Development of a rapid identification method for potato cytoplasm and its use for evaluating Japanese collections

The cytoplasm of potatoes, characterized by the presence of T-type chloroplast DNA and β-type mitochondrial DNA, is sensitive to nuclear chromosomal genes that contribute to various types of male sterility. Past breeding efforts with various potato varieties have resulted in several different cytoplasms other than T/β. Varieties with Solanum stoloniferum-derived cytoplasm (W/γ) show complete male sterility, while those with S. demissum-derived cytoplasm (W/α) produce abundant, but non-functional pollen. Thus, identification of cytoplasmic types is important for designing efficient mating combinations. To date, only T-type chloroplast DNA can be accurately identified by a PCR marker. Here, we report a rapid identification technique by multiplex PCR, followed by restriction digestion with BamHI in one reaction tube, and propose a new nomenclature for potato cytoplasm types (T, D, P, A, M, and W). Using this new technique, our collections of 748 genotypes, including 84 Japanese named varieties, 378 breeding lines and 26 landraces, and 260 foreign varieties and breeding lines, were grouped into cytoplasm types: T (73.9?%), D (17.4?%), P (4.5?%), A (1.5?%), M (0.3?%), and W (2.4?%). The utility of this marker system for breeding is discussed.     

Development of a fluorescent light technique for evaluating microdamage in bone subjected to fatigue loading.

A new method using fluorescent light microscopy has been developed to visualize and evaluate bone microdamage. We report the findings of two different experiments with a common aim of comparing the fluorescent light technique to the brightfield method for quantifying microdamage in bone. In Experiment 1, 36 canine femurs were tested in four-point cyclic bending until they had lost between 5 and 43% of their stiffness. The loaded portion of the bone was stained en bloc with basic fuchsin for the presence of damage. Standard point counting techniques were used to calculate fractional damaged area (Dm.Ar = Cr.Ar/B.Ar, mm2/mm2) under brightfield and fluorescent microscopy. In Experiment 2, bone microdamage adjacent to endosseous implants, subjected to fatigue loading (150,000 cycles, 2 Hz and 37 degrees C) ex vivo was examined. The bone around the implant was either allowed to heal (adapted specimen) for 12 weeks after placement in dog mid-femoral diaphyses prior to testing or was loaded immediately to simulate non-healed bone surrounding endosseous implants (non-adapted). Crack numerical density (Cr.Dn = Cr.N/B.Ar, #/mm2), crack surface density (Cr.S.Dn = Tt.Cr.Le/B.Ar, mm/mm2) and fractional damaged area were calculated separately by both techniques in the adapted and non-adapted specimens. In both Experiments 1 and 2, significantly more microdamage was detected by the fluorescent technique than by the brightfield method. Also, there was a trend towards higher intraobserver repeatability when using the fluorescent method. These results suggest that the brightfield technique underestimates microdamage accumulation and that the fluorescent technique better represents the actual amounts of microdamage present. The results demonstrate that the fluorescent method provides an accurate and precise approach for bone microdamage evaluation, and that it improves the prediction of stiffness loss from damage accumulation.     

Development of a bioreactor for evaluating novel nerve conduits

We describe an experimental closed bioreactor device for studying novel tissue engineered peripheral nerve conduits in vitro. The system integrates a closed loop system consisting of one, two, or three experimental nerve conduits connected in series or parallel, with the ability to study novel scaffolds within guidance conduits. The system was established using aligned synthetic microfiber scaffolds of viscose rayon and electrospun polystyrene. Schwann cells were seeded directly into conduits varying from 10 to 80 mm in length and allowed to adhere under 0 flow for 1 h, before being cultured for 4 days under static or continuous flow conditions. In situ viability measurements showed the distribution of live Schwann cells within each conduit and enabled quantification thereafter. Under static culture viable cells only existed in short conduit scaffolds (10 mm) or at the ends of longer conduits (20-80 mm) with a variation in viable cell distribution. Surface modification of scaffold fibers with type-1 collagen or acrylic acid increased cell number by 17% and 30%, respectively. However, a continuous medium flow of 0.8 mL/h was found to increase total cell number by 2.5-fold verses static culture. Importantly, under these conditions parallel viability measurements revealed a ninefold increase compared to static culture. Fluorescence microscopy of scaffolds showed cellular adhesion and alignment on the longitudinal axis. We suggest that such a system will enable a rigorous and controlled approach for evaluating novel conduits for peripheral nerve repair, in particular using hydrolysable materials for the parallel organization of nerve support cells, prior to in vivo study.     

