A novel semiautomated method for the estimation of free fatty acid in serum or plasma

A modification of the semiautomated assay method of Antonis (1965. J. Lipid Res. 6:307-312) for free fatty acid is presented. Free fatty acids are extracted from serum or plasma into di-n-butyl ether-2-methoxyethanol; the extract is almost free from phospholipids. The acids are analyzed in a portion of extract by a copper soap method using diphenylcarbazide. The extractant, being less dense than water, is easily separated from an aqueous phase both in the extraction of samples and in the assay of copper soaps. The assay is comparable in accuracy with well-tried titrimetric methods and is quicker and easier to operate.     

Semiautomated preparation of DNA templates for large-scale sequencing projects

The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates. We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day. The technique can be carried out manually or can be semiautomated using a robot pipetting device. We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.     

Quantification of vascular density using a semiautomated technique for immunostained specimens

OBJECTIVE: To develop a semiautomated, quantitative techniquefor the assessment of vascular density in immunohistochemically stained tissue sections using diaminobenzidine tetrahydrochloride (DAB) and hematoxylin as chromagens. STUDY DESIGN: A semiautomated thresholding technique was developed to quantitate vascular density in tissue sections stained with anti-CD31 (1 degrees antibody). The immunohistochemically stained specimens were digitally imaged using a 24-bit color camera. The blue component of the RGB image was segmented using a variable high-pass filter. After thresholding, the segmented areas (CD31 positive) were quantified and vascular density determined. The validity of the method was verified by calculating the precision of the technique using the coefficient of repeatability and by quantifying its agreement with manual analysis according to the Bland-Altman approach. RESULTS: Vascular endothelial cells were specifically selected using anti-CD31 as the primary antibody and the appropriate horseradish peroxidase-conjugated secondary antibody. Utilizing the semiautomated thresholding technique, the separation of DAB-stained tissuefrom non-DAB-stained tissue was achieved. The method developed possesses a low coefficient of repeatability (0.49%), agrees well with manual assessment (mean difference = -0.29 +/- 0.92%), is highly automated and is user friendly. CONCLUSION: A novel semiautomated technique for the quantification of vascular density was developed. This technique provides a method for reproducible measurement of immunostaining procedures (immunohistochemistry, immunocytochemistry and in situ hybridization) utilizing immunoperoxidase techniques with DAB as a chromagen.     

Quantitative comparative analysis of complex two-dimensional electropherograms

A protocol is described to detect and assess differences between complex electrophoretic patterns. A semiautomated method is used to collect accurate absolute mobility data from many two-dimensional electropherograms and a computer algorithm has been developed which normalizes and averages these data. The program generates refined numerical maps consisting of the mean electrophoretic mobilities and corresponding confidence limits for each component protein represented in the original two-dimensional electrophoretic pattern. Tests of statistical significance of apparent differences between averaged numerical maps are carried out to evaluate electrophoretic polymorphisms between the ribosomal proteins of two different plant species. Furthermore, using a nonlinear function relating log molecular weight to mobility, precise molecular weight estimates are obtained from measurements of electrophoretic mobilities of proteins in the presence of sodium dodecyl sulfate. Several examples are presented which demonstrate application of these semiautomated analyses to quantitative comparison and interpretation of two dimensional gel electropherograms.     

Improving marginal likelihood estimation for Bayesian phylogenetic model selection

The marginal likelihood is commonly used for comparing different evolutionary models in Bayesian phylogenetics and is the central quantity used in computing Bayes Factors for comparing model fit. A popular method for estimating marginal likelihoods, the harmonic mean (HM) method, can be easily computed from the output of a Markov chain Monte Carlo analysis but often greatly overestimates the marginal likelihood. The thermodynamic integration (TI) method is much more accurate than the HM method but requires more computation. In this paper, we introduce a new method, steppingstone sampling (SS), which uses importance sampling to estimate each ratio in a series (the "stepping stones") bridging the posterior and prior distributions. We compare the performance of the SS approach to the TI and HM methods in simulation and using real data. We conclude that the greatly increased accuracy of the SS and TI methods argues for their use instead of the HM method, despite the extra computation needed.     

Estimation of population growth or decline in genetically monitored populations

This article introduces a new general method for genealogical inference that samples independent genealogical histories using importance sampling (IS) and then samples other parameters with Markov chain Monte Carlo (MCMC). It is then possible to more easily utilize the advantages of importance sampling in a fully Bayesian framework. The method is applied to the problem of estimating recent changes in effective population size from temporally spaced gene frequency data. The method gives the posterior distribution of effective population size at the time of the oldest sample and at the time of the most recent sample, assuming a model of exponential growth or decline during the interval. The effect of changes in number of alleles, number of loci, and sample size on the accuracy of the method is described using test simulations, and it is concluded that these have an approximately equivalent effect. The method is used on three example data sets and problems in interpreting the posterior densities are highlighted and discussed.     

