140 Development of nanoscale aptamer films for controlled release of encapsulated payloads

Aptamers are short, single-stranded nucleic acids that fold into well-defined 3D structures which bind to a single target molecule (from small molecules to cells) with affinities and specificities that can rival those of antibodies (Jeong et al., 2009). Unlike antibodies, aptamers can be chemically synthesized eliminating the need for animals or cell culture, which also allows for selection under non-physiological conditions and broadens potential targets to include toxic molecules (Banka & Stockley, 2006). The compatibility of aptamers with nanomaterials, in combination with their affinity, selectivity, and conformational changes upon target interaction, have allowed for the development of a large number of therapeutic and targeted delivery systems in recent years exploiting these properties. Despite this, many challenges still exist as unprotected DNA is readily degraded by nucleases prevalent in biological and environmental systems (Bouchard et al., 2010). Embedding aptamers within multilayer polyelectrolyte films could provide a biodegradable shelter, while allowing the detection of diffusible small molecules. An understanding of these materials will allow for the eventual encapsulation of relevant payloads into aptamer–polyelectrolyte microcapsules towards the development of a controlled release system. In this work, films composed of natural polyelectrolytes chitosan and hyaluronan are employed due to their biocompatibility, strong presence in current literature, and amiability to layer-by-layer film construction. Initial progress towards the development of an aptamer-embedded polyelectrolyte film system will be presented.     

Plant nutrition in the 20th and perspectives for the 21st century

This paper briefly presents the knowledge of plant nutrition in 1900 and its expansion since then in two areas - the discovery of the micronutrients and the absorption of nutrients from soils.Application of macro- and micronutrient fertilizers has contributed substantially to the huge increase in world food production experienced this century. In developed countries, excessive fertilizer use has led to serious problems of nutrient pollution; here, plant nutritionists will be concerned with monitoring nutrient status of crops and soils to maintain crop production with minimum loss of nutrients to the environment, and development of cultivars with high nutrient efficiency in soils with luxury supplies of nutrients.In many developing countries, soil infertility limits productivity; here, plant nutritional research can raise productivity by diagnosis of nutrient deficiencies and toxicities of crops on previously unfertilized soils, their correction with minimal fertilizer and treatment costs, and development of cultivars with high nutrient efficiency in deficient soils and high tolerance of natural toxicities.The pre-occupation of developed countries with pollution is blinding them to the urgent needs of developing countries for fertilizers and fertilizer research to increase crop production ha-1 as an alternative to clearing more land.     

Functional RNA microarrays for high-throughput screening of antiprotein aptamers

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.     

Interactions of Nitrogen, Phosphorus, and Potassium supplied in Leaf Sprays or in Fertilizer added to the Soil

When sugar-beet plants grown in pots were sprayed daily withnutrient solutions supplying nitrogen, phosphorus, and potassiumseparately or in all combinations, with precautions to preventspray falling on the soil in which the plants were grown, allthree nutrients were absorbed through the leaves. In one experimentnitrogen and potassium, and in another only nitrogen, causedincreases in plant dry weight and leaf area. Swedes absorbedphosphorus from leaf sprays and from fertilizer applied to thesoil, but only the fertilizer caused an increase in dry weight. Absorption of any of the nutrients tested from a spray containingmore than one nutrient was unaffected by the presence of othersin the spray, but spraying with nitrogen-containing solutionsincreased the absorption of phosphorus and potassium from thesoil, and potassium in sprays increased the uptake of phosphorusfrom the soil. Nitrogenous fertilizer applied to the soil increased the leafarea of sugar-beet plants, and hence it also increased the amountsof nitrogen, phosphorus, and potassium deposited on the leaveswhen they were sprayed with solutions of these nutrients, andthe amounts absorbed from the spray into the plants. Phosphaticfertilizer had no effect on uptake from leaf sprays. Potassicfertilizer did not affect leaf area or the estimated volumeof spray solution retained on the leaves, but it appeared toreduce uptake of potassium from the spray. Dry weight per plant was increased by all three nutrients infertilizer, and sugar yield of the roots was increased by nitrogenand potassium in fertilizer, and by nitrogen in spray. Applicationof a nutrient in leaf spray reduced the responses in dry weightand sugar yield to the same nutrient applied in fertilizer tothe soil. Less nitrogen, but more phosphorus, was taken up from the leafsprays than from fertilizer. Nutrients from sprays producedsmaller increases in total dry weight and in dry weight perunit of absorbed nutrient than the same nutrient from fertilizer. The apparent percentage recovery of nitrogen applied in spray,based on estimates of the volumes of solution retained on theleaves, was unaffected by fertilizer treatment, that of phosphoruswas increased by nitrogen fertilizer, and that of potassiumwas increased by nitrogen fertilizer and reduced by potassiumfertilizer. The volume of spray solution held on the leaveswas probably overestimated, so that the highest apparent recovery,about 60 per cent., may represent an almost complete true recovery,because only trivial amounts of the nutrients that had beenapplied in spray remained on the leaf surface to be removedby washing before harvest. Lower apparent recoveries may bedue to reduced uptake from the soil of the nutrient suppliedin spray.     