Soil textural class plays a major role in evaluating the effects of land use on soil quality indicators

Inappropriate land use that negatively affects ecological processes and soil quality is generally considered to be the primary cause of soil degradation in tropical agroecosystems. We hypothesized that in addition to land use, soil textural class also has an important effect on ecological processes and soil quality. To test our hypothesis, effects of land use change on soil organic fractions as well as microbial and biochemical indicators were quantified for clayey and sandy-clay-loam soils within the native Cerrado biome, pasture (Brachiaria brizantha) and sugarcane (Saccharum officinarum) agroecosystems in southwestern Brazil (Minas Gerais state). Labile carbon, humic substances, soil microbial respiration (SMR), microbial biomass carbon (MBC), metabolic quotient (qCO₂), hydrolysis of fluorescein diacetate (FDA), beta-glucosidase, urease, phosphatase and arylsulphatase activities were measured for each sample. Labile carbon concentrations were not affected by land use but were lower in sandy-clay-loam soil than clayey soil. Humic substances were at the highest concentrations in the native Cerrado and the lowest in sugarcane agroecosystems. Sandy-clay-loam soil had lower humic acid concentrations than clayey soil. Soil microbial indicators (SMR, MBC and FDA) showed lower values in pasture and sugarcane agroecosystems than in the native Cerrado. FDA was a more sensitive microbial indicator than SMR and MBC for detecting land use and textural class differences. The qCO₂ indices were greater in sugarcane systems than in either pasture or native Cerrado systems. The activity of exocellular hydrolytic enzymes, such as beta-glucosidase, urease, phosphatase and arylsulphatase, was smaller in sugarcane and pasture agroecosystems than in native Cerrado ecosystems. Within the same land use, the activity of these enzymes was always greater in clayey soil than in sandy-clay-loam soil, indicating a higher impact of land uses on enzyme activities in clayey soils. Results for the measured indicators support the hypothesis that soil textural class plays a major role in assessing differences between land use systems in the Brazilian Cerrado biome.     

Effects of short-term magnesium supplementation on ionized,total magnesium and other relevant electrolytes levels

BioMetals - This study aimed to investigate the short-term effects of three magnesium (Mg) dietary supplements containing mineral immediately available for absorption on Mg biochemical status...     

The Skill Plot: a graphical technique for evaluating continuous diagnostic tests

Summary .   We introduce the Skill Plot, a method that it is directly relevant to a decision maker who must use a diagnostic test. In contrast to ROC curves, the skill curve allows easy graphical inspection of the optimal cutoff or decision rule for a diagnostic test. The skill curve and test also determine whether diagnoses based on this cutoff improve upon a naive forecast (of always present or of always absent). The skill measure makes it easy to directly compare the predictive utility of two different classifiers in an analogy to the area under the curve statistic related to ROC analysis. Finally, this article shows that the skill-based cutoff inferred from the plot is equivalent to the cutoff indicated by optimizing the posterior odds in accordance with Bayesian decision theory. A method for constructing a confidence interval for this optimal point is presented and briefly discussed.     

Evaluation of the use of a model rhizodeposition technique to separate root and microbial respiration in soil

A model rhizodeposition technique to estimate the root and microbial components of ¹⁴C soil/root respiration in pulse-labelling experiments is described. The method involves the injection of model rhizodeposits, consisting of ¹⁴C-labelled glucose, root extract or root cell wall material, into the rooted soil of an unlabelled plant, simultaneously with the pulse-labelling of a separate but similar plant with ¹⁴CO₂. In a growth chamber experiment with 30 day old wheat and barley the contribution of direct root respiration to ¹⁴C soil/root respiration over a 26 day period after labelling was estimated 89–95%. Estimates of direct root respiration in field-grown wheat and barley at different development stages in most cases accounted for at least 75% of ¹⁴C soil/root respiration over a 21 day period after labelling. The mineralization rate of injected ¹⁴C-glucose was positively correlated with the concentration of glucose-C established in soil. The use of the method in rhizosphere carbon budget estimations is evaluated. Communication No. 73 of the Dutch Programme on Soil Ecology of Arable Farming Systems. Communication No. 73 of the Dutch Programme on Soil Ecology of Arable Farming Systems.     

