Abstract: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable.
Abstract: In nature the aerial trace of pheromone used by male moths to find a female appears as a train of discontinuous pulses separated by gaps among a complex odorant background constituted of plant volatiles. We investigated the effect of such background odor on behavior and coding of temporal parameters of pheromone pulse trains in the pheromone olfactory receptor neurons of Spodoptera littoralis. Effects of linalool background were tested by measuring walking behavior towards a source of pheromone. While velocity and orientation index did drop when linalool was turned on, both parameters recovered back to pre-background values after 40 s with linalool still present. Photo-ionization detector was used to characterize pulse delivery by our stimulator. The photo-ionization detector signal reached 71% of maximum amplitude at 50 ms pulses and followed the stimulus period at repetition rates up to 10 pulses/s. However, at high pulse rates the concentration of the odorant did not return to base level during inter-pulse intervals. Linalool decreased the intensity and shortened the response of receptor neurons to pulses. High contrast (>10 dB) in firing rate between pulses and inter-pulse intervals was observed for 1 and 4 pulses/s, both with and without background. Significantly more neurons followed the 4 pulses/s pattern when delivered over linalool; at the same time the information content was preserved almost to the control values. Rapid recovery of behavior shows that change of perceived intensity is more important than absolute stimulus intensity. While decreasing the response intensity, background odor preserved the temporal parameters of the specific signal.
Abstract: Object. The first aim of this study was to diagnose more aggressive and potentially recurrent meningiomas using an in vitro embryonic chick heart invasiveness assay in which lysosomal enzyme cathepsin B was used as the invasiveness marker. The second aim was to confirm if cathepsin B and/or cathepsin L and their endogenous inhibitors were also prognostic parameters in the clinical study of 119 patients with meningioma. Methods. Primary meningioma cultured spheroids were “confronted†with embryonic chick heart spheroids in vitro, and cathepsin B was used as molecular marker to immunolabel the invasive tumor cells. In vitro invasion assays of the malignant meningioma cells were used to assess the invasive potential related to the cysteine cathepsins. As to the second aim, the possible association of cathepsin B along with selected molecular markers, cathepsin L, and endogenous cysteine protease inhibitors (stefins A and B and cystatin C) with meningioma malignancy was determined using enzyme-linked immunosorbent assays in tumor homogenates. Univariate and multivariate analyses were used to compare these parameters with established biological markers of meningioma recurrence in 119 patients with meningiomas. Results. The more invasive tumors, which characteristically overgrew the normal tissue, were identified even within a group of histologically benign meningiomas. More intensive staining of cathepsin B in these tumors was not only found at the tumor front, but also in the invading pseudopodia of a single migrating tumor cells. Matrigel invasion of malignant meningioma cells was significantly altered by modulating cathepsin B activity and by stefin B silencing. In the clinical samples of meningioma, the levels of cathepsins B and L, stefin B, and cystatin C were highest in the tumors of higher histological grades, whereas stefin A and progesterone receptor were the only markers that were significantly increased and decreased, respectively, in WHO Grade III lesions. With respect to the prognosis of relapse, cathepsin L (p = 0.035), stefin B (p = 0.007), cystatin C (p = 0.008), and progesterone receptor (p = 0.049) levels were significant, whereas cathepsin B was not a prognosticator. As expected, WHO grade, age, and Simpson grade (complete tumor resection) were prognostic, with Simpson grade only relevant in the short term (up to 90 months) but not in longer-term follow-up. Of note, the impact of all these parameters was lost in multivariate analysis, due to overwhelming prognostic impact of stefin B (p = 0.039). Conclusions. The data indicate that the cysteine cathepsins and their inhibitors are involved in a process related to early meningioma recurrence, regardless of their histological classification. Of note, the known tumor invasiveness marker cathepsin B, measured in whole-tumor homogenates, was not prognostic, in contrast to its endogenous inhibitor stefin B, which was highly significant and the only independent prognostic factor to predict meningioma relapse in multivariate analysis and reported herein for the first time. Stefin B inhibition of local invasion was confirmed by in vitro invasion assay, although its other functions cannot be excluded. (DOI: 10.3171/2009.7.JNS081729)
Abstract: A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.