Nutritional modulation of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly worldwide. It affects 30-50 million individuals and clinical hallmarks of AMD are observed in at least one third of persons over the age of 75 in industrialized countries (Gehrs et al., 2006). Costs associated with AMD are in excess of $340 billion US (American-Health-Assistance-Foundation, 2012). The majority of AMD patients in the United States are not eligible for clinical treatments (Biarnes et al., 2011; Klein et al., 2011). Preventive interventions through dietary modulation are attractive strategies because many studies suggest a benefit of micro- and macronutrients with respect to AMD, as well as other age-related debilities, and with few, if any, adverse effects (Chiu, 2011). Preservation of vision would enhance quality of life for millions of elderly people, and alleviate the personal and public health financial burden of AMD (Frick et al., 2007; Wood et al., 2011). Observational studies indicate that maintaining adequate levels of omega-3 fatty acids (i.e. with 2 servings/week of fish) or a low glycemic index diet may be particularly beneficial for early AMD and that higher levels of carotenoids may be protective, most probably, against neovascular AMD. Intervention trials are needed to better understand the full effect of these nutrients and/or combinations of nutrients on retinal health. Analyses that describe effects of a nutrient on onset and/or progress of AMD are valuable because they indicate the value of a nutrient to arrest AMD at the early stages. This comprehensive summary provides essential information about the value of nutrients with regard to diminishing risk for onset or progress of AMD and can serve as a guide until data from ongoing intervention trials are available.     

土壤微生物在植物获得养分中的作用

大量施用化肥是当今农业的一个重要特征。化肥为粮食增产做出了巨大贡献,同时也带来一系列问题,如土壤酸化、水体富营养化、温室气体排放、资源耗竭等,直接威胁着农业可持续发展。土壤微生物是陆地生态系统植物多样性和生产力的重要驱动者,直接参与了植物获得养分和土壤养分循环两个过程。因此,通过调控土壤微生物的功能,有望降低农业对化肥的过分依赖。介绍了共生固氮菌、菌根真菌和根际促生菌对植物获得养分能力的影响及其机制,分析了土壤微生物对土壤氮、磷循环的影响及其与土壤养分生物有效性、养分损失的关系。依据这些知识,提出了改善植物营养、降低化肥施用的土壤微生物途径。虽然大量试验已证明了土壤微生物在改善植物营养中的重要作用,但是大面积应用土壤微生物技术来改善植物营养还存在不少问题。随着以后对这方面研究的加强以及上述问题的不断解决,土壤微生物有望在降低化肥施用量和维持农业可持续发展中做出重要贡献。     

Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines

Aptamers are synthetic, short nucleic acid molecules capable of specific target recognition. Aptamers are selected using a screening method termed Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently have introduced a variant of SELEX called “Ligand-Guided-Selection” (LIGS) that allows the identification of specific aptamers against known cell-surface proteins. Utilizing LIGS, we introduced three specific aptamers against membrane-bound IgM (mIgM), which is the hallmark of B cells. Out of the three aptamers selected against mIgM, an aptamer termed R1, in particular, was found to be interesting due to its ability to recognize mIgM on target cells and then block anti-IgM antibodies binding their antigen. We systematically truncated parent aptamer R1 to design shorter variants with enhanced affinity. Importantly, herein we show that the specificity of the most optimized variant of R1 aptamer is similar to that of anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, we report that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature, demonstrating that LIGS-generated aptamers could be re-optimized into higher affinity variants. Collectively, these findings show the significance of LIGS in generating highly specific aptamers with potential applications in biomedicine.     