Methods for evaluating the acrosomal status of mammalian sperm

A full understanding of the acrosome reaction is central to understanding sperm function. Acrosomal status can be determined on living, motile sperm in only a few mammalian species. For other species, many light microscopic methods have been developed, including colored stains for bright-field microscopy, and probes for fluorescence microscopy. We review the existing methods and the criteria that should be considered in the choice of an assay.     

Development of a technique based on the affinity resins for evaluation of DNA methylation status in vertebrates

The methods for synthesis and application of resins based on the functional domains of Kaiso and CpG-binding protein (CGBP), which can bind methylated and unmethylated CpG-dinucleotides, respectively, are shown. Kaiso resin was obtained by the affinity interaction of glutathione-sepharose with a chimeric protein, which is expressed in Escherichia coli and contain glutathione S-transferase (GST) and zinc finger domain of methyl-DNA-binding Kaiso protein within the same translation frame. Kaiso resin, like MBD-domain based resin, has an ability to bind methylated DNA. Experiments with the short DNA fragments demonstrated that methylated DNA is eluted from the resin by 0.7 M KCl, whereas unmethylated DNA is washed out by 0.2–0.5 M KCl after binding. Quantitative PCR showed that the enrichment with methylated p16 promoter region and the absence of accumulation of γ-actin unmethylated promoter were observed due to the binding of genomic DNA, isolated from the colo 320 cell line (human colorectal adenocarcinoma), with the Kaiso resin. The CGBP resin based on the CxxC domain of CGBP protein binds to the sequences which contain unmethylated CpG-dinucleotides. Our experiments also showed no effect of MBD3L1 protein on MBD2-resin capacity of binding with methylated DNA. The obtained resins can be applied to study methylation status of both specific DNA sequences and the whole genome.     

Mab-ZAP: a tool for evaluating antibody efficacy for use in an immunotoxin

Immunotoxins, consisting of antibodies coupled to toxins, are extremely useful tools in the elimination of specific cell populations in vitro and in vivo for research and therapeutic applications. The antibody is used to target the toxin to a specific cell population, which is distinguished by its cell-surface antigen. Not all antibodies are suitable for creating an immunotoxin, and large numbers of antibodies may need to be screened. This is a time-consuming and expensive process if each potential candidate must be conjugated to the toxin and purified. A faster and more economical way to identify potential targeting antibodies is to use a second immunotoxin, an anti-IgG antibody that is coupled to the toxin. The second immunotoxin eliminates the need to couple every candidate antibody to the toxin because it can simply be added to cells in culture with the antibody of interest. Using this method, many antibodies can be screened quickly and efficiently for their ability to internalize.     

罗浮栲林土壤微生物碳利用效率对短期氮添加的响应

作为调节土壤碳矿化过程的重要参数,微生物碳利用效率(CUE)对理解陆地生态系统中的碳循环至关重要。本研究在戴云山罗浮栲林设置对照(0 kg N·hm^-2·a^-1)、低氮(40 kg N·hm^-2·a^-1)和高氮(80 kg N·hm^-2·a^-1) 3个氮添加水平以模拟氮沉降,测定了表层(0～10 cm)土壤基本理化性质、有机碳组分、微生物生物量和酶活性;并利用¹⁸O标记水方法测定土壤微生物CUE,以更好地理解氮沉降加剧对微生物CUE的影响及其影响因素。结果表明: 短期氮添加显著降低了土壤微生物的呼吸速率、碳和氮获取酶活性,但显著增加了土壤微生物CUE。β-N-乙酰氨基酸葡糖苷酶(NAG)/微生物生物量碳(MBC)、微生物呼吸速率、β-葡萄糖苷酶(BG)/MBC、纤维素水解酶(CBH)/MBC和土壤有机碳含量是影响CUE的主要因素,且CUE与NAG/MBC、微生物呼吸速率、BG/MBC和CBH/MBC呈显著负相关,与土壤有机碳呈显著正相关。综上,短期氮添加导致土壤微生物获取碳和氮的成本降低,减少微生物呼吸,从而提高了土壤微生物CUE,这将有助于提高罗浮栲林土壤碳固存潜力。     

Development of an agar lift-DNA/DNA hybridization technique for use in visualization of the spatial distribution of Eubacteria on soil surfaces.

