Validation of a food quantification picture book and portion sizes estimation applying perception and memory methods.

We tested the applicability of EPIC-SOFT food picture series used in the context of a Hungarian food consumption survey gathering data for exposure assessment, and investigated errors in food portion estimation resulted from the visual perception and conceptualisation-memory. Sixty-two participants in three age groups (10 to <74 years) were presented with three different portion sizes of five foods. The results were considered acceptable if the relative difference between average estimated and actual weight obtained through the perception method was ≤25%, and the relative standard deviation of the individual weight estimates was <30% after compensating the effect of potential outliers with winsorisation. Picture series for all five food items were rated acceptable. Small portion sizes were tended to be overestimated, large ones were tended to be underestimated. Portions of boiled potato and creamed spinach were all over- and underestimated, respectively. Recalling the portion sizes resulted in overestimation with larger differences (up to 60.7%).

Pancreatic neuroendocrine tumors in twelve baboons (Papio spp.).

BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) are rare in nonhuman primates and in humans. METHODS: Twenty-one PNETs from twelve female baboons (Papio spp.) from the Southwest National Primate Research Center were evaluated using histopathology and immunohistochemistry. RESULTS: Histologically, all tumors were benign and had neuroendocrine packeting. Immunohistochemical staining for synaptophysin and chromogranin was positive in all tumors evaluated (17/17). Insulin was positive in 16 of 21 tumors. Somatostatin was positive in 9 of 20 tumors. Multifocal staining for glucagon and pancreatic polypeptide was evident in a minority of tumors (6/20 and 2/17, respectively). Gastrin and vasoactive intestinal peptide were negative in all tumors evaluated. Nine tumors expressed more than one hormone marker. CONCLUSIONS: This is the first detailed pathologic study of pancreatic endocrine tumors in the baboon. The findings suggest that these tumors are generally benign and have similar morphologic and immunohistochemical features as those described in people, including the ability to express multiple hormones.

Measurement of ROS homeostasis in isolated mitochondria.

In this chapter, we describe the currently most advanced methods applied for the quantitative assessment of ROS homeostasis inside the mitochondrion. These techniques are of particular interest in the field of oxidative stress. After discussing the importance of quantifying mitochondrial ROS homeostasis, three major aspects of this phenomenon and the pertinent methodologies for detection are delineated in detail. First the most important methods, based on fluorimetric or spectrophotometric approaches, for the detection of mitochondrial ROS are described. Elimination of ROS generated inside the mitochondrion is another crucial mechanism that also needs to be quantified accurately to estimate the antioxidant capacity of mitochondria under specific conditions. Since ROS generation and elimination manifest in concert, there needs to exist independent methods for the estimation of the net effect. Such a sensitive biochemical marker in the mitochondrion is aconitase, a citric acid cycle enzyme which is greatly sensitive to ROS. We describe two procedures for the precise determination of aconitase activity. A few auxiliary techniques and good practices having relevance in the successful accomplishment of the more delicate approaches are also mentioned. All other relevant technical considerations including advantages/disadvantages of the various methods and the most common artifacts are also discussed.

Immunohistochemical expression of ionized calcium binding adapter molecule 1 in cutaneous histiocytic proliferative, neoplastic and inflammatory disorders of dogs and cats.

Ionized calcium-binding adapter molecule 1 (Iba1) has been used widely as a marker for microglial cells and, recently, was also recognized as a 'pan-macrophage marker', as it is expressed by all subpopulations of cells of the monocyte/macrophage lineage. To determine the specificity of Iba1 as an immunohistochemical marker for canine and feline histiocytic proliferative, neoplastic and inflammatory disorders of the skin, we evaluated its expression in two types of histiocytic tumours, two non-neoplastic histiocytic proliferative conditions, one case of granulomatous dermatitis and four non-histiocytic tumours. Cells of the monocyte/macrophage lineage in all cases of canine cutaneous histiocytoma (9/9), reactive histiocytosis (9/9), histiocytic sarcomas (5/5), feline progressive dendritic cell histiocytosis (3/3) and macrophages in cutaneous mycobacteriosis (7/7) showed strong cytoplasmic expression of Iba1. Neoplastic cells of melanomas (10/10), lymphomas (7/7), mast cell tumours (7/7) and plasmacytomas (4/4) did not express Iba1. Iba1 is therefore a useful marker of cells of the monocyte/macrophage lineage in canine and feline inflammatory, proliferative and neoplastic conditions and can be used to identify these cells in formalin-fixed, paraffin wax-embedded tissues. Iba1 is not able to differentiate between macrophages and dendritic antigen presenting cells and expression does not allow classification of these histiocytic disorders.