Nutrient Constraints in Arsenic Phytoremediation

Arsenic contamination has increased due to several environmental and anthropogenic activities. It is considered a carcinogen by the International Agency for Research on Cancer. It affects human health and causes various ailments and nervous system disorders. An environmental concern arises as arsenic enters the food chain through consumption of crops grown in arsenic affected areas. It has been observed that uptake of arsenic in plant parts is affected by the concentration of nutrients. Addition of nutrients either enhances the uptake of arsenic or the uptake of arsenic is reduced. Arsenic influences the nutrient uptake and distribution of nutrients in plants by either competing directly with nutrients and/or altering metabolic processes. The role played by nutrients has a direct bearing on the arsenic remediation of the crops and hence, it will be of significance to crop growers in reducing the arsenic content in crops. This review reports about the mobility, bioavailability and plant response to the presence of nutrients and their effect on arsenic phytoremediation. In this review, major emphasis has been made to contemplate the effects of nutrients like phosphorus, nitrogen, ferrous, calcium, potassium, sulphur and selenium in arsenic phytoremediation.     

Soil nutrient status determines how elephant utilize trees and shape environments

1. Elucidation of the mechanism determining the spatial scale of patch selection by herbivores has been complicated by the way in which resource availability at a specific scale is measured and by vigilance behaviour of the herbivores themselves. To reduce these complications, we studied patch selection by an animal with negligible predation risk, the African elephant. 2. We introduce the concept of nutrient load as the product of patch size, number of patches and local patch nutrient concentration. Nutrient load provides a novel spatially explicit expression of the total available nutrients a herbivore can select from. 3. We hypothesized that elephant would select nutrient-rich patches, based on the nutrient load per 2500 m(2) down to the individual plant scale, and that this selection will depend on the nitrogen and phosphorous contents of plants. 4. We predicted that elephant would cause more adverse impact to trees of lower value to them in order to reach plant parts with higher nutrient concentrations such as bark and root. However, elephant should maintain nutrient-rich trees by inducing coppicing of trees through re-utilization of leaves. 5. Elephant patch selection was measured in a homogenous tree species stand by manipulating the spatial distribution of soil nutrients in a large field experiment using NPK fertilizer. 6. Elephant were able to select nutrient-rich patches and utilized Colophospermum mopane trees inside these patches more than outside, at scales ranging from 2500 down to 100 m(2) . 7. Although both nitrogen and phosphorus contents of leaves from C. mopane trees were higher in fertilized and selected patches, patch choice correlated most strongly with nitrogen content. As predicted, stripping of leaves occurred more in nutrient-rich patches, while adverse impact such as uprooting of trees occurred more in nutrient-poor areas. 8. Our results emphasize the necessity of including scale-dependent selectivity in foraging studies and how elephant foraging behaviour can be used as indicators of change in the availability of nutrients.     

适配体与病毒性感染诊断及治疗

适配体（Aptamers）是通过指数富集的配体系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术,从随机核酸文库中筛选出来的单链寡核苷酸,已在临床医疗及其他领域得到日益广泛的应用.与抗体相比,适配体具有很多优点,如高亲和力、高特异性、分子量小、几乎无免疫排斥反应、结构稳定、易于合成等.可用于适配体筛选的靶标范围非常广,包括有机小分子、蛋白、完整细胞及病毒颗粒等.迅速可靠的病原检测对于病毒性传染病的成功预防和治疗具有重要意义.随着严格筛选和快速分离技术的进步,适配体在病毒感染的检测治疗中显示出巨大的潜力.本文概括介绍了适配体在病毒研究方面的最新应用进展及未来前景.     

SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands

SELEX stands for systematic evolution of ligands by exponential enrichment. This method, described primarily in 1990 [Ellington, A.D., Szostak, J.W., 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822; Tuerk, C., Gold, L., 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510] aims at the development of aptamers, which are oligonucleotides (RNA or ssDNA) binding to their target with high selectivity and sensitivity because of their three-dimensional shape. Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from large targets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. Moreover, aptamers are used for the investigation of binding phenomena in proteomics. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of the aptamers selected and for automation of the process. This review is focused on the development of aptamers by use of SELEX and gives an overview about technologies, advantages, limitations, and applications of aptamers.     

