Innovative use of controlled availability fertilizers with high performance for intensive agriculture and environmental conservation

A variety of slow release fertilizers, controlled release (availability) fertilizers (CAFs), and stability fertilizers have been developed in response to the serious drawbacks of the conventional fertilizers since the early 1960's. Of these fertilizers, CAFs which are coated with resin are consumed in the largest quantity in the world. Selecting CAFs with higher performance, the author will discuss about: 1) Innovation of agro-technologies for various field crops including new concepts of fertilizer application, 2) high yielding of field crops, 3) enhancing quality and safety of farm products, and 4) controlling the adverse effect of intensive agriculture on the environment.     

Innovative use of controlled availability fertilizers with high performance for intensive agriculture and environmental conservation

A variety of slow release fertilizers, controlled release (availability) fertilizers (CAFs),and stability fertilizers have been developed in response to the serious drawbacks of the conventional fertilizers since the early 1960's. Of these fertilizers, CAFs which are coated with resin are consumed in the largest quantity in the world. Selecting CAFs with higher performance, the author will discuss about: 1) Innovation of agro-technologies for various field crops including new concepts of fertilizer application, 2) high yielding of field crops, 3) enhancing quality and safety of farm products, and 4) controlling the adverse effect of intensive agriculture on the environment.     

Autophagic secretion of HMGB1 from cancer-associated fibroblasts promotes metastatic potential of non-small cell lung cancer cells via NFκB signaling

Tumor progression requires the communication between tumor cells and tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are major components of stromal cells. CAFs contribute to metastasis process through direct or indirect interaction with tumor cells; however, the underlying mechanism is largely unknown. Here, we reported that autophagy was upregulated in lung cancer-associated CAFs compared to normal fibroblasts (NFs), and autophagy was responsible for the promoting effect of CAFs on non-small cell lung cancer (NSCLC) cell migration and invasion. Inhibition of CAFs autophagy attenuated their regulation on epithelial–mesenchymal transition (EMT) and metastasis-related genes of NSCLC cells. High mobility group box 1 (HMGB1) secreted by CAFs mediated CAFs’ effect on lung cancer cell invasion, demonstrated by using recombinant HMGB1, HMGB1 neutralizing antibody, and HMGB1 inhibitor glycyrrhizin (GA). Importantly, the autophagy blockade of CAFs revealed that HMGB1 release was dependent on autophagy. We also found HMGB1 was responsible, at least in part, for autophagy activation of CAFs, suggesting CAFs remain active through an autocrine HMGB1 loop. Further study demonstrated that HMGB1 facilitated lung cancer cell invasion by activating the NFκB pathway. In a mouse xenograft model, the autophagy specific inhibitor chloroquine abolished the stimulating effect of CAFs on tumor growth. These results elucidated an oncogenic function for secretory autophagy in lung cancer-associated CAFs that promotes metastasis potential, and suggested HMGB1 as a novel therapeutic target.Subject terms: Cancer microenvironment, Non-small-cell lung cancer, Metastasis, Translational research     

长期不同施肥方式对麦田杂草群落的影响

以太湖地区农业科学研究所31a的长期肥料定位试验田为材料,分别于2011—2012年小麦苗期、拔节期和收获期进行了杂草群落调查,研究杂草类型与密度的分布、杂草多样性指数的变化,并对杂草种群分布与土壤养分因子进行冗余分析。结果表明:小飞蓬、看麦娘、大巢菜、稻槎菜、通泉草是本地区小麦生长期的主要杂草类型;随着小麦的生长以及氮肥、有机肥的施入,杂草密度呈下降趋势;施入有机肥降低了麦季杂草的群落多样性指数,在小麦生长的不同时期均衡施肥的CNPK处理以及不施肥的C0处理的群落各项多样性指数能维持在一个较高的水平。RDA分析显示土壤氮含量以及有机质含量与第一排序轴相关性大,是对杂草分布影响最大的两个土壤养分因子。太湖地区稻麦两熟制条件下,长期有机无机肥料单一或配合投入可显著影响麦田杂草的群落组成,其中氮肥和有机肥的施入能显著降低杂草密度;土壤养分的差异影响田面杂草密度和优势种群,均衡施肥能降低优势杂草种群的优势地位,抑制其发生危害程度,提高农田生态系统的生产力及稳定性。     