Abstract: In order to comply with the European Union regulatory threshold for the adventitious presence of genetically modified organisms (GMOs) in food and feed, it is important to trace GMOs from the field. Appropriate sampling methods are needed to accurately predict the presence of GMOs at the field level. A 2-year field experiment with two maize varieties differing in kernel colour was conducted in Slovenia. Based on the results of data mining analyses and modelling, it was concluded that spatial relations between the donor and receptor field were the most important factors influencing the distribution of outcrossing rate (OCR) in the field. The approach for estimation fitting function parameters in the receptor (non-GM) field at two distances from the donor (GM) field (10 and 25 m) for estimation of the OCR (GMO content) in the whole receptor field was developed. Different sampling schemes were tested; a systematic random scheme in rows was proposed to be applied for sampling at the two distances for the estimation of fitting function parameters for determination of OCR. The sampling approach had already been validated with some other OCR data and was practically applied in the 2009 harvest in Poland. The developed approach can be used for determination of the GMO presence at the field level and for making appropriate labelling decisions. The importance of this approach lies in its possibility to also address other threshold levels beside the currently prescribed labelling threshold of 0.9% for food and feed.
Abstract: This article presents an approach to microarray data analysis using discretised expression values in combination with a methodology of closed item set mining for class labeled data (RelSets). A statistical 2 x 2 factorial design analysis was run in parallel. The approach was validated on two independent sets of two-color microarray experiments using potato plants. Our results demonstrate that the two different analytical procedures, applied on the same data, are adequate for solving two different biological questions being asked. Statistical analysis is appropriate if an overview of the consequences of treatments and their interaction terms on the studied system is needed. If, on the other hand, a list of genes whose expression (upregulation or downregulation) differentiates between classes of data is required, the use of the RelSets algorithm is preferred. The used algorithms are freely available upon request to the authors.
Abstract: OBJECT: The first aim of this study was to diagnose more aggressive and potentially recurrent meningiomas using an in vitro embryonic chick heart invasiveness assay in which lysosomal enzyme cathepsin B was used as the invasiveness marker. The second aim was to confirm if cathepsin B and/or cathepsin L and their endogenous inhibitors were also prognostic parameters in the clinical study of 119 patients with meningioma. METHODS: Primary meningioma cultured spheroids were "confronted" with embryonic chick heart spheroids in vitro, and cathepsin B was used as molecular marker to immunolabel the invasive tumor cells. In vitro invasion assays of the malignant meningioma cells were used to assess the invasive potential related to the cysteine cathepsins. As to the second aim, the possible association of cathepsin B along with selected molecular markers, cathepsin L, and endogenous cysteine protease inhibitors (stefins A and B and cystatin C) with meningioma malignancy was determined using enzyme-linked immunosorbent assays in tumor homogenates. Univariate and multivariate analyses were used to compare these parameters with established biological markers of meningioma recurrence in 119 patients with meningiomas. RESULTS: The more invasive tumors, which characteristically overgrew the normal tissue, were identified even within a group of histologically benign meningiomas. More intensive staining of cathepsin B in these tumors was not only found at the tumor front, but also in the invading pseudopodia of a single migrating tumor cells. Matrigel invasion of malignant meningioma cells was significantly altered by modulating cathepsin B activity and by stefin B silencing. In the clinical samples of meningioma, the levels of cathepsins B and L, stefin B, and cystatin C were highest in the tumors of higher histological grades, whereas stefin A and progesterone receptor were the only markers that were significantly increased and decreased, respectively, in WHO Grade III lesions. With respect to the prognosis of relapse, cathepsin L (p = 0.035), stefin B (p = 0.007), cystatin C (p = 0.008), and progesterone receptor (p = 0.049) levels were significant, whereas cathepsin B was not a prognosticator. As expected, WHO grade, age, and Simpson grade (complete tumor resection) were prognostic, with Simpson grade only relevant in the short term (up to 90 months) but not in longer-term follow-up. Of note, the impact of all these parameters was lost in multivariate analysis, due to overwhelming prognostic impact of stefin B (p = 0.039). CONCLUSIONS: The data indicate that the cysteine cathepsins and their inhibitors are involved in a process related to early meningioma recurrence, regardless of their histological classification. Of note, the known tumor invasiveness marker cathepsin B, measured in whole-tumor homogenates, was not prognostic, in contrast to its endogenous inhibitor stefin B, which was highly significant and the only independent prognostic factor to predict meningioma relapse in multivariate analysis and reported herein for the first time. Stefin B inhibition of local invasion was confirmed by in vitro invasion assay, although its other functions cannot be excluded.