Factors affecting the estimated probabilistic acute dietary exposure to captan from apple consumption.

The effect of the number of pesticide residue values below the LOQ/LOD of analytical methods, the variability of residues in individual fruits, mass of fruit units and the number of bootstrap iterations was studied on the probabilistically estimated acute exposure of consumers. The 4720 daily apple consumption data and the results of 1239 apple sample analyses for captan residues, performed within the Hungarian monitoring programme between 2005 and 2011, were used in this study as model matrix. Up to about 95th percentile exposure (µg/(kg bw·day)), simply multiplying each residue in composite samples with each consumption value gave similar estimates to those obtained with the complex procedure taking also into account the mass of and residues in individual fruits. However, the exposure above the 95th percentile calculated with the complex procedure gradually increased with increasing percentile level compared to the simple procedure. Including the high number of non-detects reduced the estimated exposure, which was the highest when only the residues measured in treated fruits were taken into account. The number of bootstrap iterations between 100 and 10,000 did not significantly affect the calculated exposure. The 99.99th percentile exposure amounted to 17.9% of the acute reference dose of 300 µg/(kg bw·day) for women of childbearing age.

Uncertainty of the sample size reduction step in pesticide residue analysis of large-sized crops.

To estimate the uncertainty of the sample size reduction step, each unit in laboratory samples of papaya and cucumber was cut into four segments in longitudinal directions and two opposite segments were selected for further homogenisation while the other two were discarded. Jackfruit was cut into six segments in longitudinal directions, and all segments were kept for further analysis. To determine the pesticide residue concentrations in each segment, they were individually homogenised and analysed by chromatographic methods. One segment from each unit of the laboratory sample was drawn randomly to obtain 50 theoretical sub-samples with an MS Office Excel macro. The residue concentrations in a sub-sample were calculated from the weight of segments and the corresponding residue concentration. The coefficient of variation calculated from the residue concentrations of 50 sub-samples gave the relative uncertainty resulting from the sample size reduction step. The sample size reduction step, which is performed by selecting one longitudinal segment from each unit of the laboratory sample, resulted in relative uncertainties of 17% and 21% for field-treated jackfruits and cucumber, respectively, and 7% for post-harvest treated papaya. The results demonstrated that sample size reduction is an inevitable source of uncertainty in pesticide residue analysis of large-sized crops. The post-harvest treatment resulted in a lower variability because the dipping process leads to a more uniform residue concentration on the surface of the crops than does the foliar application of pesticides.

Estimation of the uncertainties of extraction and clean-up steps in pesticide residue analysis of plant commodities.

Extraction and clean-up constitute important steps in pesticide residue analysis. For the correct interpretation of analytical results, uncertainties of extraction and clean-up steps should be taken into account when the combined uncertainty of the analytical result is estimated. In the scope of this study, uncertainties of extraction and clean-up steps were investigated by spiking (14)C-labelled chlorpyrifos to analytical portions of tomato, orange, apple, green bean, cucumber, jackfruit, papaya and starfruit. After each step, replicate measurements were carried out with a liquid scintillation counter. Uncertainties in extraction and clean-up steps were estimated separately for every matrix and method combination by using within-laboratory reproducibility standard deviation and were characterised with the CV of recoveries. It was observed that the uncertainty of the ethyl acetate extraction step varied between 0.8% and 5.9%. The relative standard uncertainty of the clean-up step with dispersive SPE used in the method known as QuEChERS was estimated to be around 1.5% for tomato, apple and green beans. The highest variation of 4.8% was observed in cucumber. The uncertainty of the clean-up step with gel permeation chromatography ranged between 5.3% and 13.1%, and it was relatively higher than that obtained with the dispersive SPE method.

Exposure of consumers to morphine from poppy seeds in Hungary.

Poppy seed-containing foods are popular dishes in Hungary and some other Central European countries. The alkaloids of poppy are used in the production of medicines. Poppy seeds used as food may also contain considerable amounts of alkaloids, which raises the question of food safety. Morphine, codeine, thebaine and noscapine concentrations of poppy seed samples from the period 2001-2010 and consumption data from two Hungarian surveys, carried out in 2003 and 2009, were evaluated. Exposure calculations were made for morphine intake by both point estimate and probabilistic methods, and the uncertainty of the calculated values was estimated. The point estimate for the acute consumer exposure, calculated using the 97.5th percentiles of morphine concentration and of poppy seed consumption and taking into account the reduction of morphine content by processing, was 78.64 µg (kg bw)⁻¹ day⁻¹ for adults, and 116.90 µg (kg bw)⁻¹ day⁻¹ for children. Based on probabilistic estimations, the 97.5th and 99th percentile exposures ranged between 18.3-25.4 and 25.6-47.4 µg (kg bw)⁻¹ day⁻¹ for adults, and between 32.9 and 66.4 µg (kg bw)⁻¹ day⁻¹ for children, respectively. As a no observed effect level (NOEL) had not been established, the significance of exposure could not be assessed.

