Comparison of two methods of tear sampling for protein quantification by Bradford method

The aim of this study was to compare two methods of tear sampling for protein quantification. Tear samples were collected from 29 healthy dogs (58 eyes) using Schirmer tear test (STT) strip and microcapillary tubes. The samples were frozen at -80ºC and analyzed by the Bradford method. Results were analyzed by Student's t test. The average protein concentration and standard deviation from tears collected with microcapillary tube were 4.45mg/mL ±0.35 and 4,52mg/mL ±0.29 for right and left eyes respectively. The average protein concentration and standard deviation from tears collected with Schirmer Tear Test (STT) strip were and 54.5mg/mL ±0.63 and 54.15mg/mL ±0.65 to right and left eyes respectively. Statistically significant differences (p<0.001) were found between the methods. In the conditions in which this study was conducted, the average protein concentration obtained with the Bradford test from tear samples obtained by Schirmer Tear Test (STT) strip showed values higher than those obtained with microcapillary tube. It is important that concentration of tear protein pattern values should be analyzed according the method used to collect tear samples.

Immunoblotting and tear sampling techniques for the study of contact lens-induced variations in tear protein profiles

More information on the biochemical interactions taking place between the tear film and the contact lens is required to further our understanding of the causative mechanisms behind the symptoms of dryness and grittiness often experienced by contact lens wearers. These symptoms can often lead to an intolerance to contact lens wear.

Biochemical markers and contact lens wear

The project objective was to develop a reliable selection procedure to match contact lens materials with individual wearers by the identification of a biochemical marker for assessment of in-eye performance of contact lenses. There is a need for such a procedure as one of the main reasons for contact lens wearers ceasing wearing contact lenses is poor end of day comfort i.e. the lenses become intolerable to the wearer as the day progresses. The selection of an optimal material for individual wearers has the potential benefit to reduce drop Qut, hence increasing the overall contact lens population, and to improve contact lens comfort for established wearers. Using novel analytical methods and statistical techniques, we were able to investigate the interactions between the composition of the tear film and of the biofilm deposited on the contact lenses and contact lens performance. The investigations were limited to studying the lipid components of the tear film; the lipid layer, which plays a key role in preventing evaporation and stabilising the tear film, has been reported to be significantly thinner and of different mixing characteristics during contact lens wear. Different lipid families were found to influence symptomatology, in vivo tear film structure and stability as well as ocular integrity. Whereas the symptomatology was affected by both the tear film lipid composition and the nature of the lipid deposition, the structure of the tear film and its stability were mainly influenced by the tear film lipid composition. The ocular integrity also appeared to be influenced by the nature of the lipid deposition. Potential markers within the lipid species have been identified and could be applied as follows: When required in order to identify a problematic wearer or to match the contact lens material to the contact lens wearer, tear samples collected by the clinician could be dispatched to an analytical laboratory where lipid analysis could be carried out by HPLC. A colorimetric kit based on the lipid markers could also be developed and used by clinician directly in the practice; such a kit would involve tear sampling and classification according to the colour into "Problem", "Border line" and "Good" contact lens wearers groups. A test kit would also have wider scope for marketing in other areas such as general dry-eye pathology.

Albumin in tears

Albumin is not endogenous to the tear film and is present as a product of plasma leakage. It is used as a diagnostic marker of ocular insult and inflammation. Tear albumin is, however, poorly understood, with large variations in reported concentrations between studies. There is also no authoritative information on whether its presence in tears is responsive or part of an adaptive reaction.The presented research aimed to resolve the disparities in published tear albumin concentrations and investigate the role of albumin in the tear film. Collation and evaluation of the available literature identified collection method, stimulus, assay technique, and disease state as factors able to influence quoted tear albumin to different extents. Difference in sampling technique exhibited the largest variations in mean tear albumin concentrations. Review of the literature also highlighted that little systematic investigations of the daily cycle of tear albumin levels, and subject-to-subject-variation, had been carried out. In order to remedy this shortcoming, variations in tear albumin concentration were investigated in 13 subjects throughout the waking day. Results identified a time period where albumin levels are relatively stable (2-6 hours post-waking). This was designated a suitable baseline for the determinations of tear albumin concentrations and subject-to-subject comparisons. Significantly, a previously unrecognised progressive increase in albumin concentration during the latter part of the day was also identified in the population. This increase suggests that albumin may play a more active and dynamic role in the ocular environment than is commonly perceived. To facilitate the collection of additional tear albumin data, tear sampling and point-of-care analysis in contact lens clinics were investigated. Two instruments were evaluated and were found to be suitable for the analysis of tear albumin in commercial institutions. Collectively, the described research has provided new insight into tear albumin and a strong foundation for further studies.