The Stone Brusher, a New Sampler for Submerged Epilithic Material in Shallow Streams and Lakes

The Stone Brusher is designed to take qualitative or semi-quantitative samples of material attached to stones at 7–50 cm depth in running or stagnant waters. The epilithic material is dislodged from the stone surface with a rotating brush enclosed in a chamber and the material is drawn up directly into the sample bottle with an air-cylinder. The operator takes a sample quickly and without putting hands into the water. The sampling area is about 28 cm². The sampler is made of plastic, stainless steel and aluminium and weighs 3.1 kg. The equipment is robust and easily handled and it is constructed to meet the demand for standardized sampling for research and environmental monitoring and to improve working conditions for sampling personnel. The equipment allows sampling from bedrock and large stones that cannot be lifted from the bottom and it can be used for reliable sampling also in fast-flowing streams where the dislodged material is easily flushed away. Using Near-Infrared Spectroscopy and diatom analyses, this new sampler is evaluated in comparison to the recognized toothbrush method, which indicates that the Stone Brusher reduces sampling variability compared with the toothbrush method.     

An optimized protocol for large‐scale in situ sampling and analysis of volatile organic compounds

Chemical ecology is an ever‐expanding field with a growing interest in population‐ and community‐level studies. Many such studies are hindered due to lack of an efficient and accelerated protocol for large‐scale sampling and analysis of chemical compounds. Here, we present an optimized protocol for such large‐scale study of volatiles. A large‐scale in situ study to understand role of semiochemicals in variation in mating success of lekking blackbuck was conducted. Suitable methods for sampling and statistical analysis were identified by testing and comparing the efficiencies of available techniques to reduce analysis time while retaining sensitivity and comprehensiveness. Solid‐phase extraction using polydimethylsiloxane, analysis using a semiautomated detection of retention time and base peak, and statistical analysis using random forest algorithm were identified as the most efficient methods for large‐scale in situ sampling and analysis of volatiles. The protocol for large‐scale volatile analysis can facilitate evolutionary and metaecological studies of volatiles in situ from all types of biological samples. The protocol has potential for wider application with the analysis and interpretation methods being suitable for all kinds of semiochemicals, including nonvolatile chemicals.     

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

Molecular dynamics (MD) simulations using all-atom and explicit solvent models provide valuable information on the detailed behavior of protein–partner substrate binding at the atomic level. As the power of computational resources increase, MD simulations are being used more widely and easily. However, it is still difficult to investigate the thermodynamic properties of protein–partner substrate binding and protein folding with conventional MD simulations. Enhanced sampling methods have been developed to sample conformations that reflect equilibrium conditions in a more efficient manner than conventional MD simulations, thereby allowing the construction of accurate free-energy landscapes. In this review, we discuss these enhanced sampling methods using a series of case-by-case examples. In particular, we review enhanced sampling methods conforming to trivial trajectory parallelization, virtual-system coupled multicanonical MD, and adaptive lambda square dynamics. These methods have been recently developed based on the existing method of multicanonical MD simulation. Their applications are reviewed with an emphasis on describing their practical implementation. In our concluding remarks we explore extensions of the enhanced sampling methods that may allow for even more efficient sampling.     

Modification of Lofland's colorimetric semiautomated serum triglyceride determination, assessed by an enzymatic glycerol determination

Erroneously high values for serum triglyceride levels obtained with the semiautomated method of Lofland were found to be due to contamination of the isopropanol extracts with glucose or other carbohydrate. Treatment of the isopropanol extracts with a mixture of copper sulfate and calcium hydroxide removed the contaminating glucose. Analysis of the glucose-free extracts by either the semiautomated or manual colorimetric method gave values in good agreement with each other and with those obtained by a new specific enzymatic method. The simple modification described in this paper obviates the necessity for the more expensive automated fluorometric apparatus.     

Determination of acetaldehyde in blood using automated distillation and fluorometry

A sensitive enzymic method for the determination of acetaldehyde in human blood has been developed. The method may be operated in a semiautomated or fully automated mode and involves continuous-flow distillation of samples with fluorometry. Levels of acetaldehyde between 0.5 and 20 μmol/liter in blood may be determined, using either yeast or sheep liver aldehyde dehydrogenases.     