Exposure of consumers to deoxynivalenol from consumption of white bread in Hungary.

In view of the frequent occurrence of mycotoxins in cereals, a study was initiated to assess the exposure of the Hungarian adult population. Consumption data for 1360 individuals, based on a 3-day questionnaire, indicated that white bread accounted for the major intake of cereal-based products. Various cereal products were analysed for 16 mycotoxins by a LC/MS/MS multi-toxin method with LOD of 16 µg kg⁻¹ and LOQ of 50 µg kg⁻¹. Deoxynivalenol (DON) was most frequently detected, but no acetyl-deoxynivalenol was present in detectable concentrations. Consumer exposure was calculated with standard Monte Carlo probabilistic modelling and point estimates, taking into account bread consumption and DON contamination in independently taken wheat flour and wheat grain samples. Over 55% of cases the DON intake were below 15% of the provisional maximum tolerable daily intake (PMTDI) of 1 µg/(kg bw)/day. However, in 5-15% of cases, the intake from bread consumption alone exceeded the PMTDI. Wheat grain data led to the higher percentage. Intakes estimated from both data sets were at or below the acute reference dose (ARfD) of 8 µg/(kg bw)/day in 99.94-99.97% of cases.

Giant cell glioblastoma in the cerebrum of a Pembroke Welsh corgi.

A 6-year-old, neutered female Pembroke Welsh corgi was presented with a 1-month history of ataxia and panting. The clinical signs progressed until the dog became anorexic, obtunded and exhibited circling to the left. At necropsy examination, a mass was detected in the left forebrain, impinging on the cribriform plate. Microscopically, the mass was composed of sheets of round to pleomorphic neoplastic cells with vacuolated cytoplasm. Nuclear atypia, anisocytosis and anisokaryosis were common. Numerous bizarre, multinucleated giant cells containing 60 or more nuclei and giant mononuclear cells were present. The matrix contained abundant reticulin. Immunohistochemistry revealed the neoplastic cells uniformly to express vimentin, and a small number of neoplastic cells expressed glial fibrillary acid protein. A diagnosis of giant cell glioblastoma was made. Although well recognized in man, this tumour has been documented rarely in the veterinary literature.

Enteric dysganglionosis resembling intestinal neuronal dysplasia in a foal with bacterial colitis.

A 5-day-old quarter horse colt with a history of hypothermia, agonal breathing, and diarrhea was euthanized. At necropsy, numerous slightly raised, discrete, closely approximated submucosal nodules were observed in the colon and small intestine. Histologically, these nodules were composed of expanded submucosal mesenchyme that contained numerous neurons either individually or in ganglia. Thirty-two percent of these ganglia included 8 or more neurons, in contrast to 6% in an age-matched foal. Some nodules had necrosuppurative inflammation with vasculitis, thrombosis, and bacterial colonization. A few heterotopic neurons were randomly distributed in the mucosa and the muscularis mucosa. Histologic changes were most consistent with intestinal neuronal dysplasia, a disease of the submucosal plexus described in humans.

Immunohistochemical evaluation of mx protein expression in canine encephalitides.

Mx proteins are a group of interferon-induced GTPases whose expression has been demonstrated in a number of human viral infections and in some idiopathic inflammatory diseases. In this study, the expression of Mx protein was evaluated in known viral, nonviral, and idiopathic encephalitides in the dog via immunohistochemistry using an antibody against human MxA. All 12 cases of confirmed viral encephalitis, including 7 cases of canine distemper, 4 cases of canine herpesvirus, and 1 case of rabies, were Mx positive. In canine distemper cases, staining was particularly strong and a variety of cell types were positive, including astrocytes, macrophages/microglia, and neurons. Immunoreactivity for Mx protein was evident in a few cases of nonviral infectious encephalitis, including neosporosis (1/1), Chagas disease (2/3), aspergillosis (1/2), and encephalitozoonosis (1/1). Consistent staining was observed in most cases of idiopathic encephalitis, including granulomatous meningoencephalomyelitis (7/7), necrotizing meningoencephalitis of pug dogs (6/7), and necrotizing encephalitis of the Yorkshire Terrier (3/3) and Maltese (1/1) breeds. Mx staining was negative in 5 normal dog brains; 3 cases of cryptococcosis; and single cases of blastomycosis, protothecosis, and bacterial meningitis.