Abstract: A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.
Abstract: In order to comply with the European Union regulatory threshold for the adventitious presence of genetically modified organisms (GMOs) in food and feed, it is important to trace GMOs from the field. Appropriate sampling methods are needed to accurately predict the presence of GMOs at the field level. A 2-year field experiment with two maize varieties differing in kernel colour was conducted in Slovenia. Based on the results of data mining analyses and modelling, it was concluded that spatial relations between the donor and receptor field were the most important factors influencing the distribution of outcrossing rate (OCR) in the field. The approach for estimation fitting function parameters in the receptor (non-GM) field at two distances from the donor (GM) field (10 and 25 m) for estimation of the OCR (GMO content) in the whole receptor field was developed. Different sampling schemes were tested; a systematic random scheme in rows was proposed to be applied for sampling at the two distances for the estimation of fitting function parameters for determination of OCR. The sampling approach had already been validated with some other OCR data and was practically applied in the 2009 harvest in Poland. The developed approach can be used for determination of the GMO presence at the field level and for making appropriate labelling decisions. The importance of this approach lies in its possibility to also address other threshold levels beside the currently prescribed labelling threshold of 0.9% for food and feed.
Abstract: This article presents an approach to microarray data analysis using discretised expression values in combination with a methodology of closed itemset mining for class labeled data (RelSets). A statistical 2 x 2 factorial design analysis was run in parallel. The approach was validated on two independent sets of two-color microarray experiments using potato plants. Our results demonstrate that the two different analytical procedures, applied on the same data, are adequate for solving two different biological questions being asked. Statistical analysis is appropriate if an overview of the consequences of treatments and their interaction terms on the studied system is needed. If, on the other hand, a list of genes whose expression (upregulation or downregulation) differentiates between classes of data is required, the use of the RelSets algorithm is preferred. The used algorithms are freely available upon request to the authors.
Abstract: BACKGROUND: Phytoplasmas are bacteria without cell walls from the class Mollicutes. They are obligate intracellular plant pathogens which cause diseases in hundreds of economically important plants including the grapevine (Vitis vinifera). Knowledge of their biology and the mechanisms of their interactions with hosts is largely unknown because they are uncultivable and experimentally inaccessible in their hosts. We detail here the global transcriptional profiling in grapevine responses to phytoplasmas. The gene expression patterns were followed in leaf midribs of grapevine cv. 'Chardonnay' naturally infected with a phytoplasma from the stolbur group 16SrXII-A, which is associated with the grapevine yellows disease 'Bois noir'. RESULTS: We established an on field experimental system in a productive vineyard that allowed application of molecular tools in a plant natural environment. Global transcription profiles of infected samples were compared with the healthy ones using microarray datasets and metabolic pathway analysis software (MapMan). The two-year-long experiment revealed that plant genes involved in primary and secondary metabolic pathways were changed in response to infection and that these changes might support phytoplasma nutrition. A hypothesis that phytoplasmas interact with the plant carbohydrate metabolism was proven and some possibilities how the products of this pathway might be utilized by phytoplasmas are discussed. In addition, several photosynthetic genes were largely down-regulated in infected plants, whereas defense genes from the metabolic pathway leading to formation of flavonoids and some PR proteins were significantly induced. Few other genes involved in defense-signaling were differentially expressed in healthy and infected plants. A set of 17 selected genes from several differentially expressed pathways was additionally analyzed with quantitative real-time PCR and confirmed to be suitable for a reliable classification of infected plants and for the characterization of susceptibility features in the field conditions. CONCLUSION: This study revealed some fundamental aspects of grapevine interactions with the stolbur 'Bois noir' phytoplasma in particular and some plant interactions with phytoplasmas in general. In addition, the results of the study will likely have an impact on grape improvement by yielding marker genes that can be used in new diagnostic assays for phytoplasmas or by identifying candidate genes that contribute to the improved properties of grape.