Immunological and biochemical techniques in the analysis of tear proteins

This study is concerned with the analysis of tear proteins, paying particular attention to the state of the tears (e.g. non-stimulated, reflex, closed), created during sampling, and to assess their interactions with hydrogel contact lenses. The work has involved the use of a variety of biochemical and immunological analytical techniques for the measurement of proteins, (a), in tears, (b), on the contact lens, and (c), in the eluate of extracted lenses. Although a diverse range of tear components may contribute to contact lens spoilation, proteins were of particular interest in this study because of their theoretical potential for producing immunological reactions. Although normal host proteins in their natural state are generally not treated as dangerous or non-self, those which undergo denaturation or suffer a conformational change may provoke an excessive and unnecessary immune response. A novel on-lens cell based assay has been developed and exploited in order to study the role of the ubiquitous cell adhesion glycoprotein, vitronectin, in tears and contact lens wear under various parameters. Vitronectin, whose levels are known to increase in the closed eye environment and shown here to increase during contact lens wear, is an important immunoregulatory protein and may be a prominent marker of inflammatory activity. Immunodiffusion assays were developed and optimised for use in tear analysis, and in a series of subsequent studies used for example in the measurement of albumin, lactoferrin, IgA and IgG. The immunodiffusion assays were then applied in the estimation of the closed eye environment; an environment which has been described as sustaining a state of sub-clinical inflammation. The role and presence of a lesser understood and investigated protein, kininogen, was also estimated, in particular, in relation to contact lens wear. Difficulties arise when attempting to extract proteins from the contact lens in order to examine the individual nature of the proteins involved. These problems were partly alleviated with the use of the on-lens cell assay and a UV spectrophotometry assay, which can analyse the lens surface and bulk respectively, the latter yielding only total protein values. Various lens extraction methods were investigated to remove protein from the lens and the most efficient was employed in the analysis of lens extracts. Counter immunoelectrophoresis, an immunodiffusion assay, was then applied to the analysis of albumin, lactoferrin, IgA and IgG in the resultant eluates.

The fall and rise of tear albumin levels:a multifactorial phenomenon

Albumin in tears is used as a diagnostic marker of ocular insult and inflammation, but whether its presence in tears is responsive or part of an adaptive reaction remains unresolved. A review of the literature on tear albumin concentration emphasizes that variables such as collection method, stimulus, assay technique, and disease state influence the quoted values to different extents. Influence of assay technique is negligible in comparison to variation in sampling conditions. Ocular disease increases albumin concentrations but not in a specific manner. The literature review also highlighted that little systematic research has been carried out on the daily cycle of tear albumin levels. In order to remedy this shortcoming, we investigated variations in tear albumin concentration during the waking day. The concentration of albumin in 400 tear samples collected from 13 subjects was assessed at 2-hourly intervals throughout the waking day. Highest daytime albumin concentrations were obtained within 10 minutes of waking, with a mean concentration of >50 ± 22 µg/ml. Albumin levels were at their lowest, but most consistent, 2-6 hours post-waking. This pattern was followed by a progressive increase in albumin concentration during the latter part of the day. Although individual subject-to-subject concentration differences were observed, this distinctive pattern of diurnal variation was found in all subjects. The results presented suggest a regulated, not random, pattern of variation within the period of study. © 2013 Elsevier Inc. All rights reserved.

Temporal variation of the phytoplankton community at short sampling intervals in the Mundaú reservoir, Northeastern Brazil

The aim of this study was to determine how abiotic factors drive the phytoplankton community in a water supply reservoir within short sampling intervals. Samples were collected at the subsurface (0.1 m) and bottom of limnetic (8 m) and littoral (2 m) zones in both the dry and rainy seasons. The following abiotic variables were analyzed: water temperature, dissolved oxygen, electrical conductivity, total dissolved solids, turbidity, pH, total nitrogen, nitrite, nitrate, total phosphorus, total dissolved phosphorus and orthophosphate. Phytoplankton biomass was determined from biovolume values. The role abiotic variables play in the dynamics of phytoplankton species was determined by means of Canonical Correspondence Analysis. Algae biomass ranged from 1.17×10(4) to 9.21×10(4) µg.L-1; cyanobacteria had biomass values ranging from 1.07×10(4) to 8.21×10(4) µg.L-1. High availability of phosphorous, nitrogen limitation, alkaline pH and thermal stability all favored cyanobacteria blooms, particularly during the dry season. Temperature, pH, total phosphorous and turbidity were key factors in characterizing the phytoplankton community between sampling times and stations. Of the species studied, Cylindrospermopsis raciborskii populations were dominant in the phytoplankton in both the dry and rainy seasons. We conclude that the phytoplankton was strongly influenced by abiotic variables, particularly in relation to seasonal distribution patterns.