Application of TLC for confirmation and screening of pesticide residues in fruits, vegetables, and cereal grains: Part 2. Repeatability and reproducibility of Rf and MDQ values.

This paper illustrates the effect of major factors influencing the reproducibility of thin layer chromatography (TLC) separation and detection under largely differing environmental and laboratory conditions. The optimum conditions for reproducibility and detection sensitivity was obtained on 20 x 20 cm layer in the retention factor (Rf) range of 0.2-0.7 by applying the sample in spots of 3-4 mm diameter at 2 cm from the edge of the plate. The reproducibility rapidly decreased below Rf = 0.2. Above Rf = 0.2 the within-laboratory reproducibility of 219 pesticides obtained in ethyl acetate silica gel elution system by several laboratories was typically below 10%. The among-laboratories reproducibility of the average retention factors was generally below 12%. The minimum detectable quantities (MDQ) of 219 pesticide residues were determined with nine detection methods. The MDQ values largely varied depending on the mode of detection. Bioassay methods enabled the detection down to 0.1-10 ng, while 20-100 ng could be achieved with the chemical reagents. Higher MDQ values are also reported in order to assist the identification of compounds potentially present. The between-laboratories reproducibility of MDQ values was typically 1-5 MDQmin.

A cost-effective screening method for pesticide residue analysis in fruits, vegetables, and cereal grains.

This paper reports the results of studies performed to investigate the potential of applying thin layer chromatography (TLC) detection in combination with selected extraction and cleanup methods, for providing an alternative cost-effective analytical procedure for screening and confirmation of pesticide residues in plant commodities. The extraction was carried out with ethyl acetate and an on-line extraction method applying an acetone-dichloromethane mixture. The extracts were cleaned up with SX-3 gel, an adsorbent mixture of active carbon, magnesia, and diatomaceous earth, and on silica micro cartridges. The Rf values of 118 pesticides were tested in eleven elution systems with UV, and eight biotest methods and chemical detection reagents. Cabbage, green peas, orange, and tomatoes were selected as representative sample matrices for fruits and vegetables, while maize, rice, and wheat represented cereal grains. As an internal quality control measure, marker compounds were applied on each plate to verify the proper elution and detection conditions. The Rf values varied in the different elution systems. The best separation (widest Rf range) was achieved with silica gel (SG)--ethyl acetate (0.05-0.7), SG--benzene, (0.02-0.7) and reverse phase RP-18 F-254S layer with acetone: methanol: water/30:30:30 (v/v) (0.1-0.8). The relative standard deviation of Rf values (CV(Rf)) within laboratory reproducibility was generally less than 20%, except below 0.2 Rf, where the CVRf rapidly increased with decreasing Rf values. The fungi spore inhibition, chloroplast inhibition, and enzyme inhibition were found most suitable for detection of pesticides primarily for confirming their identity or screening for known substances. Their use for determination of pesticide residues in samples of unknown origin is not recommended.

A heritable keratinization defect of the superficial epidermis in norfolk terriers.

Although well-characterized in man, abnormal cornification secondary to heritable superficial keratin defects is rarely reported in animals. This report describes a mild cornification defect in seven related Norfolk terrier dogs. Lesions were present at birth and pedigree analysis suggested an autosomal recessive mode of inheritance. The affected dogs had hyperpigmented skin with scaling following mild trauma. The lesions were generalized but most prominent in the glabrous skin of the axillary and inguinal regions-areas where the epidermis is not protected by hair and is subject to frequent trauma. The most striking histological change was vacuolation in the upper epidermis, which often resulted in epidermolysis and blister formation. All of the affected dogs showed similar gross and histological changes. Ultrastructural changes included abnormal keratin filament clumping, prominent clear spaces in the cytoplasm of suprabasal keratinocytes, and abnormal keratohyaline granules. Immunohistochemical labelling for keratin 10 demonstrated a lack of expression in the superficial epidermis of affected dogs. All of the morphological changes noted in the Norfolk terriers were consistent with a mild form of a heritable defect in superficial keratin synthesis.

Structural and biochemical characterization of neuronal calretinin domain I-II (residues 1-100). Comparison to homologous calbindin D28k domain I-II (residues 1-93).