Organic fertilizers alter the composition of pathogens and arbuscular mycorrhizal fungi in maize roots

Roots of agricultural crops, including maize, are hosts of different microorganisms, many beneficial, like plant growth and health‐promoting arbuscular mycorrhizal fungi (AMF), as well as pathogens including Pythium, Polymyxa and Microdochium. To improve crop nutrition and health, profound knowledge is required regarding how agricultural practices affect field populations of root‐associated microorganisms. Hence, the objective of this work was to evaluate the effect of crop genotype and organic fertilizers on the plant growth performance of maize and their root‐associated microorganisms. The experiment was conducted as a fully factorial greenhouse pot experiment with maize cultivars (two land races and two hybrids) and organic fertilizers (green manure, cow manure and compost) as the two main factors. Plants were harvested 8 weeks after sowing. In general, the different maize cultivars responded similarly to the applications of the organic fertilizers. Cow manure and compost increased plant growth, whereas green manure had limited effect on plant growth. Root colonization with AMF was reduced by green manure with rape. Infection with the root pathogens Pythium and Polymyxa was reduced by all organic fertilizers, whereas in contrast, infection with Microdochium increased with the majority of the organic fertilizers applied. In conclusion, both maize genotype and organic fertilizers affect the abundance of AMF and root pathogens in maize, which should be considered when developing management strategies of these root‐inhabiting microorganisms.     

Sulphur supply and infection with Pyrenopeziza brassicae influence L-cysteine desulphydrase activity in Brassica napus L

Different field surveys have shown that sulphur (S) fertilization can increase the resistance of agricultural crops against fungal pathogens. The mechanisms of this sulphur-induced resistance (SIR) are, however, not yet known. Volatile S compounds are thought to play an important role because H(2)S is toxic to fungi. A field experiment was conducted to analyse the influence of S fertilization and the activity of H(2)S-releasing enzymes on fungal infections. Two levels of N and S fertilizers and two varieties of oilseed rape were investigated with respect to their potential to release H(2)S by the enzymatic activity of L-cysteine desulphydrase (LCD) and O-acetyl-L-serine(thiol)lyase (OAS-TL). LCD releases H(2)S during cysteine degradation, while OAS-TL consumes H(2)S during cysteine synthesis and free H(2)S is only released in a side reaction. All plots of the field trial showed an infection with Pyrenopeziza brassicae and leaf disc samples were taken from visibly infected leaf areas and apparently uninfected areas to investigate the reaction to the infection in relation to the treatments. Different S fractions and the activities of LCD and OAS-TL were measured to evaluate the potential to release H(2)S in relation to S nutrition and fungal infection. S fertilization significantly increased the contents of total S, sulphate, organic S, cysteine, and glutathione in the plants, but decreased LCD activity. Infection with P. brassicae increased cysteine and glutathione contents, as well as the activity of LCD. Therefore crops were able to react to a fungal infection with a greater potential to release H(2)S, which is reflected by an increasing LCD activity with fungal infection.     

Autophagy protects ovarian cancer-associated fibroblasts against oxidative stress

RNA-Seq and gene set enrichment anylysis revealed that ovarian cancer associated fibroblasts (CAFs) are mitotically active compared with normal fibroblasts (NFs). Cellular senescence is observed in CAFs treated with H₂O₂ as shown by elevated SA-β-gal activity and p21 (WAF1/Cip1) protein levels. Reactive oxygen species (ROS) production and p21 (WAF1/Cip1) elevation may account for H₂O₂-induced CAFs cell cycle arrest in S phase. Blockage of autophagy can increase ROS production in CAFs, leading to cell cycle arrest in S phase, cell proliferation inhibition and enhanced sensitivity to H₂O₂-induced cell death. ROS scavenger NAC can reduce ROS production and thus restore cell viability. Lactate dehydrogenase A (LDHA), monocarboxylic acid transporter 4 (MCT4) and superoxide dismutase 2 (SOD2) were up-regulated in CAFs compared with NFs. There was relatively high lactate content in CAFs than in NFs. Blockage of autophagy decreased LDHA, MCT4 and SOD2 protein levels in CAFs that might enhance ROS production. Blockage of autophagy can sensitize CAFs to chemotherapeutic drug cisplatin, implicating that autophagy might possess clinical utility as an attractive target for ovarian cancer treatment in the future.     

