Terminal Restriction Fragment Length Polymorphism Data Analysis for Quantitative Comparison of Microbial Communities

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.     

Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.     

Peak quantification in surface-enhanced laser desorption/ionization by using mixture models

Surface-enhanced laser desorption/ionization (SELDI) time of flight (TOF) is a mass spectrometry technology for measuring the composition of a sampled protein mixture. A mass spectrum contains peaks corresponding to proteins in the sample. The peak areas are proportional to the measured concentrations of the corresponding proteins. Quantifying peak areas is difficult for existing methods because peak shapes are not constant across a spectrum and because peaks often overlap. We present a new method for quantifying peak areas. Our method decomposes a spectrum into peaks and a baseline using so-called statistical finite mixture models. We illustrate our method in detail on 8 samples from culture media of adipose tissue and globally on 64 samples from serum to compare our method to the standard Ciphergen method. Both methods give similar estimates for singleton peaks, but not for overlapping peaks. The Ciphergen method overestimates the heights of such peaks while our method still gives appropriate estimates. Peak quantification is an important step in pre-processing SELDI-TOF data and improvements therein will pay off in the later biomarker discovery phase.     

Population-level variation in the expression of heterostyly in three species of Rubiaceae: does reciprocal placement of anthers and stigmas characterize heterostyly?

Heterostyly (i.e., reciprocal placement of anthers and stigmas between two or three floral morphs) is hypothesized to enhance outcrossing and reduce selfing. However, few studies have documented reciprocity among individual plants; instead, mean anther and stigma heights for floral morphs are usually reported, masking interindividual variation. We measured eight floral dimensions for individuals in five populations of three heterostylous Rubiaceae. The three methods used to quantify reciprocity yielded different conclusions regarding the degree to which populations conformed to expectations for heterostylous plants. Only Psychotria poeppigiana had stigma and, to a lesser degree, anther heights in discrete classes. Variation among plants of Bouvardia ternifolia and Psychotria chiapensis yielded a continuum of anther and stigma heights across populations. Comparison of distances between stigma and anthers indicated that only flowers of B. ternifolia had, as expected, a constant value for this distance. Finally, regression relationships between anther and stigma heights and corolla length showed that only in one population each of B. ternifolia and P. poeppigiana, and in P. chiapensis, was distance between anthers and stigmas the same across the range of corolla sizes for both floral morphs. Variation among these species in expression of heterostyly was not clearly linked to phylogenetic relationship or pollinator syndromes. Two approach herkogamous (AH) species were studied for comparison. Flowers of Psychotria brachiata were consistently AH, but flowers of P. pittieri were highly variable. Determining fitness consequences of population-level variation in sexual systems requires studies linking floral morphology to pollinator behavior and pollen transfer.     

Effects of various midsole densities of basketball shoes on impact attenuation during landing activities

The purpose of this study was to examine effects of shoe midsole densities and mechanical demands (landing heights) on impact shock attenuation and lower extremity biomechanics during a landing activity. Nine healthy male college athletes performed 5 trials of step-off landing in each of 9 test conditions, i.e., a combination of landings in shoes of 3 midsole densities (soft, normal, hard) from each of 3 landing potential energy (PE) levels (low, median, high). Ground reaction forces (GRF), accelerations (ACC) of the tibia and forehead, and sagittal kinematic data were sampled simultaneously. A 3 x 3 two-way (surface x height) repeated-measures analysis of variance (ANOVA) was performed on selected kinematic, ACC, and GRF variables; a 3 x 3 x 3 three-way (surface x height x joint) ANOVA was performed on variables related to eccentric muscular work. The GRF results showed that the forefoot peak GRF in the normal and hard midsoles was significantly greater than the soft midsole at the low and median PEs. Rearfoot peak GRF was significantly greater for the hard midsole than for the soft and normal midsoles at the median and high PEs, respectively. The peak head and tibia peak ACC were also attenuated in similar fashion. Kinematic variables did not vary significantly across different midsoles, nor did energy absorbed through lower extremity extensors in response to the increased shoe stiffness. Knee joint extensors were shown to be dominant in attenuating the forefoot impact force across the landing heights. The results showed limited evidence of impact-attenuating benefits of the soft midsole in the basketball shoes.     

