Classification accuracy in multiple color fluorescence imaging microscopy

BACKGROUND: The discriminatory power and imaging efficiency of different multicolor FISH (M-FISH) analysis systems are key factors in obtaining accurate and reproducible classification results. In a recent paper, Garini et al. put forth an analytical technique to quantify the discriminatory power ("S/N ratio") and imaging efficiency ('excitation efficiency') of multicolor fluorescent karyotyping systems. METHODS: A parametric model of multicolor fluorescence microscopy, based on the Beer-Lambert law, is analyzed and reduced to a simple expression for S/N ratio. Parameters for individual system configurations are then plugged into the model for comparison purposes. RESULTS: We found that several invalid assumptions, which are used to reduce the complex mathematics of the Beer-Lambert law to a simple S/N ratio, result in some completely misleading conclusions about classification accuracy. The authors omit the most significant noise source, and consider only one highly abstract and unrepresentative situation. Unwisely chosen parameters used in the examples lead to predictions that are not consistent with actual results. CONCLUSIONS: The earlier paper presents an inaccurate view of the M-FISH situation. In this short communication, we point out several inaccurate assumptions in the mathematical development of Garini et al. and the poor choices of parameters in their examples. We show results obtained with different imaging systems that indicate that reliable and comparable results are obtained if the metaphase samples are well-hybridized. We also conclude that so-called biochemical noise, not photon noise, is the primary factor that limits pixel classification accuracy, given reasonable exposure times. Copyright Wiley-Liss, Inc.     

Gene selection for classification of cancers using probabilistic model building genetic algorithm

Recently, DNA microarray-based gene expression profiles have been used to correlate the clinical behavior of cancers with the differential gene expression levels in cancerous and normal tissues. To this end, after selection of some predictive genes based on signal-to-noise (S2N) ratio, unsupervised learning like clustering and supervised learning like k-nearest neighbor (k NN) classifier are widely used. Instead of S2N ratio, adaptive searches like Probabilistic Model Building Genetic Algorithm (PMBGA) can be applied for selection of a smaller size gene subset that would classify patient samples more accurately. In this paper, we propose a new PMBGA-based method for identification of informative genes from microarray data. By applying our proposed method to classification of three microarray data sets of binary and multi-type tumors, we demonstrate that the gene subsets selected with our technique yield better classification accuracy.     

短期氮、水添加对放牧背景下荒漠草原短花针茅叶片属性的影响

叶片属性是反映植物对环境变化敏感程度的重要特征,可在一定程度上预测植物对放牧干扰后的恢复能力。短花针茅（Stipa breviflora）是内蒙古荒漠草原的主要建群种。在不同放牧强度背景下的短花针茅草原开展了围封模拟放牧持续利用的实验,同时进行添加氮素和水分的恢复措施,测定了7月和9月中旬建群种短花针茅叶片的比叶面积、叶干物质含量,以及叶片全氮、叶片全磷和叶片全碳含量,分析水分和氮素添加对建群种短花针茅叶片的影响,探讨不同放牧强度下短花针茅可持续利用的氮水调控机制。结果显示,氮素和水分添加显著地增加了短花针茅叶片氮含量,降低了叶片碳氮比;放牧强度也显著地增加了叶片氮含量,且轻度放牧下的叶片氮含量（20.36 g/kg）显著高于对照（18.80 g/kg）;生长末期短花针茅的比叶面积、叶片碳含量、叶片碳氮比和叶片碳磷比显著高于生长盛期,叶片氮含量和磷含量显著低于生长盛期;在生长盛期和生长末期,不同放牧强度背景下对短花针茅所采取的氮素和水分的供给措施也不同。研究结果表明在放牧背景下短期氮、水添加提高了短花针茅的叶片氮含量,特别是在生长季后期水分添加增加了叶片氮和磷含量,可进一步促进短花针茅的生长。我们的结果也表明了资源供给水平的改善有助于短花针茅的迅速恢复。     