Abstract: Background: Phytoplasmas are bacteria without cell walls from the class Mollicutes. They are obligate intracellular plant pathogens which cause diseases in hundreds of economically important plants including the grapevine (Vitis vinifera). Knowledge of their biology and the mechanisms of their interactions with hosts is largely unknown because they are uncultivable and experimentally inaccessible in their hosts. We detail here the global transcriptional profiling in grapevine responses to phytoplasmas. The gene expression patterns were followed in leaf midribs of grapevine cv. ‘Chardonnay’ naturally infected with a phytoplasma from the stolbur group 16SrXII-A, which is associated with the grapevine yellows disease ‘Bois noir’. Results: We established an on field experimental system in a productive vineyard that allowed application of molecular tools in a plant natural environment. Global transcription profiles of infected samples were compared with the healthy ones using microarray datasets and metabolic pathway analysis software (MapMan). The two-year-long experiment revealed that plant genes involved in primary and secondary metabolic pathways were changed in response to infection and that these changes might support phytoplasma nutrition. A hypothesis that phytoplasmas interact with the plant carbohydrate metabolism was proven and some possibilities how the products of this pathway might be utilized by phytoplasmas are discussed. In addition, several photosynthetic genes were largely down-regulated in infected plants, whereas defense genes from the metabolic pathway leading to formation of flavonoids and some PR proteins were significantly induced. Few other genes involved in defense-signaling were differentially expressed in healthy and infected plants. A set of 17 selected genes from several differentially expressed pathways was additionally analyzed with quantitative real-time PCR and confirmed to be suitable for a reliable classification of infected plants and for the characterization of susceptibility features in the field conditions. Conclusion: This study revealed some fundamental aspects of grapevine interactions with the stolbur ‘Bois noir’ phytoplasma in particular and some plant interactions with phytoplasmas in general. In addition, the results of the study will likely have an impact on grape improvement by yielding marker genes that can be used in new diagnostic assays for phytoplasmas or by identifying candidate genes that contribute to the improved properties of grape.
Abstract: Postembryonic development of nine species of Niphargus (Crustacea: Amphipoda) was studied, with spine development and shifts in allometric growth being combined in developmental sequences that were compared across species. The developmental sequences show high diversity with respect to the position of individual events in the sequence, as well as a high frequency of events being inapplicable in some species. Within Niphargus, the highest degree of independence between events occurs mainly in early mid-aged instars, where the shifts in position are both the largest in magnitude and also the most frequent in occurrence. Constructive troglomorphic features of subterranean species were inferred to develop more because of accelerated growth rather than a delayed offset of growth. Shifts in both relative timing and growth rate appeared to have played a role in the evolution of sexually dimorphic elongated appendages. Growth patterns differed greatly between species for individual sexually dimorphic and troglomorphic traits, hinting at their possible independent origin. The independence between developmental events that was generally apparent might indicate the existence of an important genetic basis for the extreme intrageneric morphological variation arising from numerous highly variable body parts being combined in a ‘mosaic’ manner. Moreover, we suggest that the overall diversity of the genus might actually be underestimated in light of possible convergent features accompanied by ‘cryptic’ speciation. We conclude that both sequential and growth heterochrony appear to have played a key role in the evolution of Niphargus, the most diverse genus of freshwater amphipods. A preliminary list of heterochronic characters is provided.
Abstract: Analysis of ecological dates of the arboricolous nephilid spicier Clitaetra irenae, Kuntner 2006, endemic to Maputaland forests, South Africa, indicates the species’ dependence on this highly threatened habitat. We tested C irenae habitat dependence via GIS analysis by plotting the known distribution against southern African ecoregions. In the southern part of its range, C. irenae inhabits almost exclusively one ecoregion, the Maputaland coastal plain forests; but further north, in tropical southern Africa, it continues inland into Malawi’s woodlands. We test and refute the hypotheses that C. irenae inhabits exclusively mature trees, trees of a particular species, trees with a smooth bark. tree habitats at certain height above ground, and only closed canopy forest stands. The ecological niche of C. irenae is flexible as long as suitable trees under at least partially closed canopy are available. We quantify the C irenae ontogenetic web changes from orb to ladder and the simultaneous hub displacement towards the top frame. Such web allometry allows the web to increase vertically but not horizontally, which enables the spider to remain on the same tree throughout its development and thus the ladder web architecture is an adaptation to an arboricolous life style. Adult hub displacement. common in spiders with vertical webs, is explained by gravity. Clitaetra irenae web orientation on trees correlates with forest closure, and might indicate the Maputaland forest quality. We argue for utilization of the ecology of arboricolous nephilid orb-weaving spiders (Clitaetra and Herennia) in systematic conservation assessments in the Old World tropics.