Synchronizing N release from organic residues: opportunities for integrated management of N

In intensive cropping systems, mineral nitrogen (N) fertilizers represent the largest component of the N cycle because the indigenous N supply is not adequate. The requirement for mineral fertilizer may be reduced with the use of organic nutrient sources. A more realistic use of organic matter, particularly in sub-Saharan Africa due to limited amounts and availability, is the combined use of organic nutrient sources and mineral fertilizers. The beneficial effects of integrated use of inorganic fertilizers and organic manures on improved nutrient recoveries, soil moisture retention, cation exchange capacity, and erosion control have been reported. However, there are as many reports indicating negligible benefits or even disadvantages of combining nutrient sources on crop production. This is not surprising given the combination of organic residue sources, soils, climatic, crops, and management factors that influence nutrient dynamics. The most widely accepted function of organic materials is improving the nutrient availability to crops by supplying N. The key to both improving efficiency of N use and reducing N losses is synchronization of N supply from soil, biological N2 fixation, organic residues, and inorganic fertilizers with the crop N demand. Organic materials are not magic; N losses also result from their use. Controlling N release from organic sources depends on their nutrient content and quality, soil properties, and the environmental and management factors. This paper will synthesize the information generated from integrated nutrient management trials in sub-Saharan Africa and the Philippines. Management strategies based on an organic resources database and a dynamic soil-crop simulation model are used to identify organic sources as N fertilizers or soil amendments. The decision support tools are also used to attain optimum synchrony between release from organic sources and soils with crop N demand.     

Investigating the response of soil and vegetable crops to poultry and cow manure using ground and satellite data

Based on the massive production of cow and poultry manures, farmers in Saudi Arabia are moving towards the application of organic fertilizers in their farms. Therefore, the present work was conducted to study the response of soil and selected vegetable crops to poultry and cow manures, using ground data and Landsat-8 and Hyperion images. The studied vegetable crops are cabbage, cauliflower, broccoli, and lettuce. A total of 100 t ha^?1 organic manures were applied as a pre-planting treatment. A 12.5 ha field in Tawdeehiya Farms, 200 km southeast of Riyadh, was earmarked for this study. The field was divided into sectors cultivated with the above-mentioned vegetable crops. Soil characteristics, including the soil pH, the electric conductivity (EC), the nitrogen (N), the phosphorus (P) and the potassium (K), were examined before the application of manures and 25 days after the transplanting process. Observations on crops chlorophyll content, number of leaves, the diameter of merchantable products and yield were also investigated. Furthermore, the relationship between the crop performance and yield was investigated through the satellite images generated vegetation indices (VIs). This study revealed the better performance of poultry manure compared to cow manure in terms of development and production parameters of the experimental crops. Dynamics of the chlorophyll content across the crop growth period revealed that all the tested crops responded significantly (R² = 0.69; P = 0.001) to the poultry manure treatments. Among the tested crops, the chlorophyll content, curd or head sizes and crop yields were quite better in poultry manure applied plots. The investigation of crop yield was significant with poultry manure (R² = 0.64; P = 0.001) than cow manure (R² = 0.57; P = 0.001) using the OSAVI and mNDVI, respectively.     

Plant maintenance and environmental stress. Summarising the effects of contrasting elevation,soil, and latitude on Quercus ilex respiration rates

Aims

Rice (Oryza sativa L.), wheat (Triticum aestivum L.) and common bean (Phaseolus vulgaris L.) are major staple food crops consumed worldwide. Zinc (Zn) deficiency represents a common micronutrient deficiency in human populations, especially in regions of the world where staple food crops are the main source of daily calorie intake. Foliar application of Zn fertilizer has been shown to be effective for enriching food crop grains with Zn to desirable amounts for human nutrition. For promoting adoption of this practice by growers, it is important to know whether foliar Zn fertilizers can be applied along with pesticides to wheat, rice and also common bean grown across different soil and environmental conditions.