Tissue analyses guidelines for diagnosing sulfur deficiency in white wheat

Summary Visual identification of S deficiency in white wheat is difficult since deficiency symptoms are nearly identical with those of N deficiency. In this study, S deficiency was best identified by determining the total N/S ratio rather than S concentration in vegetative tissue. Vegetative growth generally decreased from tillering to boot when the whole plant N/S ratio exceeded 17. The N/S ratio in S-sufficient plants declined gradually with age, implying that the critical N/S ratio may decline with advancing growth. Changes in stem: leaf ratio could have been responsible for the decline since the N/S ratio in stem tissue at heading was less than that of green leaf tissue.Sulphur concentration less reliably indicated S-deficiency, because differences in S levels between S-deficient and S-sufficient wheat, were often less than year-to-year variation of S concentration of plants sampled at the same growth stage. In addition, S concentration in whole plants declined sharply between tillering and heading. These factors make it difficult to designate a critical S level. Sulfur distribution among various plant organs suggests that critical S levels might best be obtained by utilizing green leaf tissue.Nitrogen concentration in S-sufficient wheat plants also decreased quite rapidly with growth, which indicates a similar difficulty for determining critical N percentages. Consequently, the most reliable distinction between N and S deficiency in wheat was accomplished by evaluation of the total N/S ratio in whole plant tissue.Contribution from the Agricultural Research Service, USDA, in cooperation with the Agricultural Experiment Station, Oregon State University. Technical Paper No.3953 of the latter.     

枯草蛋白酶E的定点突变及其对酶性质的影响

用定点突变的方法研究S221C/P225A,N118S/S221C/P225A,D60N/S221C/P225A和Q103R/S221C/P225A突变对蛋白酶活性,酯酶活性与蛋白酶活性之比的影响。结果表明：S221C/P225A突变使蛋白酶活性比枯草蛋白酶E低73000多倍,酯酶活性与蛋白酶活性之比是Subtiligase的3倍;N118S/S221C/P225A突变使蛋白酶活性和酯酶活性分别比S221C/P225A突变下降3.6倍和15倍,酯酶与蛋白酶活性之比下降4倍,同时增加变体酶的热稳定性;D60N/N118S/S221C/P225A突变使蛋白酶活性比N118S/S221C/P225A突变体下降15倍,但对酯酶活性几乎没有影响,酯酶与蛋白酶活性之比增加14倍,分别是S221C/P225A突变体和Subtiligase的3.3倍和10.3倍;但是,Q103R/N118S/S221C/P225A突变使蛋白酶活性比N118S/S221C/P225A突变体增加5倍,酯酶活性下降55倍,酯酶与蛋白酶活性之比下降1000倍。     

硫水平对玉米氮、硫代谢特性及根系活性的影响

通过Hoagland营养液水培玉米,定量研究了供S水平对玉米根系活性和植株S、N代谢特性的影响．当玉米缺S时。玉米根系活性和叶片硝酸还原酶(NR)活性显著降低,植株冠／根比增大;根系与地上部对S、N的吸收均减少。S在根系和新生叶中分配相对减少、在衰老叶和茎鞘中分配相对增多;N在根系中分配增多,新生叶和完全展开叶中分配相对减少;植株蛋白质、蛋白氮、蛋白硫、无机硫的含量均显著降低,非蛋白氮比率增多,无机硫占总S的比率变小,全N／全S比增大;蛋白质中的N／S比在地上部无显著变化。而在根系中显著增大;根系对S缺乏的反应更为敏感．研究表明。缺S植株蛋白质含量与NR活性之间呈极显著正相关;S的缺乏或过量均影响玉米的N、S代谢;以根系作为S素营养诊断器官。以N／S比或无机S／全S比作为诊断指标较灵敏。正常条件下其数值分别为10．7和0．302．     

Rapid evolution of parental rDNA in a synthetic tobacco allotetraploid line

Unidirectional gene conversion of rDNA units has occurred in the evolution of natural tobacco (Nicotiana tabacum). In this paper we report the use of the synthetic tobacco line Th37, 4n (N. sylvestris × N. tomentosiformis), to study early rDNA evolution associated with allopolyploidy. At least three classes of newly amplified rDNA unit variants were identified (17/20 plants). Their presence was often accompanied by near-complete elimination of N. tomentosiformis-donated rDNA units (15/20 plants). Novel rDNA units were of N. tomentosiformis-type and contained rearranged subrepeats in the intergenic spacer. The maternal N. sylvestris-derived units were unchanged, except for some alteration in the ratio of individual gene family members. A cytogenetic analysis revealed rDNA sites on N. sylvestris-derived chromosomes S10, S11, and S12 and N. tomentosiformis-derived chromosomes T3 and in some cases T4. An rDNA locus does not occur on N. tomentosiformis chromosome 4. The locus on chromosome T4 of some hybrids correlates with the occurrence of the novel units that probably amplified at the locus. Combined with an analysis of tobacco cultivars, the data indicate that an initial burst of rDNA evolution associated with allopolyploidy was followed by a slower process that led towards reduced complexity and a decreased number of rDNA variants.     