Precision of distances and ordering of microsatellite markers in consensus linkage maps of chromosomes 1, 3 and 4 from two reciprocal chicken populations using bootstrap sampling

Some factors complicate comparisons between linkage maps from different studies. This problem can be resolved if measures of precision, such as confidence intervals and frequency distributions, are associated with markers. We examined the precision of distances and ordering of microsatellite markers in the consensus linkage maps of chromosomes 1, 3 and 4 from two F 2 reciprocal Brazilian chicken populations, using bootstrap sampling. Single and consensus maps were constructed. The consensus map was compared with the International Consensus Linkage Map and with the whole genome sequence. Some loci showed segregation distortion and missing data, but this did not affect the analyses negatively. Several inversions and position shifts were detected, based on 95% confidence intervals and frequency distributions of loci. Some discrepancies in distances between loci and in ordering were due to chance, whereas others could be attributed to other effects, including reciprocal crosses, sampling error of the founder animals from the two populations, F(2) population structure, number of and distance between microsatellite markers, number of informative meioses, loci segregation patterns, and sex. In the Brazilian consensus GGA1, locus LEI1038 was in a position closer to the true genome sequence than in the International Consensus Map, whereas for GGA3 and GGA4, no such differences were found. Extending these analyses to the remaining chromosomes should facilitate comparisons and the integration of several available genetic maps, allowing meta-analyses for map construction and quantitative trait loci (QTL) mapping. The precision of the estimates of QTL positions and their effects would be increased with such information.

Phylogenetic relationships within the speciose family Characidae (Teleostei: Ostariophysi: Characiformes) based on multilocus analysis and extensive ingroup sampling

Background: With nearly 1,100 species, the fish family Characidae represents more than half of the species of Characiformes, and is a key component of Neotropical freshwater ecosystems. The composition, phylogeny, and classification of Characidae is currently uncertain, despite significant efforts based on analysis of morphological and molecular data. No consensus about the monophyly of this group or its position within the order Characiformes has been reached, challenged by the fact that many key studies to date have non-overlapping taxonomic representation and focus only on subsets of this diversity. Results: In the present study we propose a new definition of the family Characidae and a hypothesis of relationships for the Characiformes based on phylogenetic analysis of DNA sequences of two mitochondrial and three nuclear genes (4,680 base pairs). The sequences were obtained from 211 samples representing 166 genera distributed among all 18 recognized families in the order Characiformes, all 14 recognized subfamilies in the Characidae, plus 56 of the genera so far considered incertae sedis in the Characidae. The phylogeny obtained is robust, with most lineages significantly supported by posterior probabilities in Bayesian analysis, and high bootstrap values from maximum likelihood and parsimony analyses. Conclusion: A monophyletic assemblage strongly supported in all our phylogenetic analysis is herein defined as the Characidae and includes the characiform species lacking a supraorbital bone and with a derived position of the emergence of the hyoid artery from the anterior ceratohyal. To recognize this and several other monophyletic groups within characiforms we propose changes in the limits of several families to facilitate future studies in the Characiformes and particularly the Characidae. This work presents a new phylogenetic framework for a speciose and morphologically diverse group of freshwater fishes of significant ecological and evolutionary importance across the Neotropics and portions of Africa.

Comparative analysis of two sampling techniques for pollen gathered by Nannotrigona testaceicornis Lepeletier (Apidae, Meliponini)

Pollen counts from samples taken from storage pots throughout one year (from October to September) were adjusted by Tasei's volumetric correction coefficient for the determination of pollen sources exploited by two colonies of Nannotrigona testaceicornis in Sao Paulo, Brazil. The results obtained by this sampling technique for seven months (December to June) were compared with those from corbicula load samples taken within the same period. This species visited a large variety of plant species, but few of them were frequently used. As a rule, pollen sources that appeared at frequencies greater than 1% were found with both sampling methods and significant positive correlations (Spearman correlation coefficient) were found between their values. The pollen load sample data showed that N. testaceicornis gathered pollen throughout the external activity period.

