10^-5.同化性硝酸盐还原的功能基因在红壤中总丰度要高于黑土和潮土,其它过程相关酶的功能基因总丰度以潮土最高.编码氮代谢过程相关酶的功能基因丰度主要受土壤细菌丰富度、全钾（TK）和TP含量的驱动.在土壤磷转化方面,催化有机磷矿化的碱性磷酸酶基因（phoD）数目为1093个,酸性磷酸酶基因（PHO）数目为42个.phoD丰度高出PHO丰度2个数量级,此外,同种土壤类型下施肥对phoD和PHO丰度没有显著影响.随机森林分析表明phoD和PHO丰度均受土壤水分、有机质（OM）和全氮（TN）显著影响,但AP含量对PHO丰度影响最大.从功能基因组水平研究了玉米根际微生物的氮磷转化特征,为利用微生物功能提高农田生态系统氮磷利用率提供了科学依据.     

Prenatal diagnosis in a family with X-linked hydrocephalus

The neural cell adhesion molecule L1 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs). Its expression is essential during embryonic development of the nervous system and it is involved in cognitive function and memory. Mutations in the L1CAM gene are responsible for four related L1 disorders; X-linked hydrocephalus/HSAS (H ydrocephalus as a result of S tenosis of the A queduct of S ylvius), MASA (M ental retardation, A phasia, S huffling gait, and A dducted thumbs) syndrome, X-linked complicated spastic paraplegia type I (SPG1) and X-linked A genesis of the C orpus C allosum (ACC). These four disorders represent a clinical spectrum that varies both between and within families. The main clinical features of this spectrum are C orpus callosum hypoplasia, mental R etardation, A dducted thumbs, S pastic paraplegia and H ydrocephalus (CRASH syndrome). Since there is no biochemically assayed disease marker, molecular analysis of the L1CAM gene is the only means of confirming a clinical diagnosis. Most L1CAM mutations reported to date are point mutations (missense, nonsense, splice site) and only a few patients with larger rearrangements have been documented. We have characterised a rare intragenic deletion of the L1CAM gene in a sample of DNA extracted from a chorionic villus biopsy (CVB) performed at 12 weeks' gestation. Copyright © 2005 John Wiley & Sons, Ltd.     

Prenatal diagnosis of mucopolysaccharidosis type VII facilitating treatment in neonatal period

Duo exome testing was performed on a fetus conceived via in vitro fertilization with an egg donor. The fetus presented with non-immune hydrops fetalis (NIHF) at 20 + 0 weeks gestation. Two variants were detected in the GUSB gene. Biallelic pathogenic variants cause mucopolysaccharidosis type VII (MPS-VII), which can present with NIHF prenatally. At the time of analysis and initial report, one variant was classified as likely pathogenic and the other as of uncertain clinical significance. Biochemical testing of the amniotic fluid supernatant showed elevated glycosaminoglycans and low β-glucuronidase activity consistent with the diagnosis of MPS-VII. This evidence allowed the upgrade of the pathogenicity for both variants, confirming the diagnosis of MPS-VII. The infant was born at 36 + 5 weeks and enzyme replacement therapy (ERT) using vestronidase was initiated at 20 days with planning for hematopoietic stem cell transplant ongoing. The ERT therapy has been well tolerated, with decreasing quantitative urine glycosaminoglycans. Long-term follow up is required to determine whether treatment has been successful. This case demonstrates the utility of alternative testing methods to clarify the pathogenicity of variants and the clinical utility of obtaining a diagnosis antenatally in facilitating treatment in the neonatal period, and specifically highlights MPS-VII as a treatable cause of NIHF.     

Identification of two novel MYH3 variants causing different phenotypes in prenatal diagnosis

The MYH3 gene encodes the embryonic myosin heavy chain, which is crucial for the skeletal and muscular development. The MYH3 variants are associated with distal arthrogryposis type 2A (Freeman-Sheldon syndrome), distal arthrogryposis type 2B3 (Sheldon-Hall syndrome), CPSFS1A (Contractures, pterygia, and spondylocarpostarsal fusion syndrome 1A) and CPSFS1B, which have some shared characteristics and great variability of clinical phenotypes. In this study, we report two novel MYH3 missense variants c.1024T>G (p.Phe342Val) and c.3872A>C (p.Gln1291Pro), demonstrating different phenotypes in the prenatal setting. This study expands the spectrum of MYH3 variants and supports the domain-specific genotype-phenotype correlation of MYH3.     

Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued. Copyright © 2003 John Wiley & Sons, Ltd.     

