Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

An apparatus for the simultaneous demineralization of fifty-four specimens.

An apparatus designed to demineralize 54 specimens simultaneously is described. A drum with built-in specimen holders rotates continuously through a bath of acid, allowing a free exchange of demineralizing fluid over the specimens. Individual specimens can be easily introduced or withdrawn from the apparatus without disturbing others.     

An Apparatus for the Simultaneous Demineralization of Fifty-Four Specimens

An apparatus designed to demineralize 54 specimens simultaneously is described. A drum with built-in specimen holders rotates continuously through a bath of acid, allowing a free exchange of demineralizing fluid over the specimens. Individual specimens can be easily introduced or withdrawn from the apparatus without disturbing others.     

The relevance of time series in molecular ecology and conservation biology

The genetic structure of a species is shaped by the interaction of contemporary and historical factors. Analyses of individuals from the same population sampled at different points in time can help to disentangle the effects of current and historical forces and facilitate the understanding of the forces driving the differentiation of populations. The use of such time series allows for the exploration of changes at the population and intraspecific levels over time. Material from museum collections plays a key role in understanding and evaluating observed population structures, especially if large numbers of individuals have been sampled from the same locations at multiple time points. In these cases, changes in population structure can be assessed empirically. The development of new molecular markers relying on short DNA fragments (such as microsatellites or single nucleotide polymorphisms) allows for the analysis of long‐preserved and partially degraded samples. Recently developed techniques to construct genome libraries with a reduced complexity and next generation sequencing and their associated analysis pipelines have the potential to facilitate marker development and genotyping in non‐model species. In this review, we discuss the problems with sampling and available marker systems for historical specimens and demonstrate that temporal comparative studies are crucial for the estimation of important population genetic parameters and to measure empirically the effects of recent habitat alteration. While many of these analyses can be performed with samples taken at a single point in time, the measurements are more robust if multiple points in time are studied. Furthermore, examining the effects of habitat alteration, population declines, and population bottlenecks is only possible if samples before and after the respective events are included.     

A Trimming Stage for Stereomicroscopes

Scott and Thurston (1975) have described a simple, useful modification of ultratome specimen holders which permits transillumination of plastic embedded specimens for hand trimming.¹ We have refined their original concept by fabricating a Lucite chuck-mounting disc which, in addition to allowing transillumination of embedded specimens, may also be rotated 360 degrees for specimen orientation or locked in place in any desired trimming position. When used in circular stage mounts of stereomicroscopes such as the Nikon SMZ-10, the disc transforms the microscope into a highly effective trimming instrument.     

Peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display

The human Plasma Proteome Project pilot phase aims to analyze serum and plasma specimens to elucidate specimen characteristics by various proteomic techniques to ensure sufficient sample quality for the HUPO main phase. We used our proprietary peptidomics technologies to analyze the samples distributed by HUPO. Peptidomics summarizes technologies for visualization, quantitation, and identification of the low-molecular-weight proteome (<15 kDa), the "peptidome." We analyzed all four HUPO specimens (EDTA plasma, citrate plasma, heparin plasma, and serum) from African- and Asian-American donors and compared them to in-house collected Caucasian specimens. One main finding focuses on the most suitable method of plasma specimen collection. Gentle platelet removal from plasma samples is beneficial for improved specificity. Platelet contamination or activation of platelets by low temperature prior to their removal leads to distinct and multiple peptide signals in plasma samples. Two different specimen collection protocols for platelet-poor plasma are recommended. Further emphasis is placed on the differences between plasma and serum on a peptidomic level. A large number of peptides, many of them in rather high abundance, are only present in serum and not detectable in plasma. This ex vivo generation of multiple peptides hampers discovery efforts and is caused by a variety of factors: the release of platelet-derived peptides, other peptides derived from cellular components or the clot, enzymatic activities of coagulation cascades, and other proteases. We conclude that specimen collection is a crucial step for successful peptide biomarker discovery in human blood samples. For analysis of the low-molecular-weight proteome, we recommend the use of platelet-depleted EDTA or citrate plasma.     

Reliable genotyping of samples with very low DNA quantities using PCR.

Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.     

Mechanical study of potential ceramic implant materials for minimal invasive anterior lumbar interbody fusion]

While autologous bone grafts are highly suitable for use in spinal arthrodesis, their use is also associated with problems (traumatization, complications). Ceramic bone substitute materials provide an attractive alternative for lumbar interbody spinal fusion. The aim of the present study was to investigate the mechanical properties of various types of ceramic using a specific fusion method. Ten specimens each of 7 different types of ceramic were tested using a hydraulic testing machine with two different sample holders: polyurethane foam (mechanical properties similar to cancellous bone) and aluminium. The parameters axial compression and axial torque were investigated. With the polyurethane foam holders, none of the ceramic implants failed under compression, while under axial rotation, two types of ceramic failed. With the aluminium holders, 3 ceramics showed no failure up to 25 kN under compression, while under torsion all the ceramics failed. One type of ceramic showed specific fracture properties with a higher load-bearing capacity after failure in comparison with all the other types studied.     