A densitometric nondestructive microassay for DNA quantitation

A nondestructive assay for small quantities of nonradioactive DNA has been developed. Submicroliter volumes of DNA samples of concentrations as low as 10 micrograms/ml in 10 mM Tris, pH 8, containing 10 micrograms/ml ethidium bromide are photographed in 1-microliter microcapillary tubes on a near-uv transilluminator. The negatives are scanned with a densitometer, producing a sharp peak for each capillary; samples whose peak heights show apparent linearity with DNA content are compared to the linear portion of a standard curve constructed from DNA samples of known concentrations. The samples may be recovered undamaged, as introduction into capillaries and illumination for photography do not introduce nicks into the DNA. The DNA samples may be of heterogeneous molecular weight, but all DNAs should be of similar base composition and in the same buffer. Traces of phenol do not interfere with the determination.     

An off-line data processing system for biochemistry profiling based on a 2K programmable calculator.

An off-line data processing system based on a Hewlett Packard 2K programmable calculator to be used with a biochemistry profiling system is described. The program is in two sections. A Data Acquisition phase calculates results from Auto Analyser II peak heights after corrections for drift and stores them on magnetic tape cassettes. Quality control statistics are produced. A Reporting phase types the profile results on self-adhesive pre-printed labels to be attached to the test-request form and also prepares a laboratory record sheet. The system is routinely used to process up to 2000 peak heights per day. Non-profile heights may also be read using this program.     

Robust protein quantitation in chromatographic fractions using MALDI-MS of tryptic peptides

A method is introduced to evaluate protein concentrations using the height sum of all MALDI-MS peaks that unambiguously match theoretic tryptic peptide masses of the protein sought after. The method uses native chromatographic protein fractionation prior to digestion but does not require any depletion, labeling, derivatization, or preparation of a compound similar to the analyte. All peak heights of tryptic peptides are normalized with the peak height of a unique standard peptide added to the MALDI-MS samples. The sum of normalized peak heights, S(n), or the normalized mean peak height, M(n), reflects the concentration of the respective protein. For fractions containing various proteins, S(n) and M(n) can be used to compare concentrations of a protein between different fractions. For fractions with one predominating protein, they can be used to estimate concentration ratios between fractions, or to quantify the fractional protein concentration after calibration with pure protein solutions. Initial native fractionation retains the possibility to apply all conventional analytic procedures. Moreover, it renders the method relatively robust to MS mass accuracy. The method was validated with albumin, transferrin, alpha1-antitrypsin, and immunoglobulin G within highly complex chromatographic fractions of pathological and normal sera, which contained the respective intact native protein in dominating as well as minor concentrations. The correlation found between S(n) and the protein concentration as determined with ELISA showed that the method can be applied to select markers for distinguishing between normal and pathological serum samples.     

Effect of sampling interval on serum concentrations of luteinizing hormone, follicle-stimulating hormone, and prolactin in prepubertal, ovariectomized, and cycling gilts.

Three experiments were conducted to determine the effect of sampling interval on serum concentrations of LH, FSH, and prolactin (PRL) in prepubertal, ovariectomized, and cycling gilts. In all experiments, blood samples were drawn at 2-min intervals for 4 h from indwelling jugular catheters. Mean serum hormone concentrations, mean number of peaks, and mean and maximum peak heights of LH, FSH, and PRL were calculated using values reflecting 2-, 6-, 10-, 20-, 30-, and 60-min sampling intervals. For LH, FSH, and PRL, mean serum concentrations can be obtained through blood samples drawn at hourly intervals. Since LH peaks are very distinct in pigs, the number of secretory peaks and mean peak height can be obtained via samples drawn at 20-min intervals. Since FSH and PRL peaks are less well defined, a more frequent sampling interval (10 min) is needed to determine number of peaks and mean peak height. To obtain the maximum peak height or the number of minutes for LH, FSH, or PRL to rise from its nadir to zenith, blood samples need to be drawn at 2-min intervals. Regardless of reproductive state, these data indicate that the sampling interval needed to characterize serum concentrations of LH, FSH, and PRL in the gilt is dependent upon the parameter in question.     