While microbial growth is well-understood in pure culture systems, less is known about growth in intact soil systems. The objective of this work was to develop a technique to allow visualization of the two-dimensional spatial distribution of bacterial growth on a homogenous soil surface. This technique is a two-step process wherein an agar lift is taken and analyzed using a universal gene probe. An agar lift is comprised of a thin layer of soil that is removed from a soil surface using an agar slab. The agar is incubated to allow for microbial growth, after which, colonies are transferred to a membrane for conventional bacterial colony DNA/DNA hybridization analysis. In this study, a eubacterial specific probe was used to demonstrate that growing bacterial populations on soil surfaces could be visualized. Results show that microbial growth and distribution was nonuniform across the soil surface. Spot supplementation of the soil with benzoate or glucose resulted in a localized microbial growth response. Since only growing colonies are detected, this technique should facilitate a greater understanding of the microbial distribution and its response to substrate addition in more heterogenous soil systems.     

A direct observation technique for evaluating sclerotium germination by Macrophomina phaseolina and effects of biocontrol materials on survival of sclerotia in soil

Germination of sclerotia of Macrophomina phaseolina was quantified by direct microscopic observation following application of experimental treatments in vitro and incubation of sclerotia in soil. To assay germination, pieces of agar containing sclerotia were macerated in dilute, liquid cornmeal agar on glass slides; thinly spread; and incubated in a saturated atmosphere for 18–22 h. Germinated sclerotia then were identified by morphological features of germ hyphae. Frequencies of germination were similar in three dilute agar media. Germination was not affected by air-drying sclerotia for 2 weeks, but it was significantly reduced after 4 weeks and greatly reduced or eliminated after 6 or 8 weeks. Survival of sclerotia for 14 days in soil was greatest at 50, 75, and 100% moisture-holding capacity, less at 0 and 25%, and least at 125% (flooded soil). Incorporation of ground poultry litter into soil at 5% by weight reduced survival of sclerotia after 13 days, and incorporation of litter at 10% nearly eliminated it. These results indicate that the direct-observation technique may be used to evaluate animal wastes and other agricultural byproducts for biocontrol activity against sclerotia of M. phaseolina in soil.     

油气田土壤甲烷氧化菌实时荧光定量PCR检测技术的 建立与应用

【目的】建立快速检测甲烷氧化菌含量的SYBR GreenⅠ实时荧光定量PCR技术,用于油气微生物勘探。【方法】以含有甲烷氧化菌功能基因pmoA片段的重组质粒为标准品,优化实验条件,建立标准曲线,进行敏感性、重复性和特异性评价,并将该技术用于实际样品的检测。【结果】该技术标准曲线的相关系数R2为0.999 9,扩增效率为99.976%,检测范围为3.897×101-3.897×109 copies/μL,检出限约为40 copies/μL,重复性实验中CT值的变异系数优于3%,对12种非甲烷氧化菌均没有扩增,显示该技术具有很好的敏感性、重复性和特异性。利用该技术对气田、油田和非油气田土样进行了检测,发现气田具有明显的异常高值。【结论】为油气田的勘探建立了一种高效、特异、灵敏、准确的甲烷氧化菌荧光定量PCR检测技术,同时为其它指示菌检测技术的建立提供了参考。     

半干旱草原土壤呼吸组分区分与菌根呼吸的贡献

土壤呼吸组分的区分对于理解地下碳循环过程非常重要。而菌根真菌在地下碳循环过程中扮演着重要的角色, 但是有关菌根呼吸在草原生态系统中的研究相对较少。该研究在内蒙古半干旱草原应用深浅环网孔法, 结合浅环、深环(排除根系)和一个带有40 μm孔径窗口的土壤环(排除根系但是有菌根菌丝体)将根和菌丝物理分离, 来区分不同的呼吸组分。结果表明: 异养呼吸对总呼吸的贡献比例为51%, 根呼吸的贡献比例为26%, 菌根呼吸的贡献比例为23%, 菌根呼吸的比例3年变化范围为21%-26%。与国内外研究相比, 此方法提供了一个相对稳定的菌根呼吸测量精度范围, 在草原生态系统中切实可行。对菌根呼吸的准确定量将有助于预测草原生态系统土壤碳释放过程对未来气候变化的响应。