Naturally Acquired Picornavirus Infections in Primates at the Dhaka Zoo

The conditions in densely populated Bangladesh favor picornavirus transmission, resulting in a high rate of infection in the human population. Data suggest that nonhuman primates (NHP) may play a role in the maintenance and transmission of diverse picornaviruses in Bangladesh. At the Dhaka Zoo, multiple NHP species are caged in close proximity. Their proximity to other species and to humans, both zoo workers and visitors, provides the potential for cross-species transmission. To investigate possible interspecies and intraspecies transmission of picornaviruses among NHP, we collected fecal specimens from nine NHP taxa at the Dhaka Zoo at three time points, August 2007, January 2008, and June 2008. Specimens were screened using real-time PCR for the genera Enterovirus, Parechovirus, and Sapelovirus, and positive samples were typed by VP1 sequencing. Fifty-two picornaviruses comprising 10 distinct serotypes were detected in 83 fecal samples. Four of these serotypes, simian virus 19 (SV19), baboon enterovirus (BaEV), enterovirus 112 (EV112), and EV115, have been solely associated with infection in NHP. EV112, EV115, and SV19 accounted for 88% of all picornaviruses detected. Over 80% of samples from cages housing rhesus macaques, olive baboons, or hamadryas baboons were positive for a picornavirus, while no picornaviruses were detected in samples from capped langurs or vervet monkeys. In contrast to our findings among synanthropic NHP in Bangladesh where 100% of the picornaviruses detected were of human serotypes, in the zoo population, only 15% of picornaviruses detected in NHP were of human origin. Specific serotypes tended to persist over time, suggesting either persistent infection of individuals or cycles of reinfection.     

Improved Thermocouple Psychrometer for the Measurement of Plant and Soil Water Potential: I. THERMOCOUPLE PSYCHROMETRY AND AN IMPROVED INSTRUMENT DESIGN

This paper is the first of a series which describes: (a) thedesign and operation of a thermocouple psychrometer which providesimproved range of measurement, accuracy, and equilibration rateof water potential determinations of plant and soil samples,and which avoids or minimizes six sources of large error, oneor more of which occur with previous psychrometers and (b) studiesthat provide a better understanding of the thermocouple psychrometricmethod. The present paper describes the main difficulties encounteredwith the method, examines those due to absorption phenomena,and describes an improved thermocouple psychrometer which overcomesor reduces some of these difficulties. Improvements include: (i) chamber walls of stainless steel type316 and samples arranged in shielding geometries to reduce delaysin equilibration due to adsorption, (ii) sample holders of geometriesthat avoid or reduce errors of leaf resistance, adsorption andtissue damage when appropriate cooling periods and tissue segmentsof adequate size are used, (iii) shielding arrangement of respiringtissue samples to permit thermal equilibration in the regionsensed by the ‘active’ thermojunction, a new thermocoupleassembly for increased range of measurement of water potential,(v) sample holders that permit plant or soil samples to be accommodatedin the one chamber.     

Statistical Considerations in the Sampling of Escherichia coli from Intestinal Sources for Serotyping

A theoretical study of the expected distribution of Escherichia coli serotypes among samples randomly drawn from populations containing different numbers, ranging from 2 to 40, was compared with experimental data based on the examination of 10 and 100 coiony samples from the same specimens. From these studies it was possible to lay down guide lines for the planning of efficient sampling programmes for determining most or all serotypes present in various animal species.     

A label-free light-scattering method to resolve assembly and disassembly of DNA nanostructures

DNA self-assembly, and in particular DNA origami, has evolved into a reliable workhorse for organizing organic and inorganic materials with nanometer precision and with exactly controlled stoichiometry. To ensure the intended performance of a given DNA structure, it is beneficial to determine its folding temperature, which in turn yields the best possible assembly of all DNA strands. Here, we show that temperature-controlled sample holders and standard fluorescence spectrometers or dynamic light-scattering setups in a static light-scattering configuration allow for monitoring the assembly progress in real time. With this robust label-free technique, we determine the folding and melting temperatures of a set of different DNA origami structures without the need for more tedious protocols. In addition, we use the method to follow digestion of DNA structures in the presence of DNase I and find strikingly different resistances toward enzymatic degradation depending on the structural design of the DNA object.     

Factors affecting success of PCR amplification of microsatellite loci from otter faeces

We investigated the effect of multiple variables on the amplification success rate of microsatellite DNA extracted from faeces of wild Eurasian otters. The success rate was affected by (i) type of sample, with higher success rates in anal jelly samples than faeces, and (ii) temperature, with a negative effect of increased temperature at time of collection. To increase the effectiveness of microsatellite genotyping of otter faeces, we recommend collecting samples in cold months and early in the morning, preferably in a frozen state, and the collection of anal jelly samples, or the jelly part from faeces, whenever possible.     