Method for cadmium and lead longitudinal profiles determination in hair by solid sampling graphite furnace atomic absorption spectrometry

This paper describes methods for the direct determination of Cd and Pb in hair segments (c.a. 5 mm similar to 80 mu g) by solid sampling graphite furnace atomic absorption spectrometry, becoming possible longitudinal profiles in a single strand of hair. To distinguish endogenous and exogenous content. strands of hair were washed by using two different procedures: IAEA protocol (acetone + water + acetone) and the combination of IAEA protocol with HCl washing (acetone + water + acetone + 0.1 mol l(-1) HCl). The concentration of Cd and Pb increased from the root Until the tip of hair washed according to IAEA protocol. However, when the strand of hair was washed using the combination of IAEA protocol and 0.1 mol l(-1) HCl, Cd concentrations decreased in all segments, and Pb concentrations decreased drastically near to the root (5 to 12 mm) and was systematically higher ill the end. The proposed method showed to be useful to assess the temporal variation to Cd and Pb exposure and call be Used for toxicological and environmental investigations. The limits of detection were 2.8 ng g(-1) for Cd and 40 ng g(-1) for Pb. The characteristic masses based oil integrated absorbance were 2.4 pg for Cd and 22 pg for Pb.

Simultaneous determination of Cr, Fe, Ni and V in crude oil by emulsion sampling graphite furnace atomic absorption spectrometry

In this work a simple and reliable method for the simultaneous determination of Cr, Fe, Ni and V in crude oil, using emulsion sampling graphite furnace atomic absorption spectrometry is proposed. Under the best conditions, sample masses around 50 mg were weighed in polypropylene tubes and emulsified in a mixture of 0.5% (v v(-1)) hexane + 6% (m v(-1)) Triton X-100 (R). Considering the compromised conditions, the pyrolysis an atomization temperatures for the simultaneous determination of Cr, Fe, Ni and V were 1400 degrees C and 2500 degrees C, respectively. Aliquots of 20 mu L of reference solution and sample emulsion were co-injected into the graphite tube with 10 mu L of 1.0 g L(-1) Mg(NO(3))(2) as chemical modifier. The detection limits (n = 10, 3 sigma) and characteristic masses were, respectively: 0.07 mu g g(-1) and 19 pg for Cr; 2.15 mu g g(-1) and 31 pg for Fe; 1.25 mu g g(-1) and 44 pg for Ni; and 1.15 mu g g(-1) and 149 pg for V. The reliability of the proposed method was checked by fuel oil Standard Reference Material (SRMTriton X-100 (R) 1634c - NIST) analysis. The concentrations found presented no statistical differences compared to the certified values at 95% confidence level.

A sampling device to quantify offspring release of sessile marine invertebrates

Quantifying the rate of propagule release is of most importance to estimate reproductive output of natural populations, but simple methods to obtain such data are seldom reported. We designed and tested an inexpensive apparatus capable of reliably measure the release of gametes, eggs or larvae of sessile marine invertebrates in vertical walls. A population of the acom barnacle Chthamalus bisinuatus was sampled with this trap over 68d to obtain a time series of naupliar release. An apparent semilunar trend is shown, indicating the effectiveness of this sampling method.

Sampling study in milk storage tanks by INAA

This study investigated the representativeness of samples for assessing chemical elements in milk bulk tanks. Milk samples were collected from a closed tank in a dairy plant and from an open top tank in a dairy farm. Samples were analyzed for chemical elements by instrumental neutron activation analysis (INAA). For both experiments, Br, Ca, Cs, K, Na, Rb and Zn did not present significant differences between samples thereby indicating the appropriateness of the sampling procedure adopted to evaluate the analytes of interest.

Feasibility of using solid sampling graphite furnace atomic absorption spectrometry for preparation of spiked filter papers with Cu and Zn as standards for direct solid analysis

An approach was developed for the preparation of cryogenic ground spiked filter papers with Cu and Zn for use as synthetic calibrating standards for direct solid microanalysis. Solid sampling graphite furnace atomic absorption spectrometry was used to evaluate the microhomogeneity and to check the applicability of the synthetic calibrating standards for the direct determination of Cu and Zn in vegetable certified reference materials. The found concentrations presented no statistical differences at the 95% confidence level. The homogeneity factors ranged from 2.7 to 4.2 for Cu and from 6.4 to 11.5 for Zn.