Abstract: Due to the great variety of preprocessing tools in two-channel expression microarray data analysis it is difficult to choose the most appropriate one for a given experimental setup. In our study, two independent two-channel inhouse microarray experiments as well as a publicly available dataset were used to investigate the influence of the selection of preprocessing methods (background correction, normalization, and duplicate spots correlation calculation) on the discovery of differentially expressed genes. Here we are showing that both the list of differentially expressed genes and the expression values of selected genes depend significantly on the preprocessing approach applied. The choice of normalization method to be used had the highest impact on the results. We propose a simple but efficient approach to increase the reliability of obtained results, where two normalization methods which are theoretically distinct from one another are used on the same dataset. Then the intersection of results, that is, the lists of differentially expressed genes, is used in order to get a more accurate estimation of the genes that were de facto differentially expressed.
Abstract: Postembryonic development of nine species of Niphargus (Crustacea: Amphipoda) was studied, with spine development and shifts in allometric growth being combined in developmental sequences that were compared across species. The developmental sequences show high diversity with respect to the position of individual events in the sequence, as well as a high frequency of events being inapplicable in some species. Within Niphargus, the highest degree of independence between events occurs mainly in early mid-aged instars, where the shifts in position are both the largest in magnitude and also the most frequent in occurrence. Constructive troglomorphic features of subterranean species were inferred to develop more because of accelerated growth rather than a delayed offset of growth. Shifts in both relative timing and growth rate appeared to have played a role in the evolution of sexually dimorphic elongated appendages. Growth patterns differed greatly between species for individual sexually dimorphic and troglomorphic traits, hinting at their possible independent origin. The independence between developmental events that was generally apparent might indicate the existence of an important genetic basis for the extreme intrageneric morphological variation arising from numerous highly variable body parts being combined in a ‘mosaic’ manner. Moreover, we suggest that the overall diversity of the genus might actually be underestimated in light of possible convergent features accompanied by ‘cryptic’ speciation. We conclude that both sequential and growth heterochrony appear to have played a key role in the evolution of Niphargus, the most diverse genus of freshwater amphipods. A preliminary list of heterochronic characters is provided. (c) 2008 Gesellschaft fur Biologische Systematik. Published by Elsevier GmbH. All rights reserved.
Abstract: Due to the great variety of preprocessing tools in two-channel expression microarray data analysis it is difficult to choose the most appropriate one for a given experimental setup. In our study, two independent two-channel inhouse microarray experiments as well as a publicly available dataset were used to investigate the influence of the selection of preprocessing methods (background correction, normalization, and duplicate spots correlation calculation) on the discovery of differentially expressed genes. Here we are showing that both the list of differentially expressed genes and the expression values of selected genes depend significantly on the preprocessing approach applied. The choice of normalization method to be used had the highest impact on the results. We propose a simple but efficient approach to increase the reliability of obtained results, where two normalization methods which are theoretically distinct from one another are used on the same dataset. Then the intersection of results, that is, the lists of differentially expressed genes, is used in order to get a more accurate estimation of the genes that were de facto differentially expressed.
Abstract: Quantitative characterization of nucleic acids is becoming a frequently used method in routine analysis of biological samples, one use being the detection of genetically modified organisms (GMOs). Measurement uncertainty is an important factor to be considered in these analyses, especially where precise thresholds are set in regulations. Intermediate precision, defined as a measure between repeatability and reproducibility, is a parameter describing the real situation in laboratories dealing with quantitative aspects of molecular biology methods. In this paper, we describe the top-down approach to calculating measurement uncertainty, using intermediate precision, in routine GMO testing of food and feed samples. We illustrate its practicability in defining compliance of results with regulations. The method described is also applicable to other molecular methods for a variety of laboratory diagnostics where quantitative characterization of nucleic acids is needed.