The use of multivariate techniques in the study of skeletal populations

A major problem in taxonomic or evolutionary studies (micro or macro) of populations through their skeletal remains is what to do about single specimens, or samples of half a dozen, or fragments of skeletons. Multivariate analysis makes possible finer distinctions of all sorts than does univariate analysis, including sex and population assignment, allowing such placement objectively when adequate samples of identified populations are available to form the multivariate context. The history and essential nature of multivariate analysis is described briefly, and examples of its application to single specimens (the Fish Hoek and Keilor skulls) are given.     

Characterization of mechanical behavior of a porcine pulmonary artery strip using a randomized uniaxial stretch and stretch-rate protocol

Background  

Much of the experimental work in soft tissue mechanics has been focused on fitting approximate relations for specific tissue types from aggregate data on multiple samples of the tissue. Such relations are needed for modeling applications and have reasonable predictability - especially given the natural variance in specimens. There is, however, much theoretical and experimental work to be done in determining constitutive behaviors for particular specimens and tissues. In so doing, it may be possible to exploit the natural variation in tissue ultrastructure - so to relate ultrastructure composition to tissue behavior. Thus, this study focuses on an experimental method for determining constitutive behaviors and illustrates the method with analysis of a porcine pulmonary artery strip. The method characterizes the elastic part of the response (implicitly in terms of stretch) and the inelastic part in terms of short term stretch history (i.e., stretch-rate) H _t2, longer term stretch history H _t1, and time since the start of testing T.     

Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens

Museum specimens have provided the material for a large proportion of ancient DNA studies conducted during the last 20 years. However, a major drawback of the genetic analyses is that the specimens investigated are usually damaged, as parts of skin, bone, or a tooth have to be removed for DNA extraction. To get around these limitations, we have developed a nondestructive extraction method for bone, tooth, and skin samples. We found that it is possible to amplify mitochondrial DNA (mtDNA) sequences up to at least 414 bp long from samples up to 164 years old. Using this method, almost 90% (35 of 40) of the investigated samples yielded amplifiable mtDNA. Moreover, we found that repeated extractions of the same samples allowed amplifications of the expected length for all samples at least three times and for some samples up to at least five times. Thus this method opens up the possibility to repeatedly use museum collections for mtDNA analyses without damaging the specimens and thus without reducing the value of irreplaceable collections for morphological analyses.     

Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens

BackgroundDengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis.ConclusionsAlthough the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.     

Effects of storage conditions on the stability and distribution of clinical trace elements in whole blood and plasma: Application of ICP-MS

BackgroundKnowledge of trace element stability during sample handling and preservation is a prerequisite to produce reliable test results in clinical trace element analysis.MethodAn alkaline dissolution method has been developed using inductively coupled plasma mass spectrometry to quantify eighteen trace element concentrations: vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, bromine, molybdenum, cadmium, antimony, iodine, mercury, thallium, lead, and bismuth in human blood, using a small sample volume of 0.1 mL. The study evaluated the comparative effects of storage conditions on the stability of nutritionally essential and non-essential elements in human blood and plasma samples stored at three different temperatures (4 °C, −20 °C and −80 °C) over a one-year period, and analysed at multiple time points. The distribution of these elements between whole blood and plasma and their distribution relationships are illustrated using blood samples from 66 adult donors in Queensland.ResultsThe refrigeration and freezing of blood and plasma specimens proved to be suitable storage conditions for many of the trace elements for periods up to six months, with essentially unchanged concentrations. Substantially consistent recoveries were obtained by preserving specimens at −20 °C for up to one year. Ultra-freezing of the specimens at −80 °C did not improve stability; but appeared to result in adsorption and/or precipitation of some elements, accompanied by a longer sample thawing time. A population sample study revealed significant differences between the blood and plasma concentrations of six essential elements and their relationships also varied significantly for different elements.ConclusionBlood and plasma specimens can be reliably stored at 4 °C for six months or kept frozen at −20 °C up to one year to obtain high quality test results of trace elements.     

A comparison of trace-elemental composition of natural color and gray hair

In order to get some information on the possible causes of graying of hair, we have used the technique of X-ray fluorescence (XRF) analysis for comparing the trace element contents of natural color and gray hair from a number of subjects. The technique of XRF was preferred to other analytical methods for this kind of comparative studies since it appeared to be simple, convenient, quick, and contamination free. Natural color and gray hair from each subject were obtained from the same scalp region. The hair samples were washed in the recommended fashion. The natural color and gray hair from different subjects were mounted separately on hollow plastic cylindrical sample holders, assuring that the hair were parallel to, and not on top of one another. The samples were analyzed in a commercial wave length dispersive XRF system, with different X-ray tubes being used for obtaining maximum sensitivity for different elements. The scattered X-ray peak from each sample was also monitored and gave a measure of the sample volume being investigated. So far, hair samples from 10 subjects have been analyzed. Their results are presented in the paper, and advantages of XRF, for trace element analysis on hair are discussed.