Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing.

We describe a simple and cost-effective method for the synthesis of an internal fluorescently labeled DNA standard for fragment sizing using an automated DNA sequencer. A set of primer pairs labeled with ROX was developed to amplify 12 DNA fragments, 58-417 bp, derived from a conserved region of plant chloroplast DNA. These amplified fragments were mixed together, constituting a fluorescent internal DNA size marker. The precision of the size standard was evaluated by estimating the size of 20 alleles that were amplified at four dinucleotide microsatellite loci with the synthesized size standard and the commercial internal sizing standard, GeneScan Rox500. A number of intra-gel and inter-gel comparisons were run, and an analysis of variance was carried out. No significant difference was observed between the size estimates obtained with the synthesized DNA standard and the commercial standard. This facile and general PCR-based method for the synthesis of internal standards allows for significant savings in the implementation of large genotyping experiments using microsatellite or AFLP markers.     

Structural Variation among Wild and Industrial Strains of Penicillium chrysogenum

Strain selection and strain improvement are the first, and arguably most important, steps in the industrial production of biological compounds by microorganisms. While traditional methods of mutagenesis and selection have been effective in improving production of compounds at a commercial scale, the genetic changes underpinning the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54–1255). We developed an assembly-free method of identifying chromosomal rearrangements and validated the in silico predictions with a PCR-based assay and Sanger sequencing. Despite many rounds of mutagen treatment and artificial selection, WIS 54–1255 differs from its wild progenitor at only one of the identified rearrangements. We suggest that natural variants predisposed for high penicillin production were instrumental in the success of WIS 54–1255 as an industrial strain. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be high penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known high penicillin producers may be viable candidates for further improvement efforts.     

Variation in species‐level plant functional traits over wetland indicator status categories

Wetland indicator status (WIS ) describes the habitat affinity of plant species and is used in wetland delineations and resource inventories. Understanding how species‐level functional traits vary across WIS categories may improve designations, elucidate mechanisms of adaptation, and explain habitat optima and niche. We investigated differences in species‐level traits of riparian flora across WIS categories, extending their application to indicate hydrologic habitat. We measured or compiled data on specific leaf area (SLA ), stem specific gravity (SSG ), seed mass, and mature height of 110 plant species that occur along the Colorado River in Grand Canyon, Arizona. Additionally, we measured leaf δ¹³C, δ¹⁵N, % carbon, % nitrogen, and C/N ratio of 56 species with C3 photosynthesis. We asked the following: (i) How do species‐level traits vary over WIS categories? (ii) Does the pattern differ between herbaceous and woody species? (iii) How well do multivariate traits define WIS categories? (iv) Which traits are correlated? The largest trait differences among WIS categories for herbaceous species occurred for SSG , seed mass, % leaf carbon and height, and for woody species occurred for height, SSG , and δ¹³C. SSG increased and height decreased with habitat aridity for both woody and herbaceous species. The δ¹³C and hence water use efficiency of woody species increased with habitat aridity. Water use efficiency of herbaceous species increased with habitat aridity via greater occurrence of C4 grasses. Multivariate trait assemblages differed among WIS categories. Over all species, SLA was correlated with height, δ¹³C, % leaf N, and C/N; height was correlated with SSG and % leaf C; SSG was correlated with % leaf C. Adaptations of both herbaceous and woody riparian species to wet, frequently inundated habitats include low‐density stem tissue. Adaptations to drier habitats in the riparian zone include short, high‐density cavitation‐resistant stem tissue, and high water use efficiency. The results enhance understanding about using traits to describe plant habitat in riparian systems.     

PCR-based quantitation of Cryptosporidium parvum in municipal water samples.

A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by steam pretreatment impregnated with hydrogen peroxide

Sugarcane bagasse was subjected to steam pretreatment impregnated with hydrogen peroxide. Analyses were performed using 2³ factorial designs and enzymatic hydrolysis was performed at two different solid concentrations and with washed and unwashed material to evaluate the importance of this step for obtaining high cellulose conversion. Similar cellulose conversion were obtained at different conditions of pretreatment and hydrolysis. When the cellulose was hydrolyzed using the pretreated material in the most severe conditions of the experimental design (210°C, 15 min and 1.0% hydrogen peroxide), and using 2% (w/w) water‐insoluble solids (WIS), and 15 FPU/g WIS, the cellulose conversion was 86.9%. In contrast, at a milder pretreatment condition (190°C, 15 min and 0.2% hydrogen peroxide) and industrially more realistic conditions of hydrolysis (10% WIS and 10 FPU/g WIS), the cellulose conversion reached 82.2%. The step of washing the pretreated material was very important to obtain high concentrations of fermentable sugars. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis

Recent advances in environmental DNA (eDNA) analysis using high‐throughput sequencing (HTS) enable evaluation of intraspecific genetic diversity in a population. As the intraspecific genetic diversity provides invaluable information for wildlife conservation and management, there is an increasing demand to apply eDNA analysis to population genetics and the phylogeography by quantitative evaluation of intraspecific diversity. However, quantitative evaluations of intraspecific genetic diversity using eDNA is not straightforward because the number of eDNA sequence reads obtained by HTS may not be an index of the quantity of eDNA. In this study, to quantitatively evaluate genetic diversity using eDNA analysis, we applied a quantitative eDNA metabarcoding method using the internal standard DNAs. We targeted Ayu (Plecoglossus altivelis altivelis) and added internal standard DNAs with known copy numbers to each eDNA sample obtained from three rivers during the library preparation process. The sequence reads of each Ayu haplotype were successfully converted to DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In all rivers, the calculated copy number of each haplotype showed a significant positive correlation with the haplotype frequency estimated by a capture‐based survey. Furthermore, estimates of genetic indicators such as nucleotide diversity based on the eDNA copy numbers were comparable with those estimated based on a capture‐based study. Our results demonstrate that eDNA analysis with internal standard DNAs enables reasonable quantification of intraspecific genetic diversity, and this method could thus be a promising tool in the field of population genetics and phylogeography.     

Investigating the quantitative nature of MALDI-TOF MS

MALDI-TOF MS has been applied by several groups to relative quantitative measurements. At the same time, the non-quantitative character of this method has been widely reported. We conducted experiments to test the reliability of this technique for quantitation using the statistical method of inverse confidence limit calculation for the first time in this context. The relationship between relative intensities of known amounts of standard peptides and their concentration ratios was investigated. We found that the concentration ratios determined by relative intensity measurements were highly inaccurate and strongly influenced by the molecular milieu of the sample analyzed. Thus, we emphasize the necessity of using the sample itself for calibration. We also performed experiments using an isotope-labeled derivative of the analyte as an internal standard for calibration line generation. As expected, the use of such standard led to a dramatic increase in precision and a less pronounced improvement in accuracy. We recommend performing a similar statistical analysis as a demonstration of reliability for every system where MALDI-TOF MS is used for quantitative measurements.     

The use of biomagnetic separation to recover DNA suitable for PCR from Claviceps species

DNA analysis of agriculturally important fungi using polymerase chain reaction (PCR)-based methods is becoming routine in research and for diagnostic purposes. Rapid, small-scale DNA isolation methods that take advantage of the sensitivity, speed and automation potential of PCR technology are needed for timely analysis of important plant pathogens. DNA isolated from Claviceps africana (causal agent of ergot of sorghum) using several standard DNA extraction protocols was found to be unamplifiable using PCR. The standard methods apparently failed to separate DNA from substances inhibitory to the Taq polymerase enzyme. We obtained DNA amenable to PCR analysis using a novel method involving magnetic beads and high salt extraction buffer. The biomagnetic purification method allowed us to obtain reliable PCR amplification of the internal transcribed spacer (ITS) regions of rDNA of Claviceps africana, making genetic comparisons possible.     

Determination of citalopram and escitalopram together with their active main metabolites desmethyl(es-)citalopram in human serum by column-switching high performance liquid chromatography (HPLC) and spectrophotometric detection

We established a method for automated quantitative analysis of (es-)citalopram and desmethyl(es-)citalopram in serum using column-switching high performance liquid chromatography (HPLC). For sample clean-up serum was injected onto a LiChrospher CN 20 microm precolumn using 8% acetonitrile in deionized water. Drugs were eluted by back-flush flow onto the analytical column (LiChrospher CN 5 microm) at a flow rate of 1.5 ml/min with phosphate buffer 8 mmol/l pH 6.4/acetonitrile (50/50, v/v). Haloperidol was used as internal standard. Analytes were detected by ultraviolet spectrophotometry at 210 nm. Detection limit of (es-)citalopram was 6 ng/ml. The method was found to be suitable for therapeutic drug monitoring of patients treated with citalopram or escitalopram.     

A comparison between methods for linkage disequilibrium fine mapping of quantitative trait loci

We present a maximum likelihood method for mapping quantitative trait loci that uses linkage disequilibrium information from single and multiple markers. We made paired comparisons between analyses using a single marker, two markers and six markers. We also compared the method to single marker regression analysis under several scenarios using simulated data. In general, our method outperformed regression (smaller mean square error and confidence intervals of location estimate) for quantitative trait loci with dominance effects. In addition, the method provides estimates of the frequency and additive and dominance effects of the quantitative trait locus.     

Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards: analysis of phosphoprotein dynamics in a B cell lymphoma cell line

Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a non-isotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase Cgamma2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospholipase Cgamma2 phosphorylation sites. Our results illustrate the capacity of this conceptually simple LC-MS method for quantification of gel-separated proteins and their phosphorylation sites and for quantitative profiling of biological systems.     

Rapid and sensitive static headspace gas chromatography-mass spectrometry method for the analysis of ethanol and abused inhalants in blood

A sensitive and specific method using static headspace gas chromatography coupled with mass spectrometry (GC/MS) has been developed for the quantitative determination of ethanol in biological fluids using n-propanol as internal standard. Gas chromatography was performed in isothermal mode with a GC run time of 2.6 min. The quantification was performed using scan mode abstracting a quantitative ion and a qualifier ion for ethanol and for the internal standard. The method was linear (r(2), 0.999, in the concentration range of 5-200 mg/dl), specific (no interference from methanol acetaldehyde, acetone or from endogenous materials), sensitive (limit of quantification and limit of detection of 0.2 and 0.02 mg/dl, respectively) and robust (less than 5% inter- and intra-assay coefficient of variation). A slightly modified method was also developed for the quantification of five commonly abused inhalants (dichloromethane, ethyl acetate, benzene, toluene and xylene) in blood. The method used a gradient GC program with a run time of 8 min. The quantification was performed using scan mode and integrating the area under the peak using trichloroethane as an internal standard. Without optimization, the method was linear (from 5 to 100 mg/l) and sensitive.     

The use of Lenticules for the process control of enumeration techniques in food and environmental microbiology

AIMS: Lenticules consist of control-dried plano convex discs in which biologically-active materials are contained within a water-soluble matrix. They can be produced to contain stable numbers of bacteria from 10 cfu lenticule-1 to 108 cfu lenticule-1 with a wide variety of organisms. These experiments were carried out to validate their use as a tool for internal quality control in quantitative microbiology. METHODS AND RESULTS: The Lenticules were used routinely in standard quantitative microbiological procedures across five laboratories. Results showed the materials to be stable, homogeneous and capable of identifying systematic errors. CONCLUSIONS: The Lenticules provide suitable, stable control materials. SIGNIFICANCE AND IMPACT OF THE STUDY: Routine internal quality control of quantitative measurements is greatly improved; the materials are easy to use and enable comparisons between laboratories to be made.     

Rheological characterization of dilute acid pretreated softwood

Large-scale bioethanol production from lignocellulosic biomass will require high solids loading in the enzymatic hydrolysis step. However, slurries of pretreated lignocelluloses are complex fluids due to the fibrous nature, especially at high concentrations of water insoluble solids (WIS). A prerequisite for dealing with transport issues and for developing efficient full-scale processes is a fundamental understanding of the flow properties of pretreated lignocellulose. A comprehensive rheological characterization of dilute acid pretreated spruce has been carried out in this study, accounting for the effects of WIS concentration, particle size distribution (PSD), and the degree of enzymatic hydrolysis. The rheology of pretreated spruce slurries was found to be strongly dependent on the WIS concentration. The storage modulus (G'(LVR)) and yield stress showed typical power-law dependencies on volume fraction and WIS content. Milling of the pretreated material resulted in significantly higher yield stress and viscosity, likely due to narrower PSD, which suggests that the strength of the network of the coarsest fibers determines the rheology of these materials to a large extent. During enzymatic hydrolysis, yield stress and viscosity decreased dramatically, partly due to decreasing WIS content, but possibly also due to changes in fiber properties such as the chemical composition.     

Optimization of Enzymatic Hydrolysis of Steam Pretreated Triticale Straw

Efficient conversion of the carbohydrates into fermentable sugars is crucial for industrial implementation of 2G biofuels such as bioethanol. The main objective of this study was to improve the enzymatic hydrolysis of steam pretreated triticale straw (slurry, pressed-slurry or water insoluble solids (WIS)) by optimal combination of cellulase (Cellic® CTec2) and hemicellulase (Cellic® HTec2) and incubation period for a target glucan conversion of 80 %. Among the three substrates evaluated, pressed-slurry and WIS resulted in similar sugar yields but WIS presented lower enzyme requirements. Different combinations of cellulase and endo-xylanase could provide an 80 % of glucan conversion depending on the weight assigned to constrain. The selected enzyme combination, 0.1 mL Cellic®CTec2/g WIS and 0.2 mL Cellic®HTec2/g WIS, could achieve a glucan conversion of 80 % in 45 h (desirability of 0.9). Doubling the enzyme dosage could further improve the saccharification productivity by reducing the incubation period to 37 h. The optimisation of enzymatic hydrolysis of lignocellulosic substrates, to reduce the cost of sugars production, is a compromise between substrate, enzyme dosage, incubation time and the benchmark yield, although a more favourable response can be generated with an optimised combination of enzymes.     