四环素胁迫对Shigella flexneri细菌四环素抗性基因抗性表达的影响过程

四环素(TC)抗生素在不同环境介质中已被广泛检出,为研究其对四环素抗性基因(TC-ARGs)丰度变化及表达水平的影响过程,以从活性污泥中筛选和纯化分离获得的弗氏志贺氏菌(Shigella flexneri)为研究对象,考察了不同浓度TC对其生长过程的作用影响,采用荧光定量PCR和逆转录PCR方法定量检测了不同抗性机制TC-ARGs,包括tetC、tetO和tetX基因的丰度变化及表达水平,并探讨了TC浓度与TC-ARGs丰度及其表达水平之间的相关关系.结果表明,在培养周期内(24 h),TC胁迫对Shigella flexneri细菌的生长具有抑制作用,细菌细胞浓度增长速率随TC暴露浓度的升高而降低,但对TC-ARGs丰度变化影响较小.TC胁迫能够促进Shigella flexneri细菌TC-ARGs的转录表达,tetC、tetO和tetX基因表达水平在整个培养周期内均先升高后降低.由相关性分析可知,TC浓度与TC-ARGs丰度及其表达水平之间相关关系不显著,但tetC和tetO基因丰度与其转录表达水平之间存在显著的正相关关系,表明其基因丰度一定程度上可用来衡量和评价其抗性表达水平.     

Keeping the blood flowing—plasminogen activator genes and feeding behavior in vampire bats

The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of a fetus at risk for dihydropteridine reductase deficiency by direct mutation analysis using denaturing gradient gel electrophoresis

Dihydropteridine reductase (DHPR) is an enzyme involved in the recycling of tetrahydrobiopterin (BH₄), which is an obligate co-factor of the aromatic amino acid hydroxylases. DHPR deficiency is a rare, autosomal recessive disorder caused by mutations in the QDPR gene. DHPR-deficient patients are diagnosed by a lack of response to a low phenylalanine diet and by severe neurological symptoms. Final diagnosis is made by measurements of neurotransmitters and pterin metabolites in cerebrospinal fluid (CSF) and urine, in addition to DHPR enzyme activity, which can be assessed in whole red blood cells. Treatment of DHPR deficiency can be difficult and the outcome is not always satisfying, even if all treatment strategies are followed. Therefore prenatal diagnosis is of great importance in affected families. Prenatal diagnosis is possible by measuring DHPR activity in different cell types but this is time consuming. More than 25 different mutations have to date been identified in the QDPR gene and direct identification of a mutation in a fetus would be easy and rapid. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) for the analysis of the QDPR gene. The method is useful for rapid and simultaneous scanning of all exons and flanking intronic sequences of the QDPR gene. We describe the first prenatal diagnosis conducted using this method. Copyright © 2001 John Wiley & Sons, Ltd.     

Responses of activities, abundances and community structures of soil denitrifiers to short-term mercury stress

The responses of activities, abundances and community structures of soil denitrifiers to mercury (Hg) stress were investigated through a short-term incubation experiment. Four soil treatments with different concentrations of Hg (CK, Hg25, Hg50, and Hg100, denoted as 0, 25, 50, and 100 mg Hg/kg dry soil, respectively) were incubated for 28 days. Soil denitrification enzyme activity (DEA) was measured at day 3, 7 and 28. The abundances and community structures of two denitrification concerning genes, nirS (cd₁-nitrite reductase gene) and nosZ (nitrous oxide reductase gene), were analyzed using real-time PCR and denaturing gradient gel electrophoresis (DGGE). Results showed that soil DEA was significantly stimulated in the treatments of Hg25 and Hg50 compared with others at day 7. Meanwhile, no difference in the abundances of soil nirS and nosZ was found between Hg spiked treatments and CK, except the lower abundance of nirS (P<0.05) in the Hg added treatments compared with that in the CK at day 28. The community structures of denitrifiers based on nirS gene presented obvious change at day 7 along with the Hg additions, however, no variation was found in all treatments based on the nosZ gene. The results indicated that Hg (Hg25 and Hg50) had a strongly short-term stimulation on soil DEA, and nirS gene is more sensitive than nosZ gene to Hg stress.     

Emissions corridors for reducing the risk of a collapse of the Atlantic thermohaline circulation

In this paper, we present the integrated assessment model dimrise (dynamic integrated model of regular climate change impacts and singular events). This model is designed to investigate the stability of the Atlantic thermohaline circulation (THC) and to derive related climate policy recommendations. It is written in GAMS and comprises a dynamic model of the THC coupled to a climate model and a global economy model for assessing the monetary cost of climate protection. The THC model is a dynamic four-box interhemispheric extension of the classic Stommel model calibrated against results obtained using the CLIMBER-2 climate model. The reduced-form climate model used to drive the THC model is the ICLIPS multi-gas climate model, which is a computationally efficient, globally aggregated model able to mimic the response of more sophisticated carbon cycle and atmosphere-ocean general circulation models. The THC and climate modules are coupled to a globally aggregated Ramsey-type optimal growth model of the world economy derived from the Nordhaus DICE model. Together, these components create a novel dynamic fully coupled computationally efficient integrated assessment model. Illustrative applications demonstrate that dimrise is able to derive (constrained) economically optimal emissions paths that comply with prescribed bounds on admissible THC weakening imposed in order to avoid an irrevocable breakdown. In addition, emissions corridors are presented which contain all possible emissions paths that do not endanger the stability of the THC and that simultaneously obey restrictions on welfare loss arising from mitigation efforts. The presented results show that, under worst-case conditions, the stability of the THC may be threatened within two decades if global emissions would not deviate from the business-as-usual trajectory.