Abstract: The concept of personality implies individual differences in behavior and physiology that show some degree of repeatability/consistency over time and across contexts. Most studies of animal personality, particularly studies of individuals’ variation in physiological mechanisms, have been conducted on selected individuals in controlled conditions. We attempted to detect consistent behaviors as well as physiological patterns in greylag ganders (Anser anser) from a free-roaming flock living in semi-natural conditions. We tested 10 individuals repeatedly, in a handling trial, resembling tests for characterization of “temperaments†in captive animals. We recorded the behavior of the same 10 individuals during four situations in the socially intact flock: (1) a “low density feeding conditionâ€, (2) a “high density feeding conditionâ€, (3) a “low density post-feeding situation†and (4) while the geese rested. We collected fecal samples for determination of excreted immuno-reactive corticosterone (BM) and testosterone metabolites (TM) after handling trials, as well as the “low density feeding†and the “high density feeding†conditions. BM levels were very highly consistent over the repeats of handling trials, and the “low density feeding condition†and tended to be consistent over the first two repeats of the “high density feeding conditionâ€. Also, BM responses tended to be consistent across contexts. Despite seasonal variation, there tended to be inter-test consistency of TM, which pointed to some individual differences in TM as well. Aggressiveness turned out to be a highly repeatable trait, which was consistent across social situations, and tended to correlate with an individual’s resistance during handling trials. Also, “proximity to the female partner†and “sociability†- the average number of neighboring geese in a close distance while resting - were consistent. We conclude that aggressiveness, “affiliative tendencies†and levels of excreted corticosterone and testosterone metabolites may be crucial factors of personality in geese. (c) 2006 Elsevier Inc. All rights reserved.
Abstract: Quantitative characterization of nucleic acids is becoming a frequently used method in routine analysis of biological samples, one use being the detection of genetically modified organisms (GMOs). Measurement uncertainty is an important factor to be considered in these analyses, especially where precise thresholds are set in regulations. Intermediate precision, defined as a measure between repeatability and reproducibility, is a parameter describing the real situation in laboratories dealing with quantitative aspects of molecular biology methods. In this paper, we describe the top-down approach to calculating measurement uncertainty, using intermediate precision, in routine GMO testing of food and feed samples. We illustrate its practicability in defining compliance of results with regulations. The method described is also applicable to other molecular methods for a variety of laboratory diagnostics where quantitative characterization of nucleic acids is needed.
Abstract: The concept of personality implies individual differences in behavior and physiology that show some degree of repeatability/consistency over time and across contexts. Most studies of animal personality, particularly studies of individuals' variation in physiological mechanisms, have been conducted on selected individuals in controlled conditions. We attempted to detect consistent behaviors as well as physiological patterns in greylag ganders (Anser anser) from a free-roaming flock living in semi-natural conditions. We tested 10 individuals repeatedly, in a handling trial, resembling tests for characterization of "temperaments" in captive animals. We recorded the behavior of the same 10 individuals during four situations in the socially intact flock: (1) a "low density feeding condition", (2) a "high density feeding condition", (3) a "low density post-feeding situation" and (4) while the geese rested. We collected fecal samples for determination of excreted immuno-reactive corticosterone (BM) and testosterone metabolites (TM) after handling trials, as well as the "low density feeding" and the "high density feeding" conditions. BM levels were very highly consistent over the repeats of handling trials, and the "low density feeding condition" and tended to be consistent over the first two repeats of the "high density feeding condition". Also, BM responses tended to be consistent across contexts. Despite seasonal variation, there tended to be inter-test consistency of TM, which pointed to some individual differences in TM as well. Aggressiveness turned out to be a highly repeatable trait, which was consistent across social situations, and tended to correlate with an individual's resistance during handling trials. Also, "proximity to the female partner" and "sociability" - the average number of neighboring geese in a close distance while resting - were consistent. We conclude that aggressiveness, "affiliative tendencies" and levels of excreted corticosterone and testosterone metabolites may be crucial factors of personality in geese.