This study characterizes the calcium-bound CR I-II domain (residues 1-100) of rat calretinin (CR). CR, with six EF-hand motifs, is believed to function as a neuronal intracellular calcium-buffer and/or calcium-sensor. The secondary structure of CR I-II, defined by standard NMR methods on 13C,15N-labeled protein, contains four helices and two short interacting segments of extended structure between the calcium-binding loops. The linker between the two helix-loop-helix, EF-hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I-II) confirms that the linker of CR I-II is solvent-exposed and that other potential sites are protected by regular secondary structure. 45Ca-overlay of glutathione S-transferase (GST)-CR(1-60) and GST-CR(61-100) fusion proteins confirm that both EF-hands of CR I-II have intrinsic calcium-binding properties. The primary sequence and NMR chemical shifts, including calcium-sensitive glycine residues, also suggest that both EF-hand loops of CR I-II bind calcium. NMR relaxation, analytical ultracentrifugation, chemical cross-linking and NMR translation diffusion measurements indicate that CR I-II exists as a monomer. Calb I-II (the homologous domain of calbindin D28k) has the same EF-hand secondary structures as CR I-II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur. J. Biochem. 262, 933-938]. In contrast, Calb I-II binds one calcium cation per monomeric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I-II and Calb I-II probably have distinct tertiary structure features that suggest different cellular functions for the full-length proteins.

Calcium binding of transglutaminases: a 43Ca NMR study combined with surface polarity analysis.

Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.

Polyethylene glycol enhanced refolding of the recombinant human tissue transglutaminase.

Tissue transglutaminase forms cross-links between lysine and glutamine side-chains of polypeptide chains in a Ca2+-dependent reaction; its structural basis is still not clarified. In this study, we demonstrate that the refolding of the human recombinant enzyme molecule to its catalytically active form from inclusion bodies needs the presence of a helper material with higher molecular mass, but only in the initiation phase. Ca2+ and nucleotides are ascribed as affector molecules also in the early phase of structural reconstitution. Two optimal concentrations of polyethylene glycol and a relatively long time scale for the evolution of the final structure were identified. The optimized refolding procedure is reported.

Analysis of DDT residues in soil by ELISA: an international interlaboratory study.

An international interlaboratory study was conducted to determine the performance of a group of laboratories from developing and developed countries. The study used a commercial microwell ELISA on unknown samples spiked with different levels of DDT. The study design was based on Youden pairs and balanced replicates. Two soils, differing in particle size distributions, organic matter content, and cation-exchange capacities and thought to be DDT-free, were spiked at 5 DDT levels between 0.025 and 2 mg/kg. Nineteen laboratories in 17 countries took part in the collaborative trial; of these, the majority were modestly equipped laboratories in developing countries. Samples were analyzed without filtration or cleanup and using standards of pure DDT in methanol. Data were analyzed for repeatability and reproducibility, and average recoveries at the spike levels were calculated. Mean real recoveries for both soils were similar (103% for soil A and 100% for soil B), with values between 0.1 and 2 mg/kg DDT. Precision estimates were best in the linear working range of the assay (0.1-0.5 mg/kg DDT), with reproducibility relative standard deviations (RSDR) typically averaging about 38 and 46% near the upper and lower detection limits, respectively. Corresponding repeatability relative standard deviation (RSDr) values were 20-36% and 36-57%. Thus, even though much of the trial was performed under developing country conditions, performance statistics were similar to other reported results obtained with ELISAs on small molecules of agricultural importance, such as mycotoxins and pesticide and antibiotic residues.

Within and between field variability of residue data and sampling implications.

Information on the variability of residues in individual fruits and vegetables is required to estimate the acute dietary exposure to pesticides. The distribution of residues in apples, kiwi fruits, potatoes and butter beans was studied in field experiments representing commercial farming practice. No correlation was found between the residue concentration or surface residue and the mass of apples. The relative frequency distributions of residues in crop units were continuous and skew positive. The log-normal transformation did not result in a normal distribution in any of the trials. Consequently, 299, 120 and 59 random samples should be analysed to estimate 99th, 97.5th and 95th percentile of the residues at 95% confidence level. The distribution of residues is not significantly influenced by the mean residue, pre-harvest interval, chemical and physical properties of the active ingredient, formulation of pesticide or, on top fruit, the foliar application method. However, the residue distribution is likely to be influenced by the size, shape and density of the plants, and mode of application of pesticides. The variability factor should be defined as the ratio of the highest value at a specified percentile of residues occurring in unit crops and the population mean. Generic variability factors may be determined for various groups of commodities. Variability factors of 5 and 9 are recommended for medium size fruits, and potato following granular application of pesticides, respectively.