Evaluation of two methods for quantifying passeriform lice

Two methods commonly used to quantify ectoparasites on live birds are visual examination and dust‐ruffling. Visual examination provides an estimate of ectoparasite abundance based on an observer's timed inspection of various body regions on a bird. Dust‐ruffling involves application of insecticidal powder to feathers that are then ruffled to dislodge ectoparasites onto a collection surface where they can then be counted. Despite the common use of these methods in the field, the proportion of actual ectoparasites they account for has only been tested with Rock Pigeons (Columba livia), a relatively large‐bodied species (238–302 g) with dense plumage. We tested the accuracy of the two methods using European Starlings (Sturnus vulgaris; ～75 g). We first quantified the number of lice (Brueelia nebulosa) on starlings using visual examination, followed immediately by dust‐ruffling. Birds were then euthanized and the proportion of lice accounted for by each method was compared to the total number of lice on each bird as determined with a body‐washing method. Visual examination and dust‐ruffling each accounted for a relatively small proportion of total lice (14% and 16%, respectively), but both were still significant predictors of abundance. The number of lice observed by visual examination accounted for 68% of the variation in total abundance. Similarly, the number of lice recovered by dust‐ruffling accounted for 72% of the variation in total abundance. Our results show that both methods can be used to reliably quantify the abundance of lice on European Starlings and other similar‐sized passerines.     

Functional methods for quantifying agonists and antagonists.

The development of occupancy theory has allowed the formulation of a series of mathematical models that describe the interaction of agonists and antagonists with their receptors, in terms of affinity and efficacy. These models provide a framework for the analysis and interpretation of E/[A] curve data and have proved to be useful tools in quantitative pharmacology. Unfortunately, despite the proven utility of this approach and the widespread availability of powerful computer-based curve-fitting programs [BMDP (41), Microsoft Excel. etc.], which greatly facilitate analysis, the application of mathematical modeling remains the exception rather than the rule in pharmacological studies.     

A simple technique for quantifying apoptosis in 96-well plates

Background  

Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification.     

Two simple volumetric methods for respiration measurements with oxygen supply to the sample

Summary Two easy and inexpensive procedures suitable especially for the measurements of aerobic respiration activity of numerous soil samples are described. Size of samples can be changed within broad limits. The first of the two methods is based on measurements of the sum of oxygen consumed during a prolonged period of time. Actual rate of O₂ consumption over short-time intervals is determined by the second method.     

Graphical methods for quantifying macromolecules through bright field imaging

Bright field imaging of biological samples stained with antibodies and/or special stains provides a rapid protocol for visualizing various macromolecules. However, this method of sample staining and imaging is rarely employed for direct quantitative analysis due to variations in sample fixations, ambiguities introduced by color composition and the limited dynamic range of imaging instruments. We demonstrate that, through the decomposition of color signals, staining can be scored on a cell-by-cell basis. We have applied our method to fibroblasts grown from histologically normal breast tissue biopsies obtained from two distinct populations. Initially, nuclear regions are segmented through conversion of color images into gray scale, and detection of dark elliptic features. Subsequently, the strength of staining is quantified by a color decomposition model that is optimized by a graph cut algorithm. In rare cases where nuclear signal is significantly altered as a result of sample preparation, nuclear segmentation can be validated and corrected. Finally, segmented stained patterns are associated with each nuclear region following region-based tessellation. Compared to classical non-negative matrix factorization, proposed method: (i) improves color decomposition, (ii) has a better noise immunity, (iii) is more invariant to initial conditions and (iv) has a superior computing performance.     

A simple method for quantifying Leishmania in tissues of infected animals

In experimental animals infected with Leishmania major, the size of cutaneous lesions of the parasite often does not correlate with the number of parasites within the lesion. Indeed, cutaneous lesions can heal, but still contain parasites. Thus, the ability to determine parasite burden in infected animals becomes important, especially when assessing vaccines that are intended to induce sterilizing immunity. Here, Hermenio Lima, Julie Bleyenberg and Richard Titus describe a simple technique for enumerating Leishmania in infected tissue. It is hoped that this technique will allow all researchers working with Leishmania (especially those in countries where leishmaniasis is endemic) to determine parasite burden easily in infected animals.     

Cooperativity in low-affinity Mg2+ binding to tRNA

The Pb2+-catalyzed cleavage of tRNAPhe has been used to probe the effect of Na+ and Mg2+ binding to tRNA. Na+ is a noncompetitive inhibitor of the Pb2+-catalyzed cleavage. Millimolar Mg2+ is also a noncompetitive inhibitor. Analysis of the Mg2+ data show that at least two sites are involved in binding and that there is an interaction between the sites (cooperativity). Low-affinity Mg2+ binding is thus different from "weak" and "strong" Mg2+ binding to tRNA characterized previously. We postulate that the alterations induced by low-affinity Mg2+ binding in tRNA mimic to some extent those brought about in RNA by the interaction with a protein factor and that at appropriate [Mg2+] the whole structure of tRNA is able to respond in a concerted way to a signal from the environment such as aminoacylation or codon binding.     