大气N沉降的不断增加对森林生态系统的影响

若干年代以来,大气Ｎ沉降不断增加．在一些地区,大气Ｎ沉降超过了森林生态系统的Ｎ需求．Ｎ沉降的增加对植物生长的刺激作用和对菌根的危害、过剩的ＮＨ４＋在体内对其它阳离子的交换取代和在土壤中对其它阳离子在根的养分吸收方面的竞争抑制,都可造成植物体内其它养分缺乏,导致森林营养失调．Ｎ沉降的增加将提高硝化作用,加速ＮＯ３－和盐基阳离子的淋失,引起土壤酸化和Ａｌ、Ｍｎ活化．植物体内的高Ｎ水平将增加森林对寒冷、霜冻、真菌病害及可能的虫害等胁迫的敏感性．Ｎ沉降长期而持续的增加可通过干扰演替动力学,促使植物群落发生变化．     

Parametric sensitivity analysis and reduction of a detailed nutritional model of plant cell cultures

A kinetic model of plant nutrition described by Cloutier et al. (Cloutier et al., 2008. Biotechnol Bioeng 99:189-200) is progressively simplified so as to obtain a predictive model that describes the evolution of the biomass and the extracellular and intracellular concentrations of three determining nutrients, that is, free intracellular nitrogen, phosphate, and carbohydrate compounds. Three techniques of global sensitivity analysis are successively applied to assess the model parameter influence and potential correlation. The resulting dynamic model is able to predict plant growth for the two most encountered plant bioprocesses, namely suspension cells and hairy roots.     

Assays for aptamer-based platforms

Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.     

Properties of an LNA-modified ricin RNA aptamer

'Locked nucleic acids' (LNAs) are sugar modified nucleic acids containing the 2'-O-4'C-methylene-β-D-ribofuranoses. The substitution of RNAs with LNAs leads to an enhanced thermostability. Aptamers are nucleic acids, which are selected for specific target binding from a large library pool by the 'SELEX' method. Introduction of modified nucleic acids into aptamers can improve their stability. The stem region of a ricin A chain RNA aptamer was substituted by locked nucleic acids. Different constructs of the LNA-substituted aptamers were examined for their thermostability, binding activity, folding and RNase sensitivity as compared to the natural RNA counterpart. The LNA-modified aptamers were active in target binding, while the loop regions and the adjacent stem nucleotides remained unsubstituted. The thermostability and RNase resistance of LNA substituted aptamers were enhanced as compared to the native RNA aptamer. This study supports the approach to substitute the aptamer stem region by LNAs and to leave the loop region unmodified, which is responsible for ligand binding. Thus, LNAs possess an encouraging potential for the development of new stabilized nucleic acids and will promote future diagnostic and therapeutic applications.     

Focus Issue on Roots: Root Nutrient Foraging

During a plant''s lifecycle, the availability of nutrients in the soil is mostly heterogeneous in space and time. Plants are able to adapt to nutrient shortage or localized nutrient availability by altering their root system architecture to efficiently explore soil zones containing the limited nutrient. It has been shown that the deficiency of different nutrients induces root architectural and morphological changes that are, at least to some extent, nutrient specific. Here, we highlight what is known about the importance of individual root system components for nutrient acquisition and how developmental and physiological responses can be coupled to increase nutrient foraging by roots. In addition, we review prominent molecular mechanisms involved in altering the root system in response to local nutrient availability or to the plant''s nutritional status.In natural and agricultural soils, the ability of plants to quickly and efficiently acquire nutrients may determine their competitive success and productivity. Because mineral elements interact differently with themselves and other soil constituents or are carried by water out of the rooted soil volume, their availability to plants may decrease and lead to nutrient deficiency. Under these conditions, plants activate foraging responses that include morphological changes, such as the modulation of root system architecture (RSA) or root hair formation, and physiological changes, such as the release of nutrient-mobilizing root exudates or the expression of nutrient transporters (Gojon et al., 2009; Hinsinger et al., 2009; Gruber et al., 2013). These responses are often spatially coupled to increase the root-soil interaction zone and improve the ability of the plant to intercept immobile nutrients. Noteworthy, although not discussed herein, symbiosis or associative rhizosphere microorganisms can also alter the RSA and enhance the foraging capacity of the plant (Gutjahr and Paszkowski, 2013). Here, we provide an update on the morphological responses induced by plants to forage sparingly available nutrients and some of the underlying molecular mechanisms known to date to be involved in RSA adaptations to nutrient availabilities.     