A software complement to AMDIS for processing GC-MS metabolomic data

The software package AMDIS performs gas chromatography–mass spectrometry (GC–MS) peak deconvolution but tends to produce false positives and leaves missing values where peaks are found in only a proportion of a set of chromatograms. We have developed a software complement to AMDIS that (i) allows rapid manual inspection of chromatographic peaks across all samples to confirm data quality and (ii) for a given sample set, integrates peak areas across all samples even where AMDIS deconvolution would leave missing values. The freely available package runs within the commercial Matlab environment and is useful where GC–MS is used to profile complex mixtures.     

Applications of pooled DNA samples to the assessment of population affinities: short tandem repeats

Pooled DNA samples have been used in association studies of Mendelian disease genes. This method involves combining equal quantities of DNA from patients and control subjects into separate pools and comparing the pools for distributions of genetic markers. In this study identical quantities of DNA from 300 individuals representing 6 populations were pooled and amplified for 296 loci using the touchdown polymerase chain reaction (PCR) method. The purpose of this study is to test the efficacy of pooled DNA markers in the reconstruction of the genetic structure of human populations. The populations sampled included Chuvash, Buryats, Kizhi, Native Americans, South Africans, and New York City whites. To test the accuracy of the allele-frequency distributions, we genotyped the Buryats and New York samples individually for six microsatellite markers and compared their frequencies to the allele frequencies derived from the electropherogram peak heights for the pooled DNA, producing a correlation of 0.9811 with a variance of less than 0.04. Two-dimensional scaling of genetic distances among the six populations produced clusters that reflected known historical relationships. A distance matrix was created using all 296 loci, and matrices based on individual chromosomes were correlated against the total matrix. As expected, the largest chromosomes had the highest correlations with the total matrix, whereas one of the smallest chromosomes, chromosome 22, had the lowest correlation and differed most from the combined STR distance matrix.     

Determination of O-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic assay for O⁶-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were constituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O⁶-Benzylguanine peak heights were compared to peak heights of O⁶-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O⁶-benzylguanine.     

Characterization of LH and testosterone responses to intramuscular injection of GnRH in tropical postpubertal bulls

Five Zebu x British crossbred bulls 17 months of age and of uniform liveweight (320+/-3 kg) were used to study testosterone responses to single intramuscular doses of exogenous gonadotropin-releasing hormone (GnRH). The eight dose levels used were 0, 31.25, 62.5, 125, 250, 500, 1000, and 2000 ng GnRH/kg live weight. Plasma samples for hormone responses were collected at 30-minute intervals from zero to three hours and at one-hour intervals from three to seven hours postinjection. Luteinizing hormone (LH) and testosterone responses were measured as peak heights or as areas under response curves. Increasing the dosage of GnRH increased the time to reach the peak LH response, the height and duration of the response, and the area under the response curve. The maximum LH peak height was reached by the 1 mug/kg dose. In contrast to LH, testosterone responses reached the same peak heights (two hours postinjection of GnRH) for all doses of GnRH. The only effect of increased dosage was to increase the duration of response. Testosterone responses showed repeatable differences (P<0.01) between animals, but LH responses did not. It was demonstrated that the testosterone status of bulls can be accurately assessed by simply measuring testosterone in a single plasma sample collected two to three hours after the intramuscular injection of 100 mug or more (dose unimportant) of GnRH per bull.     