Comparing two methods for quantifying soil-borne Entomophaga maimaiga resting spores

To improve usability of methods for quantifying environmentally persistent entomophthoralean resting spores in soil, we modified and tested two methods using resting spores (azygospores) of the gypsy moth pathogen Entomophaga maimaiga. Both methods were effective for recovering resting spores at concentrations >100 resting spores/g dry soil. While a modification of a method originally described by Weseloh and Andreadis (2002) recovered more resting spores than a modified method based on Percoll density gradients, the ability to estimate true densities from counts was similar for both methods. Regression equations are provided for predicting true resting spore densities from counts, with R² values for both methods ?0.90.     

Rapid methods for quantifying VAM fungal infections in maize roots

Roots of maize (Zea mays cv W64A × W182E) infected by vesicular arbuscular mycorrhizal (VAM) fungi (Glomus versiforme (Karst) Berch or a Glomus species isolated from an alfalfa soil) exhibit a bright yellow pigmentation. The percentage of pigmented roots can be quantified by a rapid visual estimate or by a grid intersect method. Both methods gave similar estimates of VAM infection to those obtained using a grid intersect count on cleared roots stained with chlorazol black E. Thus for experimental or field evaluation where speed and quantity are important, the rapid visual estimate (less than one minute for each washed root system) yields reliable results. The yellow root intersect method takes longer (5–15 minutes per root system) but gives more reproducible results. The yellow root pigmentation is light sensitive However, root systems can be reliably assayed after 1 week when stored at 5°C in the dark or after 1 year if dried.     

A simple method for quantifying biomass cell and polymer distribution in biofilms

Biofilms are ubiquitous and play an essential role in both environmental processes and hospital infections. Standard methods are not capable of quantifying biomass concentration in dilute suspensions. Furthermore, standard techniques cannot differentiate biomass composition. In this study, a user-friendly technique was developed for measuring biomass cell and polymer content in detached biofilms using a standard coulter counter. The method was demonstrated for an environmentally relevant strain of Pseudomonas aeruginosa (Schroeter) Migula grown in a bioreactor and also for a medically relevant strain of P. aeruginosa (PAO1) grown on standard growth pegs. Results were compared and validated by standard assays, including EPA method 1684 for measuring biomass, microscopic direct counts, and a crystal violet staining assay. The minimum detection limit for the coulter counter method (0.07 mg-biomass L^− 1) was significantly lower than the EPA method 1684 (1.9 ± 0.4 mg/L) and the crystal violet assay (1.1 ± 0.2 mg L^− 1). However, the coulter counter method is limited to dilute biomass samples (below 204 ± 16 mg L^− 1) due to clogging of the aperture tube. While biomass measurements are useful, the major advantage of the coulter counter method is the ability to directly determine EPS, cell, and aggregate fractions after mild chemical treatment. The rapid technique (4–5 min per sample) was used to measure biomass fractions in dispersed P. aeruginosa (Schroeter) and PAO1 biofilms. This technique will be critical for understanding biofilm formation/dispersal.     

Albumin inhibition of transferrin low-affinity binding to K562 cells

Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin.     

Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly

The onset of chromosomal DNA replication requires highly precise and reproducible interactions between initiator proteins and replication origins to assemble a pre-replicative complex (pre-RC) that unwinds the DNA duplex. In bacteria, initiator protein DnaA, bound to specific high- and low-affinity recognition sites within the unique oriC locus, comprises the pre-RC, but how complex assembly is choreographed to ensure precise initiation timing during the cell cycle is not well understood. In this study, we present evidence that higher-order DnaA structures are formed at oriC when DnaA monomers are closely positioned on the same face of the DNA helix by interaction with two oppositely oriented essential arrays of closely spaced low-affinity DnaA binding sites. As DnaA levels increase, peripheral high-affinity anchor sites begin cooperative loading of the arrays, which is extended by sequential binding of additional DnaA monomers resulting in growth of the complexes towards the centre of oriC. We suggest that this polarized assembly of unique DnaA oligomers within oriC plays an important role in mediating pre-RC activity and may be a feature found in all bacterial replication origins.     