Control of DNA Replication by the Nucleus/Cytoplasm Ratio in Xenopus

The nucleus/cytoplasm (N/C) ratio controls S phase dynamics in many biological systems, most notably the abrupt remodeling of the cell cycle that occurs at the midblastula transition in early Xenopus laevis embryos. After an initial series of rapid cleavage cycles consisting only of S and M phases, a critical N/C ratio is reached, which causes a sharp increase in the length of S phase as the cell cycle is reconfigured to resemble somatic cell cycles. How the N/C ratio determines the length of S phase has been a longstanding problem in developmental biology. Using Xenopus egg extracts, we show that DNA replication at high N/C ratio is restricted by one or more limiting substances. We report here that the protein phosphatase PP2A, in conjunction with its B55α regulatory subunit, becomes limiting for replication origin firing at high N/C ratio, and this in turn leads to reduced origin activation and an increase in the time required to complete S phase. Increasing the levels of PP2A catalytic subunit or B55α experimentally restores rapid DNA synthesis at high N/C ratio. Inversely, reduction of PP2A or B55α levels sharply extends S phase even in low N/C extracts. These results identify PP2A-B55α as a link between DNA replication and N/C ratio in egg extracts and suggest a mechanism that may influence the onset of the midblastula transition in vivo.     

Transmission ratio distortion in intraspecific hybrids of Mimulus guttatus: implications for genomic divergence

We constructed a genetic linkage map between two divergent populations of Mimulus guttatus. We genotyped an F(2) mapping population (N = 539) at 154 AFLP, microsatellite, and gene-based markers. A framework map was constructed consisting of 112 marker loci on 14 linkage groups with a total map length of 1518 cM Kosambi. Nearly half of all markers (48%) exhibited significant transmission ratio distortion (alpha = 0.05). By using a Bayesian multipoint mapping method and visual inspection of significantly distorted markers, we detected 12 transmission ratio distorting loci (TRDL) throughout the genome. The high degree of segregation distortion detected in this intraspecific map indicates substantial genomic divergence that perhaps suggests genomic incompatibilities between these two populations. We compare the pattern of transmission ratio distortion in this map to an interspecific map constructed between M. guttatus and M. nasutus. A similar level of segregation distortion is detected in both maps. Collinear regions between maps are compared to determine if there are shared genetic patterns of non-Mendelian segregation distortion within and among Mimulus species.     

Effect of sulfide to nitrate ratios on the simultaneous anaerobic sulfide and nitrate removal

Present investigation deals with the effect of sulfide to nitrate (S/N) molar ratio on the simultaneous anaerobic sulfide and nitrate removal on capacity, stability and selectivity of the process. The volumetric sulfide-sulfur and nitrate-nitrogen removal rates at molar S/N ratio of 5:2 were 4.86 kg (m³ d)⁻¹ and 0.99 kg (m³ d)⁻¹, respectively, which were higher than those at S/N molar ratios of 5:5 and 5:8. Moreover, the fluctuations in the effluent at S/N ratio of 5:2 were less than those at the other two tested ratios. During the operation, the ratio of converted sulfide to converted nitrate tended to approach 5:2. The selectivity for elemental sulfur and dinitrogen was improved when the S/N molar ratio was set at 5:2 rather than 5:5 or 5:8. The process became unstable if the influent sulfide surpassed its critical concentration. The electron balance between reactants was also analyzed for different S/N molar ratios.     