The elongation of the human cervical vertebral column

Cervical vertebral elongation has been studied using serial cephalometric radiographs of 32 children examined regularly from 0.25 to 17 years. Mean vertebral body heights increased rapidly to about 2.5 years and then decelerated except for a spurt at about the age of peak height velocity. There were only small sex differences in vertebral body elongation to 12 years. From then to 15 years, the vertebral body heights in the girls exceeded those in the boys; later this sex difference was reversed. There was no pubertal spurt in disc elongation. The correlation coefficients were negative between vertebral body heights and the heights of adjoining intervertebral discs, e.g., body C3 and disc C3–4, but those between body heights or between disc heights were positive. The heights of adjacent cervical vertebral bodies were correlated more highly than the heights of non-adjacent bodies. There was a similar pattern of differences between correlation coefficients for the heights of adjacent and non-adjacent intervertebral discs.     

A 3D unsteady flow analysis in a doubly constricted arterial vessel

The aim of this study is to examine the interaction between two mild atherosclerotic proliferations spaced apart by a distance S by analyzing their influence on flow structure, pressure drop and stress field in an arterial vessel under pulsatile flow conditions. This has been achieved numerically by employing a time accurate, cell centered finite volume method in solving the Navier-Stokes equations governing the 3D unsteady flow dynamics in a conceptual model of an multiply constricted arterial vessel. In comparison to the pressure drop across a single stenosis, nearly a 50% increase in the late systolic and early diastolic pressure drops has been observed across the two mild constrictions when they are spaced within a distance of S相似文献   

Automated Technique for the Detection of Hemagglutinins to Sheep Erythrocytes

The Technicon autoanalyzer system used for the detection of homologous human hemagglutinins has been modified for the detection of hemagglutinins to sheep red blood cells. Antibody quantitation was obtained by plotting the optical density (OD) readings of peak heights of a serially diluted reference serum versus the serum dilutions. When the OD of the peak height of an unknown sample fell within the linear portion of the plot, a direct determination of the antibody titer was made. If the OD of the sample fell outside the linear portion, dilutions of the sample were carried out until a direct reading could be made. Assay by this method of at least 140 samples was possible within a day. Titers obtained with the autoanalyzer agreed very closely with those obtained by manual titration.     

Oxalic acid stabilizes dopamine, serotonin, and their metabolites in automated liquid chromatography with electrochemical detection

Use of antioxidative agents is required in automated LC assay of microdialysis samples, due to rapid degradation of the monoamine neurotransmitters and their metabolites. Addition of oxalic acid prevented degradation of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid efficiently: after a 24-h incubation at room temperature the decreases in peak heights were less than 10%. The long-term stability of the analytes, however, was still enhanced when acetic acid and -cysteine were included in the solution. Using this antioxidative solution, the monoamine neurotransmitters and their metabolites could be determined with an automated LC assay even at room temperature.     

Ecological correlates of small mammal community structure in the semi-arid Karoo, South Africa

The understanding of the determinants of small mammal community structure in arid and semiarid ecosystems is of importance, both in the light of the role that small mammals play, and the impact of livestock grazing on the flora of these systems. In a study aimed at identifying these determinants, small mammal assemblages and environmental features were quantified at six localities (a gradient of floristic structure, with constant annual rainfall) across the southern Karoo, South Africa. Stepwise variable regression indicated that small mammal diversity was correlated with plant and rock cover, as well as plant cover and horizontal foliage diversity at intermediate heights (40–60 cm). Initially, small mammal diversity increased with increasing plant cover, but decreased at cover levels greater than 30%. This relationship is similar to that found in other desert systems, although the peak in diversity found here is at higher levels of plant cover than found previously. I suggest that this higher peak may be owing to the lack of reliance on granivory by these animals, which are relatively omnivorous. This model may therefore explain the conflicting reports on the impact of livestock grazing on desert small mammals, with small mammal diversity decreasing with grazing below the peak, and increasing with grazing above the peak.