Integrated response of intercropped maize and potatoes to heterogeneous nutrients and crop neighbours

Background and Aims

In communities, plants often simultaneously interact with intra- and inter-specific neighbours and heterogeneous nutrients. How plants respond under these conditions and then affect the structure and function of communities remain important questions.

Methods

Maize (Zea mays L.) was intercropped with potatoes (Solanum tuberosum L.). In the field experiment, we applied fertilizer both homogeneously and heterogeneously under monocropping and intercropping conditions. The heterogeneous nutrient treatment in intercropping was designed with different fertilizer placements, at intraspecific and interspecific rows, respectively. In the pot experiment, crops were grown under both homogeneous and heterogeneous nitrogen conditions with single plant, intraspecific and interspecific competition. Shoot and root biomass and yield were measured to analyse crop performance.

Results

In the field experiment, the heterogeneous nitrogen, compared with the homogenous one, enhanced the performance of the intercropped crop. Importantly, this effect of heterogeneous nitrogen was greater when fertilizer was applied at interspecific rows, rather than at intraspecific rows. Moreover, in pot experiments, the root foraging precision of the two crops was increased by interspecific neighbours, but only that of potatoes was increased by intraspecific neighbours.

Conclusions

The integrated responses of plants to heterogeneous neighbours and nutrients depend on the position of nutrient-rich patches, which deepen our understanding of the function of plant diversity, and show that fertilizer placement within multi-cropping systems merits more attention. Moreover, the enhanced utilization of heterogeneous nitrogen could drive overyielding in multi-cropping systems.     

黑土长期施肥及养分循环再利用的作物产量及土壤肥力质量变化Ⅲ.土壤养分收支

许了1985－1999年试验期间各模拟施肥模型的作物移出养分量和施肥输入养分量,结果表明,施用N肥可加剧土壤P收支赤字,而施用N,P肥则加剧土壤K收支赤字,实验展示了我国在20世纪70年代大面积贫P土壤和80年代大面积缺K土壤出现的原因,保持养分循环再利用可缓解土壤养分收支赤字,但不能满足丰产作物的养分需求,在保持养分循环利用基础上根据土壤肥力适当施用化肥,可满足丰产作物的养分需求和平衡土壤养分收支,不致发生大量过剩N进入环境。     

Temporal Asynchrony in Soil Nutrient Dynamics and Plant Production in a Semiarid Ecosystem

A central goal of ecosystem ecology is to understand how the cycling of nutrients and the growth of organisms are linked. Ecologists have repeatedly observed that nutrient mineralization and plant production are closely coupled in time in many terrestrial ecosystems. Typically, mineralization rates of limiting nutrients, particularly of nitrogen, during the growing season determine nutrient availability while pools of mineral nutrients remain low and relatively constant. Although several previous reports suggest nitrogen mineralization has the potential to vary seasonally and out of phase with plant production, such a phenomenon has been poorly documented. Here we report results from a semiarid savanna ecosystem characterized by distinct temporal asynchrony in rates of soil nitrogen cycling and plant production. Periods of positive plant growth following the onset of rains coincide with periods of low N turnover rates, whereas higher rates occur late in the wet season following plant senescence and throughout dry seasons. Plant uptake from the substantial mineral N pool present early in the growing season is sufficient to explain most of the N allocation to aboveground plant biomass during the growing season, even in the absence of any wet-season mineralization. The mineral N pool is subsequently recharged by late wet- and dry-season mineralization, plus urine inputs at sites with high levels of ungulate activity. These findings suggest fundamental changes in the quality of substrates available to decomposers over a seasonal cycle, with significant implications for the partitioning of limiting nutrients by plant species, the seasonal pattern of nutrient limitations of aboveground production, and the effective use of N fertilizers in semiarid ecosystems.