Determination of size distribution of elliptical microvessels from size distribution measurement of their section profiles

In transmission electron microscopy, microvessels (MVs) are studied as profiles on ultrathin sections. To determine MV sizes from measurements made on MV profiles, an assumption must be made about MV shape, a circular cylinder being used to approximate the latter on limited lengths. However, this model is irrelevant in case MVs have some flatness. The elliptical cylinder model is preferable, although relationships between the cylinder profile (two-dimensional; 2D) and its true (three-dimensional; 3D) sizes are not yet known. We have obtained the 2D/3D functions that express the relationships between such profile sizes as the minor radius (Y), major radius (X), axial ratio (X/Y), area (S), and perimeter (P) on the one hand, and the corresponding MV sizes (Y(0), X(0), X(0)/Y(0), S(0), and P(0)) on the other. The 2D/3D functions make it possible to derive elliptical MV sizes from section profile size distributions, probability density functions (PDFs) for the latter being determined. We have applied the 2D/3D functions in studying axial ratios of thyroid hemocapillaries. A factual X/Y frequency histogram has been constructed and fitted by theoretical X/Y PDFs plotted for different sets of capillary sizes. The thyroid capillaries have been revealed to be clustered, 72.7% of them having X(0)/Y(0) approximately 1.6, 17.6%, X(0)/Y(0) approximately 1.0, and 9.7%, X(0)/Y(0) approximately 3.2. The proposed technique is instrumental in precise modeling of microcirculatory network geometry.     

Effect of the concentration of nitrogen compounds on microbial reduction of sulphates.

A change of the N/S ratio in the reaction medium affects the degree of the microbial reduction of sulphates to sulphides (x'). The time interval between the initial and the log phase of the process (t0) also varies, depending on the N/S ratio. It was demonstrated that optimal reduction conditions by the studied strain of Desulfovibrio desulphuricans exist in media of N/S=0.33.     

Seroprevalence of Neospora caninum infection on dairy cattle in farms from southern Romania

Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, is one of the major causes of abortion in cattle worldwide. Conventional serological techniques, such as the indirect fluorescent antibody test and enzyme-linked immunosorbent assay (ELISA), are routinely used in adult animals and aborted fetuses for the detection of anti- N. caninum antibodies. In Romania, infection with N. caninum in cattle has been reported recently, but only in limited areas from the north and central parts of the country. Therefore, the aim of this study was to obtain additional seroepidemiological data on infection with N. caninum on dairy farms from the south of Romania. A total of 258 blood samples was analyzed from 230 dairy cows and 28 calves from 9 dairy farms in southern Romania; the presence of specific IgG antibodies against N. caninum was determined using an indirect ELISA test. The average seroprevalence was 40.3%, but the within-herd prevalence ranged between 11.5 and 80.0%; the seroprevalence in dairy cows was 41.7%, while in calves it was 28.6%. Of the positive samples, 74.0% (77/104) had a high positive reaction (S/P ratio more than 1.0), while 26.0% (27/104) had a low positive reaction (S/P ratio between 0.5 and 1.0). This study indicates that N. caninum infection is widespread in the south of Romania, which could explain the causes of abortions registered in some herds in the studied area. However, a serological screening across the country is planned in order to assess the actual national prevalence of N. caninum infection, followed by implementation of a prevention and control program.     

沙地植被碳氮磷化学计量特征与物种多样性的关系

植被化学计量特征表征植物营养限制.它是否会影响物种多样性需要进一步探究.本研究以宁夏哈巴湖国家级自然保护区沙地油蒿群落和沙柳群落为对象,计算了植被碳(C)、氮(N)、磷(P)含量,分析了植被C、N、P生态化学计量特征对沙地植物群落物种多样性的影响.结果表明: 在流动沙丘和半固定沙丘生境中的沙柳群落,物种多样性Simpson指数与植被C/N值存在显著的负相关关系,而与植被N/P值的相关性不显著;在半固定沙丘和固定沙丘生境中的油蒿群落,物种多样性Shannon指数与N/P值存在显著正相关关系,而与C/N值存在显著负相关关系.结合C、N、P生态化学计量特征与冗余分析可知,油蒿群落和沙柳群落的植被P含量对物种多样性的影响不同,导致两个群落的植被N/P值对物种多样性产生不同的影响.     