Methods

The feasibility of foliar application of zinc sulphate (ZnSO₄.7H₂O) to wheat, rice and common bean in combination with commonly used five fungicides and nine insecticides was investigated under field conditions at the 31 sites-years of seven countries, i.e., China, India, Pakistan, Thailand, Turkey, Brazil and Zambia.

Results

Significant increases in grain yields were observed with foliar Zn/foliar Zn?+?pesticide (5.2–7.7 % of wheat and 1.6–4.2 % of rice) over yields with no Zn treatment. In wheat, as average of all experiments, higher grain Zn concentrations were recorded with foliar Zn alone (41.2 mg kg^?1) and foliar Zn?+?pesticide (38.4 mg kg^?1) as compared to no Zn treatment (28.0 mg kg^?1). Though the magnitude of grain Zn enrichment was lesser in rice than wheat, grain Zn concentrations in brown rice were significantly higher with foliar Zn (24.1 mg kg^?1) and foliar Zn?+?pesticide (23.6 mg kg^?1) than with no Zn (19.1 mg kg^?1). In case of common bean, grain Zn concentration increased from 68 to 78 mg kg^?1 with foliar Zn alone and to 77 mg kg^?1 with foliar Zn applied in combination with pesticides. Thus, grain Zn enrichment with foliar Zn, without or with pesticides, was almost similar in all the tested crops.

Conclusions

The results obtained at the 31 experimental site-years of seven countries revealed that foliar Zn fertilization can be realized in combination with commonly-applied pesticides to contribute Zn biofortification of grains in wheat, rice and common bean. This agronomic approach represents a useful practice for the farmers to alleviate Zn deficiency problem in human populations.

     

Ethanol Tolerance in Bacteria

Abstract

One promising application of biotechnology is in making the production of energy and chemical products from plants (“biomass”) practical. Applications include new markets for crops and reduction of wastes by using field residues or “wastelands” as feedstocks in biomass energy production. In addition, engineered crops could synthesize complex compounds. However, conversion to widespread use of biomass could have undesirable impacts. For example, changes in cropping practices may reduce wildlife populations or disrupt food chains. Other negative impacts of an environmentally insensitive development might include increased release of CO₂ (worsening global warming), and production of wastes, either water, chemical or biological, that present disposal problems. Biotechnology applied to biomass and chemical production has the potential for aiding economic development while greatly improving environmental quality. Environmental concerns are expressed in this paper, in the belief that consideration of potential problems at the inception of a technology increases the likelihood that environmentally sound courses will be pursued.     

Effect of incorporated green manure crops on subsequent oat production in an acid,infertile silt loam

A field-size experiment was initiated in 1982 on an acid, low fertility Springhill silt loam to determine the effect of five unfertilized green manure crops (alsike clover, sweet clover, single- and double-cut red clover, and buckwheat) on subsequent oat production and soil fertility. The field was limed in 1982 and green manures were seeded (without fertilizer) in spring, 1983 in 1400 m² strips randomly assigned within three treatment blocks. Plant tissue samples were taken from different locations in each plot in the fall of 1983 and all crops were incorporated. In 1984 the field was separated into an upper and lower section and each section received three rates of NPK fertilizer (0; 30-36-36; 60-72-72 kg ha^-1) spread across the previous strips. Gary oats were seeded and at harvest were divided into grain and straw. The results indicated significant effects of field sample location, green manure type and fertilizer level on oat yields. Buckwheat significantly reduced oat production compared to the four clovers, while the highest fertilizer rate improved oat yields compared with the other levels of fertilizers. Elemental analysis of the green manure crops and soil fertility was compared with data of the same crops grown in more fertile, neutral soils.     