Using habitat suitability models to sample rare species in high-altitude ecosystems: a case study with Tibetan argali

Models of the distribution of rare and endangered species are important tools for their monitoring and management. Presence data used to build up distribution models can be based on simple random sampling, but this for patchy distributed species results in small number of presences and therefore low precision. Convenience sampling, either based on easily accessible units or a priori knowledge of the species habitat but with no known probability of sampling each unit, is likely to result in biased estimates. Stratified random sampling, with strata defined using habitat suitability models [estimated in the resource selection functions (RSFs) framework] is a promising approach for improving the precision of model parameters. We used this approach to sample the Tibetan argali (Ovis ammon hodgsoni) in Indian Transhimalaya in order to estimate their distribution and to test if it can lead to a significant reduction in survey effort compared to random sampling. We first used an initial sample of argali feeding sites in 2005 and 2006 based on a priori selected vantage points and survey transects. This initial sample was used to build up an initial distribution model. The spatial predictions based on estimated RSFs were then used to define three strata of the study area. The strata were randomly sampled in 2007. As expected, much more presences per hour were obtained in the high quality strata compared to the low quality strata—1.33 obs/h vs. 0.080/h. Furthermore the best models selected on the basis of the prospective sample differed from those using the first a priori sample, suggesting bias in the initial sampling effort. The method therefore has significant implications for decreasing sampling effort in terms of sampling time in the field, especially when dealing with rare species, and removing initial sampling bias.     

Could ecologists be more random? Straightforward alternatives to haphazard spatial sampling

When conducting field studies, it is common for ecologists to choose the locations of sampling units arbitrarily at the time sampling occurs, rather than using a properly randomised sampling design. Unfortunately, this ‘haphazard’ sampling approach cannot provide formal statistical inference from the sample to the population without making untestable assumptions. Here, we argue that two recent technological developments remove the need for haphazard sampling in many situations. A general approach to simple randomised sampling designs is outlined, and some examples demonstrate that even complicated designs can be implemented easily using software that is widely used among ecologists. We consider that more rigorous, randomised sampling designs would strengthen the validity of the conclusions drawn from ecological studies, to the benefit of the discipline as a whole.     

An assessment of some improved techniques for estimating the abundance (frequency) of sedentary organisms

The traditional sampling method for estimating frequency (the number of sub-quadrats containing a basal part of the organisms) is compared, using both computer simulations and direct comparison in the field, to two new methods that use a compound series of variable-sized concentric sub-quadrats. Both the new frequency-score and the new importance-score methods are closer approximations of density than is the standard frequency method, and the estimates produced by both of the new methods are less affected by the choice of sub-quadrat size and the spatial distribution (dispersion) of the organisms (i.e. clumping and regularity). Thus, the two nested-quadrat methods appear to ameliorate the usual frequency limitations associated with sub-quadrat size and organism dispersion, by the use of a range of different sub-quadrat sizes. This is important in community studies, where the component species may show a wide range of densities and dispersions. Both of the new methods are easily employed in the field. The importance-score method involves no more sampling effort than does standard qualitative (presence-absence) sampling, and it can therefore be used to sample a larger quadrat area than would normally be used for frequency sampling. This makes the method much more cost-effective as a means of estimating abundance, and it allows a greater number of the rarer species to be included in the sampling. The frequency-score method is more time-consuming, but it is capable of detecting more subtle community patterns. This means that it is particularly useful for the study of species-poor communities or where small variations in composition need to be detected.     

The Efficient Method for Simultaneous Monitoring of the Culturable as Well as Nonculturable Airborne Microorganisms

Cultivation-based microbiological methods are a gold standard for monitoring of airborne micro-organisms to determine the occupational exposure levels or transmission paths of a particular infectious agent. Some highly contagious microorganisms are not easily culturable but it is becoming evident that cultivation and molecular methods are complementary and in these cases highly relevant. We report a simple and efficient method for sampling and analyzing airborne bacteria with an impactor-type high-flow-rate portable air sampler, currently used for monitoring culturable bacteria or fungi. A method is reported for extraction of nucleic acids from impacted cells without prior cultivation and using agarose as a sampling matrix. The DNA extraction efficiency was determined in spiked samples and, samples taken from a wastewater treatment plant and an alpine area. The abundance, diversity and quantity of total bacteria were analysed by a quantitative polymerase chain reaction, and by construction and analysis of clone libraries. The method does not interfere with downstream PCR analysis and can cover the gap between traditional culture and molecular techniques of bioaerosol monitoring.     