Capillary zone electrophoresis for the study of the binding of antithrombin to low-affinity heparin

When low-affinity interactions between glycosaminoglycans and precious proteins are studied, it is imperative to design an experimental set-up that consumes as little material as possible. To evaluate the applicability of the CZE technique to this problem, we explored the interaction between antithrombin and low-affinity heparin. In a series of CZE experiments we demonstrated that the mobility of antithrombin increases gradually as increased concentrations of low-affinity heparin were added to the electrolyte. The results were, as expected, consistent with the general algorithm for monovalent binding. The binding constant was estimated at 20±6 μM in excellent agreement with the value reported in the literature. This revised version was published online in November 2006 with corrections to the Cover Date.     

Comparison of methods for quantifying the hydrolytic potential of cellulase enzymes

Summary The adequacy of -glucosidase activity in various cellulase enzyme mixtures was assessed by monitoring the accumulation of cellobiose in the reaction mixture. The influence of accumulated glucose and cellobiose on a filter paper (FP) assay indicated the relative susceptibility of the different enzyme preparations to end-product inhibition. An HPLC analysis of the profile of sugars released also provided a better means of predicting the hydrolytic potential of the various cellulase mixtures. An accurate prediction of the hydrolytic potential of a cellulase preparation could not be based on the conventional FP assay alone. The hydrolytic potential of a Celluclast/Novozym mixture was superior to that of Trichoderma harzianum even when the latter system was supplemented with increased concentrations of -glucosidase (Novozym).     

Comparison of extraction methods for quantifying vitamin E from animal tissues

Four extraction methods: (1) solvent (SOL), (2) ultrasound assisted solvent (UA), (3) saponification and solvent (SP), and (4) saponification and ultrasound assisted solvent (SP-UA), were used in sample preparation for quantifying vitamin E (tocopherols) in chicken liver and plasma samples. The extraction yields of SOL, UA, SP, and SP-UA methods obtained by adding delta-tocopherol as internal reference were 95%, 104%, 65%, and 62% for liver and 98%, 103%, 97%, and 94% for plasma, respectively. The methods with saponification significantly affected the stabilities of tocopherols in liver samples. The measured values of alpha- and gamma-tocopherols using the solvent only extraction (SOL) method were much lower than that using any of the other extraction methods. This indicated that less of the tocopherols in those samples were in a form that could be extracted directly by solvent. The measured value of alpha-tocopherol in the liver sample using the ultrasound assisted solvent (UA) method was 1.5-2.5 times of that obtained from the saponification and solvent (SP) method. The differences in measured values of tocopherols in the plasma samples by using the two methods were not significant. However, the measured value of the saponification and ultrasound assisted solvent (SP-UA) method was lower than either the saponification and solvent (SP) or the ultrasound assisted solvent (UA) method. Also, the reproducibility of the ultrasound assisted solvent (UA) method was greater than any of the saponification methods. Compared with the traditional saponification method, the ultrasound assisted solvent method could effectively extract tocopherols from sample matrix without any chemical degradation reactions, especially for complex animal tissue such as liver.     

Comparison of label-free methods for quantifying human proteins by shotgun proteomics

Measurements of mass spectral peak intensities and spectral counts are promising methods for quantifying protein abundance changes in shotgun proteomic analyses. We describe Serac, software developed to evaluate the ability of each method to quantify relative changes in protein abundance. Dynamic range and linearity using a three-dimensional ion trap were tested using standard proteins spiked into a complex sample. Linearity and good agreement between observed versus expected protein ratios were obtained after normalization and background subtraction of peak area intensity measurements and correction of spectral counts to eliminate discontinuity in ratio estimates. Peak intensity values useful for protein quantitation ranged from 10(7) to 10(11) counts with no obvious saturation effect, and proteins in replicate samples showed variations of less than 2-fold within the 95% range (+/-2sigma) when >or=3 peptides/protein were shared between samples. Protein ratios were determined with high confidence from spectral counts when maximum spectral counts were >or=4 spectra/protein, and replicates showed equivalent measurements well within 95% confidence limits. In further tests, complex samples were separated by gel exclusion chromatography, quantifying changes in protein abundance between different fractions. Linear behavior of peak area intensity measurements was obtained for peptides from proteins in different fractions. Protein ratios determined by spectral counting agreed well with those determined from peak area intensity measurements, and both agreed with independent measurements based on gel staining intensities. Overall spectral counting proved to be a more sensitive method for detecting proteins that undergo changes in abundance, whereas peak area intensity measurements yielded more accurate estimates of protein ratios. Finally these methods were used to analyze differential changes in protein expression in human erythroleukemia K562 cells stimulated under conditions that promote cell differentiation by mitogen-activated protein kinase pathway activation. Protein changes identified with p<0.1 showed good correlations with parallel measurements of changes in mRNA expression.