Inclusion of cancer-associated fibroblasts in drug screening assays to evaluate pancreatic cancer resistance to therapeutic drugs

Pancreatic ductal adenocarcinoma (PDAC) is characterised by a pro-inflammatory stroma and multi-faceted microenvironment that promotes and maintains tumorigenesis. However, the models used to test new and emerging therapies for PDAC have not increased in complexity to keep pace with our understanding of the human disease. Promising therapies that pass pre-clinical testing often fail in pancreatic cancer clinical trials. The objective of this study was to investigate whether changes in the drug-dosing regimen or the addition of cancer-associated fibroblasts (CAFs) to current existing models can impact the efficacy of chemotherapy drugs used in the clinic. Here, we reveal that gemcitabine and paclitaxel markedly reduce the viability of pancreatic cell lines, but not CAFs, when cultured in 2D. Following the use of an in vitro drug pulsing experiment, PDAC cell lines showed sensitivity to gemcitabine and paclitaxel. However, CAFs were less sensitive to pulsing with gemcitabine compared to their response to paclitaxel. We also identify that a 3D co-culture model of MIA PaCa-2 or PANC-1 with CAFs showed an increased chemoresistance to gemcitabine when compared to standard 2D mono-cultures a difference to paclitaxel which showed no measurable difference between the 2D and 3D models, suggesting a complex interaction between the drug in study and the cell type used. Changes to standard 2D mono-culture-based assays and implementation of 3D co-culture assays lend complexity to established models and could provide tools for identifying therapies that will match clinically the success observed with in vitro models, thereby aiding in the discovery of novel therapies.

     

Inhibition of PAI‐1 limits chemotherapy resistance in lung cancer through suppressing myofibroblast characteristics of cancer‐associated fibroblasts

Plasminogen activator inhibitor‐1 (PAI‐1) promotes pulmonary fibrosis through increasing myofibroblast (MF) characteristics, expressing alpha‐smooth muscle actin (α‐SMA) in fibroblasts. Fibroblasts in the tumour stroma are called cancer‐associated fibroblasts (CAFs). Some CAFs have MF characteristics and substantially promote tumour progression and chemotherapy resistance. This study determined whether inhibition of PAI‐1 suppressed MF characteristics of CAFs and limited chemotherapy resistance in lung cancer. To investigate cellular PAI‐1 expression and its correlation with α‐SMA expression of CAFs, 34 patients’ paraffin‐embedded lung adenocarcinoma tissue sections were immunohistochemically stained for PAI‐1 and α‐SMA. Immunohistochemical analysis of lung adenocarcinoma tissues showed that PAI‐1 expression was correlated with that of α‐SMA (r = 0.71, p < 0.001). Furthermore, in vitro, α‐SMA expression of CAFs was limited by PAI‐1 inhibition, and apoptosis of CAFs was increased. In addition, the effectiveness of cisplatin on lung cancer cells co‐cultured with CAFs was increased by suppressing α‐SMA expression using PAI‐1 inhibitor. In lung adenocarcinoma tissues, PAI‐1 expression was associated with T factor and TNM stage. Our data suggest that inhibition of PAI‐1 increased the chemotherapeutic effect on lung cancer through suppressing the MF characteristics of CAFs. Hence, PAI‐1 might be a promising therapeutic target for patients with chemotherapeutic‐resistant lung cancer with CAFs.     

Varying responses of insect herbivores to altered plant chemistry under organic and conventional treatments

The hypothesis that plants supplied with organic fertilizers are better defended against insect herbivores than those supplied with synthetic fertilizers was tested over two field seasons. Organic and synthetic fertilizer treatments at two nitrogen concentrations were supplied to Brassica plants, and their effects on the abundance of herbivore species and plant chemistry were assessed. The organic treatments also differed in fertilizer type: a green manure was used for the low-nitrogen treatment, while the high-nitrogen treatment contained green and animal manures. Two aphid species showed different responses to fertilizers: the Brassica specialist Brevicoryne brassicae was more abundant on organically fertilized plants, while the generalist Myzus persicae had higher populations on synthetically fertilized plants. The diamondback moth Plutella xylostella (a crucifer specialist) was more abundant on synthetically fertilized plants and preferred to oviposit on these plants. Glucosinolate concentrations were up to three times greater on plants grown in the organic treatments, while foliar nitrogen was maximized on plants under the higher of the synthetic fertilizer treatments. The varying response of herbivore species to these strong differences in plant chemistry demonstrates that hypotheses on defence in organically grown crops have over-simplified the response of phytophagous insects.     

Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment

Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy.     

Winter habitat associations of seed-eating passerines on Scottish farmland

CapsuleExtensive surveys highlighted the importance of weedy fodder brassicas, stubbles and open farmland landscapes to declining birds.

Aims To determine habitat associations of seed-eating passerines on Scottish farmland in winter, and recommend appropriate conservation measures.

Methods Transect surveys were carried out on farmland in 100 1-km squares, containing 2885 fields, across Scotland. Birds, crops, field boundary features and weeds were recorded on three visits. Bird–habitat associations were examined using logistic regression models.

Results Seed-eating passerines were highly concentrated: 100 fields (1.4% of the area surveyed) held half the total count. The highest field densities of birds were recorded in fodder brassica crops and stubbles, averaging 12 and 3.3 birds/ha respectively. In fodder crops, weedier fields held many more birds. Multiple regression analysis emphasized the importance of fodder crops, stubbles and farmyards and suggested that declining bird species preferred more open landscapes. Several bird species were associated with fields containing very weedy patches, and/or weeds of the family Chenopodiaceae. These features were commonest in fodder brassicas. Many of the crop types found to be important as bird habitats in winter have declined in area in Scotland; the availability of seed food in such habitats may also have diminished. Such changes may partly explain recent population declines.

Conclusions Agri-environment measures for seed-eating passerines should support the widespread provision of small areas of seed-rich wintering habitats, such as weedy fodder brassicas and stubbles, especially in open (0–10% woodland and scrub) landscapes in areas with mild winters.     

Variation in Yield Gap Induced by Nitrogen,Phosphorus and Potassium Fertilizer in North China Plain

A field experiment was conducted under a wheat-maize rotation system from 1990 to 2006 in North China Plain (NCP) to determine the effects of N, P and K on yield and yield gap. There were five treatments: NPK, PK, NK, NP and a control. Average wheat and maize yields were the highest in the NPK treatment, followed by those in the NP plots among all treatments. For wheat and maize yield, a significant increasing trend over time was found in the NPK-treated plots and a decreasing trend in the NK-treated plots. In the absence of N or P, wheat and maize yields were significantly lower than those in the NPK treatment. For both crops, the increasing rate of the yield gap was the highest in the P omission plots, i.e., 189.1 kg ha⁻¹ yr⁻¹ for wheat and 560.6 kg ha⁻¹ yr⁻¹ for maize. The cumulative omission of P fertilizer induced a deficit in the soil available N and extractable P concentrations for maize. The P fertilizer was more pivotal in long-term wheat and maize growth and soil fertility conservation in NCP, although the N fertilizer input was important for both crops growth. The crop response to K fertilizers was much lower than that to N or P fertilizers, but for maize, the cumulative omission of K fertilizer decreased the yield by 26% and increased the yield gap at a rate of 322.7 kg ha⁻¹ yr⁻¹. The soil indigenous K supply was not sufficiently high to meet maize K requirement over a long period. The proper application of K fertilizers is necessary for maize production in the region. Thus, the appropriate application of N and P fertilizers for the growth of both crops, while regularly combining K fertilizers for maize growth, is absolutely necessary for sustainable crop production in the NCP.     

Distribution of15N fertilizer in field-lysimeters sown with garlic (Allium sativum) and foxtail millet (Setaria italica)

We examined the distribution of residual¹⁵N and its uptake by a foxtail millet crop grown in field lysimeters following at previous garlic crops fertilized with either¹⁵N-urea or¹⁵N-ammonium sulphate. Garlic apprently removed more N from the lysimeters treated with urea-N than from those treated with (NH₄)₂SO₄. Fertilizer-N in the lysmeters was similar (ca. 32% of original) following millet harvest. About 16 per cent of both fertilizers in the lysimeters was removed by the millet.     

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis^1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA³. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment⁴. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion^5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages⁹. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment¹⁰. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture^11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)^13,14. Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs^9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting¹⁶. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.