Depth profiling of the elemental surface composition of the oral microorganism S. salivarius HB and fibrillar mutants by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy (XPS) on microbial cell surfaces requires freeze-drying of cells, and as a result, the cell surface appendages flatten out on the cell surface and form a collapsed fibrillar mass. At present, it is unclear how the density, length and composition of these fibrils influence the elemental surface composition as probed by XPS. The sampling depth of XPS can be varied by changing the electron take-off angle. In this article, we made a depth profiling of the collapsed fibrillar mass of Streptococcus salivarius HB and fibril-deficient mutants by angle-dependent XPS. Methylamine tungstate negative staining and ruthenium red staining followed by sectioning revealed distinct classes of fibrils with various lengths on each of the strains. Interpretation of the angle dependence of the oxygen/carbon (O/C) and phosphorus/carbon (P/C) surface concentration ratios of these strains was difficult. However, the angle dependence of the nitrogen/carbon (N/C) surface concentration ratio could be fully interpreted: N/C did not vary with sampling depth on a bald strain, S. salivarius HBC12 and on S. salivarius HB7, a strain with a dense array of fibrils of uniform length. N/C decreased with sampling depth in case of a sparsely fibrillated strain, S. salivarius HBV51 and eventually reached the value observed for the bald strain, HBC12. A high N/C at small sampling depth was observed for S. salivarius HB with protruding, protein rich fibrils. We conclude that elemental depth profiling of microbial cell surfaces by XPS can be interpreted to coincide with structural and biochemical information on the cell surface as obtained by electron microscopy and can therefore be considered as a useful technique to study structural features of cell surfaces in combination with electron microscopy.     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

The signal-to-noise ratio as a measure of HA oligomer concentration: a MALDI-TOF MS study

MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) was used to determine ng amounts of defined hyaluronan (HA) oligomers obtained by enzymatic digestion of high molecular weight HA with testicular hyaluronate lyase. The signal-to-noise (S/N) ratio of the positive and negative ion spectra represents a reliable concentration measure: Amounts of HA down to about 40 fmol could be determined and there is a linear correlation between the S/N ratio and the HA amount between about 0.8 pmol and 40 fmol. However, the detection limits depend considerably on the size of the HA oligomer with larger oligomers being less sensitively detectable. The advantages and drawbacks of the S/N ratio as concentration measure are discussed.     

Macronutrient content in leaves of bean plants (Phaseolus vulgaris,L.) grown with differents rates of N/S as fertilizers

Summary The effect of increasing rates of nitrogen (N) and sulphur (S) as fertilizers on the yield, leaf area and N, P, S, Ca, Mg, NO₃ ⁻ and SO₄ ⁼ content in leaves of bean (Phaseolus vulgaris, L.) were studied in a hydroponic culture experiment under greenhouse conditions. Bean plants responded significantly to all treatments with differents N/S ratios. When plants grew with high N/S ratios, the leaf content of N, Ca and NO₃ ⁻ increased while the content of K, P and SO₄ ⁼ decreased. However, optimal yield and leaf area were not obtained. Optimal leaf and fruit dry matter was obtained at N/S ratio value of 1.41. When lower N/S rates were used, optimal leaf and fruit dry matter was only observed when the leaf N/S ratio was between 15 and 16. At high sulphate levels in the nutrient solution there is no interaction with nitrate which is easily observed, resulting in an increase in yield. An interaction between nitrate and sulphate in the nutrient solution was found at a N/S ratio of 0.81 which produced in leaves a synergic effect between P-K, an antagonistic effect between N-P and N-K and a lower yield. This research was supported by Fundacion ‘Ramon Areces’.     

Overestimation of nonsynonymous/synonymous rate ratio by reverse-translation of aligned amino acid sequences

In the analysis of protein-coding nucleotide sequences, the ratio of the number of nonsynonymous substitutions to that of synonymous substitutions (d(N)/d(S)) is used as an indicator for the direction and magnitude of natural selection operating at the amino acid sequence level. The d(S) and d(N) values are estimated based on the comparison of homologous codons, which are often identified by converting (reverse-translating) aligned amino acid sequences into codon sequences. In this method, however, homologous codons may be mis-identified when frame-shifts occurred or amino acid sequences were mis-aligned, which may lead to overestimation of the d(N)/d(S) ratio. Here the effect of reverse-translating aligned amino acid sequences on the estimation of d(N)/d(S) ratio was examined through a large-scale analysis of protein-coding nucleotide sequences from vertebrate species. Apparently, 1-9% of codon sites that were identified as homologous with reverse-translation contained non-homologous codons, where the d(N)/d(S) ratio was unduly high. By correcting the d(N)/d(S) ratio for these codon sites, it was inferred that the ratio was 5-43% overestimated with reverse-translation. These results suggest that caution should be exerted in the study of natural selection using the d(N)/d(S) ratio by reverse-translating aligned amino acid sequences.