Jetski-based plankton towing as a new method of sampling larval fishes in shallow marine habitats

I tested jetski-based plankton towing as a new method of sampling larval fishes in a shallow marine environment in warm temperate South Africa. Larval fishes were collected by towing a stainless-steel ring net behind a three-man jetski. The sampling technique was designed and tested to overcome problematic sampling of shallow sandy beach surf zones and the adjacent, shallow nearshore immediately seaward of the surf zone (behind the breaker line) with the same type of equipment. The composition, density, stage of development, estuary association, and length frequency of larval fish catches obtained by the new sampling method in the surf zone and in the adjacent shallow nearshore were compared. The success of the technique was evaluated by determining the extent of replication of known trends observed in other surf studies in the same climatic region of South Africa using either a seine or pushnet. Preflexion and postflexion larval fishes were collected. Larvae of the family Sparidae dominated the catches. Trends in catch composition, estuary association, and length frequency of the larvae were similar to those found in other studies of warm temperate South African surf zones. Density of larvae varied depending on the type of equipment used, although these results are tentative given the subjectivity of calculation of the water volume sampled and the extent of nursery use by larvae in the habitats sampled in each study. Increased towing times are recommended. Jetski-based plankton towing is a useful alternative technique for sampling larval fishes in shallow habitats and can easily be applied in surf zones, shallow nearshore areas, and estuaries. The technique greatly increases sampling agility in previously problematic sampling areas.     

A Fast and Accurate Method for Glaucoma Screening from Smartphone-Captured Fundus Images

The glaucoma is an eye disease that causes blindness when it progresses in an advanced stage. Early glaucoma diagnosis is essential to prevent the vision loss. However, early detection is not covered due to the lack of ophthalmologists and the limited accessibility to retinal image capture devices.In this paper, we present an automated method for glaucoma screening dedicated for Smartphone Captured Fundus Images (SCFIs). The implementation of the method into a smartphone associated to an optical lens for retina capturing leads to a mobile aided screening system for glaucoma. The challenge consists in insuring higher performance detection despite the moderate quality of SCFIs, with a reduced execution time to be adequate for the clinical use. The main idea consists in deducing glaucoma based on the vessel displacement inside the Optic Disk (OD), where the vessel tree remains sufficiently modeled on SCFIs. Within this objective, our major contribution consists in proposing: (1) a robust processing for locating vessel centroids in order to adequately model the vessel distribution, and (2) a feature vector that relevantly reflect two main glaucoma biomarkers in terms of vessel displacement. Furthermore, all processing steps are carefully chosen based on lower complexity, to be suitable for fast clinical screening.A first evaluation of our method is performed using the two public DRISHTI-DB and DRIONS-DB databases, where 99% and 95% accuracy, 96.77% and 97,5% specificity and 100% and 95% sensitivity are respectively achieved. Thereafter, the method is evaluated using two fundus image databases respectively captured through a smartphone and retinograph for the same persons. We achieve 100% accuracy using both databases which assesses the robustness of our method. In addition, the detection is performed on 0.027 and 0.029 second when executed respectively on the Samsung-M51 on the Samsung-A70 smartphone devices. Our proposed smartphone app provides a cost-effective and widely accessible mobile platform for early screening of glaucoma in remote clinics or areas with limited access to fundus cameras and ophthalmologists.     

Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay.

The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a colorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled; and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed.     

The use of image analysis for morphological measurements on filamentous microorganisms

Characterization of mycelial morphology is important for the design and operation of filamentous fermentations. Initial investigations have been made of semiautomated image analysis as a replacement for a digitizing-table method. It was shown that the image analysis method was more precise than the digitizing-table method used, although this extra precision is unimportant in this application. On average, image analysis gave mean hyphal lengths 6% greater than digitizing, because the latter used chord lengths to represent are lengths. For short branches image analysis was less accurate. In time and convenience the image analysis method had an overwhelming advantage, and this advantage might be enhanced by full automation. The resulting ability to characterize mycelial morphology rapidly would permit such characterization to be used routinely in studies of filamentous fermentations.     

Vascular-access-port implantation for serial blood sampling in conscious swine

A method to simplify serial venous blood sampling in miniature pigs was developed. Jugular vein Vascular-Access-Ports (VAP) were implanted in four animals. The mean functional lifetime of these ports was 37.7 +/- 23.0 (S.D.) days with a range of 17 to 77 days. The VAP allowed easy serial blood sampling and intravenous drug administration. Use of these ports in animals restrained in a sling was simple and effective and caused no undue stress to the animal over a 6 to 8 hour experiment. The VAPs were implanted easily and required minimal maintenance. Subcutaneous location of the entire apparatus allowed for group housing of the pigs and decreased the susceptibility of postoperative infection and/or damage. Provided that proper maintenance and careful aseptic sampling techniques are used, the VAP is a relatively easy, safe and reliable alternative to conventional methods of serial blood sampling in swine.