基于多源遥感数据的草地净初级生产力质量评价

植被净初级生产力NPP(Net Primary Production)的遥感估算与分析对全球生态系统具有重要指导意义,不同尺度的遥感影像上占主导地位的地物景观信息是不同的,现代生态研究多尺度分析至关重要。以青海省海北藏族自治州为研究区,使用Landsat 8 OLI遥感影像、天宫二号宽波段成像仪影像、融合影像(Landsat8 OLI影像和天宫二号宽波段成像仪影像融合)联同MODIS影像,作为CASA模型的输入参数,探究不同尺度下的研究区域NPP的空间分布情况,并对比分析不同数据源数据在估算NPP时的精度。结果表明:(1)Landsat 8 OLI数据的NPP值位于150—200 g C m^-2 a^-1所占比例最高;天宫二号宽波段成像仪影像数据和融合后影像的NPP值位于50—100 g C m^-2 a^-1所占比例最高;MODIS数据的NPP值位于小于50 g C m^-2 a^-1比例最高。(2)天宫二号宽波段成像仪影像数据的均方根误差(RMSE)、平均绝对误...     

Validation of non-aqueous capillary electrophoresis for simultaneous determination of four tricyclic antidepressants in pharmaceutical formulations and plasma samples

We present the validation of a method using non-aqueous capillary electrophoresis (NACE) for quantitative analysis of four tricyclic antidepressants (TADs) in pharmaceutical formulations and plasma. The method presented high resolution allowing the separation of the TADs in 4.3 min at optimized conditions: 50 mM ammonium acetate, 1 M acetic acid in acetonitrile, capillary with 48 cm in length, 40 cm to the detector, and voltage of 30 kV. Acceptable precision (relative standard deviation R.S.D.14.1% from plasma samples) and linearity were achieved using the internal standard (IS) method. The limits of quantification determined for plasma, after liquid-liquid extraction (LLE), were between 30 and 50 ng ml-1. These values are beyond the plasmatic therapeutic concentration. Our results were found comparable or better than those described in the literature for high performance liquid chromatography (HPLC)-based methods.     

Development of an HPLC-MS/MS method for the selective determination of paracetamol metabolites in mouse urine

An HPLC-MS/MS method has been developed for the selective quantitative analysis of paracetamol and its two major metabolites. The use of tandem MS enabled the detection and quantitation of metabolites in small sample sizes with high sensitivity and selectivity. Isocratic elution using acetonitrile and water containing formic acid combined with electrospray-tandem MS enabled the separation and accurate quantitation of each analyte and the internal standard 3-acetamidophenol. The on-column limits of detection for paracetamol, paracetamol sulfate, and paracetamol glucuronide were 2.4, 1.2, and 1.2 pmol, respectively. The method was applied to quantitate paracetamol and its metabolites in mouse urine. It is highly specific, sensitive, and easily adaptable to measure these analytes in biological fluids of other animals.     

Development and validation of a gradient reversed-phase high-performance liquid chromatographic assay for S(−-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0923) from a transdermal delivery system

A gradient reversed-phase HPLC method for potency determination of N-0923 (10 mg0 from a transdermal delivery system (TDS), was developed and validated with single point calibration yusing internal standard quantitation. N-0923 and the internal standard, N-0434, are eluted from a reversed-phase C₁₈ column using a gradient which contains 0.1 M triethylamine-0.04 M citrate buffer, pH 5.9, water, and acetonitrile with UV detection at 272 nm. N-0923 is isolated from the transdermal delivery system by extraction with n-heptane followed by extraction of the resulting organic phase with 0.1 M citric acid containing the internal standard. The method weas free from matrix interferences in both untreated and forced degraded placebo delivery systems. Acceptable linearity and quantitative recovery form spiked placebo delivery systems over the range 50–150% of nominal label claim were demonstrated. Within-day assay precision from individual samples of active transdermal dlivery systems (n = 10) was 5.6% R.S.D. The detection limit was at least 0.1 μg/ml which is equivalent to 0.05% of the working standard concentration. Replicate injection precision at this level was 0.08% R.S.D. (n =4). Analysis of thermally stressed active and placebo delivery systems with this HPLC method and photodiode-array detection showed that the chromatography was stability-indicating as demonstrated by the absence of measurable interferences from principal degradation products of either the N-0923 or the delivery system excipients.     

Analyzing real-time PCR data by the comparative C(T) method

Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.