镉对鸡垂体Fas和caspase-3 mRNA表达的影响

探讨氯化镉(CdCl2)诱导鸡脑垂体细胞凋亡的发生情况及对凋亡相关基因Fas和caspase-3表达的影响.选择健康50日龄海兰白蛋鸡90只,分为3组,每组30只,以CdCl2含量为0、140、210 mg·kg -1的拌料饲喂,分别于染毒20d、40d和60d时取垂体,用原位末端标记(Tunel)和RT-PCR方法检测细胞凋亡和基因表达情况.实验结果表明,CdCl2作用后可致鸡腺垂体细胞呈现明显的凋亡征象,并可诱导垂体细胞Fas和caspase-3 mRNA表达的增加;在整个试验期的各个时间点2个剂量组的凋亡率和基因表达量与对照组相比差异显著(p<0.01),且随着染镉时间的延长,低剂量组的凋亡率以及Fas、caspase-3 mRNA表达增加较明显.这表明,一定剂量的CdCl2可诱导鸡垂体细胞凋亡,在此过程中Fas和caspase-3 mRNA表达呈现出与凋亡较为一致的趋势.     

Walking on insect paths? Early ommatidial development in the compound eye of the ancestral crustacean, Triops cancriformis

 Ommatidia (the compound eye's functional units) in insects are formed by the recruitment of undifferentiated cells under the control of signalling factors. During this process, a sequence of "preclusters" composed of specifically arranged precursor cells is followed. In the growth zone of the eye of Triops, an ancestral crustacean, we observed a patterning process that corresponds well with that of insects. In both taxa, clusters with arc-like, five-cell and eight-cell patterns are found, and the sequence in which the photoreceptor or R-cells of each ommatidium become identifiable is basically the same. The first to appear are R8-like and R2/5-like cells, second are R3/4-like, and third are R1/6- and R7-like cells (if the fly's cell-numbering system is used). Thus, the morphogenetic steps during which the cell identities and the cellular architecture of the ommatidia develop appear to be conserved between these arthropod groups. Furthermore, the individual cells and cell pairs which build an insect ommatidium seem to have their homologues in crustaceans. In the evolution of developmental processes, intercellular recruitment seems to be a mechanism operating on the level of single cells even in distantly related species. Received: 12 May 2000 / Accepted in revised form: 17 May 2000     

富里酸存在条件下铜对普通小球藻光合作用与细胞生理的影响

为探究富里酸存在条件下,铜对普通小球藻光合作用与细胞生理的影响,对普通小球藻进行富里酸及铜的复合暴露,检测相关的光合作用及细胞生理指标,并与单独暴露富里酸的普通小球藻指标水平进行比较分析.结果表明,铜的加入会影响藻细胞的光合作用,增加细胞膜通透性,使藻细胞体积增大,并导致藻细胞的死亡.富里酸可以明显减弱铜的毒性,减少藻细胞的死亡.随富里酸浓度升高,藻细胞死亡率呈下降趋势;高浓度的富里酸(74.5 mg·L~(-1)和119.2 mg·L~(-1))可以明显减弱铜对藻细胞体积增大的作用,低浓度的富里酸(20 mg·L~(-1)和54 mg·L~(-1))无明显影响.富里酸单独作用能够增强普通小球藻的光合作用,增加藻细胞的细胞膜通透性,但不具有致死性.     

Carbon dioxide fixation by Chlorella sp. USTB-01 with a fermentor-helical combined photobioreactor

A promising microalgal strain isolated from fresh water, which can grow both autotrophically on inorganic carbon under lighting and heterotrophically on organic carbon without lighting, was identified as Chlorella sp. USTB-01 with the phylogenetic analysis based on 18S ribosomal ribonucleic acid (rRNA) gene sequences. In the heterotrophic batch culture, more than 20.0 g·L⁻¹ of cell dry weight concentration (DWC) of Chlorella sp. USTB-01 was obtained at day 5, and which was used directly to seed the autotrophic culture. A novel fermentor-helical combined photobioreactor was established and used to cultivate Chlorella sp. USTB-01 for the fixation of carbon dioxide (CO₂). It showed that the autotrophic growth of Chlorella sp. USTB-01 in the combined photobioreactor was more effective than that in the fermentor alone and the maximum DWC of 2.5 g·L⁻¹ was obtained at day 6. The highest CO₂ fixation of 95% appeared on day 1 in the exponential growth phases of Chlorella sp. USTB-01 and 49.8% protein was found in the harvested microalgal cells.