Abstract: Quantitative characterization of nucleic acids is becoming a frequently used method in routine analysis of biological samples, one use being the detection of genetically modified organisms (GMOs). Measurement uncertainty is an important factor to be considered in these analyses, especially where precise thresholds are set in regulations. Intermediate precision, defined as a measure between repeatability and reproducibility, is a parameter describing the real situation in laboratories dealing with quantitative aspects of molecular biology methods. In this paper, we describe the top-down approach to calculating measurement uncertainty, using intermediate precision, in routine GMO testing of food and feed samples. We illustrate its practicability in defining compliance of results with regulations. The method described is also applicable to other molecular methods for a variety of laboratory diagnostics where quantitative characterization of nucleic acids is needed.
Abstract: A series of sampling sites in 6 caves was established in order to assess the importance of the epikarst as a habitat of high diversity of meiofauna. Special attention was given to the stygobiotic species of copepods (Crustacea), which was the most abundant group of animals in water percolating from the epikarst. In total, 37 species of Copepoda were identified from nearly 4000 individuals. To understand epikarst as a habitat, 14 structural and physico-chemical factors were analysed and compared for two types of habitats, i.e., trickles and pools filled with percolation water. Results of canonical correspondence analysis indicated that the significant factors influencing faunal composition in trickles were the thickness of the cave ceiling and the concentrations of sodium, nitrate, and potassium ions, while in pools correlations between individual species and pH, temperature, volume, conductivity and thickness of the cave ceiling were statistically significant. Aquatic copepod assemblages in epikarst encompass species of different taxonomic affinities, living in severe environmental conditions. The pattern of the system reflects both high taxonomic diversity and environmental severity.
Abstract: We present a simple statistical method for detection and identification of firing rate changes in spontaneously active neurons. Spontaneously active neurons (such as olfactory neurons) can be, in response to stimulation, either excited or suppressed and thus increase or decrease the spike firing rate. The described method is based on the detection of changes in slope of cumulative spike time distribution and efficiently detects excitations and suppressions. Using the simulated spike trains we examined the method’s power in relation to response strength and duration.
Abstract: We present a simple statistical method for detection and identification of firing rate changes in spontaneously active neurons. Spontaneously active neurons (such as olfactory neurons) can be, in response to stimulation, either excited or suppressed and thus increase or decrease the spike firing rate. The described method is based on the detection of changes in slope of cumulative spike time distribution and efficiently detects excitations and suppressions. Using the simulated spike trains we examined the method’s power in relation to response strength and duration. (c) 2004 Published by Elsevier B.V.
Abstract: The primary patterns of racial differentiation in the water louse, Asellus aquaticus (L.) sensu RACOVITZA, were assessed by the analysis of geographic variation in 59 morphometric characters. Mates from 20 localities in Slovenia and 18 in other countries were analyzed. In Multivariate Discriminant Function Analysis, Minimum Spanning Tree and in Cluster Analysis, samples comprising specimens without eye and body pigmentation are separated from the samples with pigmented specimens, even thou-h these two troglomorphic traits are not being considered. Only 7 traits are needed for the separation. In the cline of the samples with pigmented specimens, 3 groups of samples with the concentric distribution ranges may be distinguished. The results confirm the existence of extensive racial diversification in Slovenia and indicate some inconsistencies in the presently applied classification. The results of separate analysis of male gonopod structure are consistent with the results of multivariate statistical analysis.
Abstract: Cysteine, serine and metalloproteinases and their respective inhibitors are involved in tumor cell invasion and may have prognostic value for the outcome of malignant disease. The aim of the study was to compare the expression of new potential biological tumor markers, the lysosomal cysteine proteinases and their endogenous inhibitors, with that of the serine proteinases and their inhibitors in breast cancinoma and to relate their levels to the clinicopathological factors of the disease. Enzyme-linked immunosorbent assays (ELISAs) were used to measure cysteine cathepsin B (CatB) and cathepsin L (CatL) and their inhibitors, stefin A (StA) and stefin B (StB), together with urokinase (u-PA) and plasminogen activator inhibitor-1 (PAI-1), in 150 cytosols of primary invasive breast carcinoma. A good correlation was found between the levels of the two cysteine proteinases but only a moderate one between those of the cysteine and serine proteinases. u-PA and PAI-1 levels correlated positively with histological grade and negatively with estrogen receptor (ER) status. PAI-1 correlated with most clinicopathological factors that indicate the progression of the disease, while cathepsins and stefins were independent of these factors. In the total group of patients, high u-PA and PAI-1 and low StB levels correlated significantly with shorter disease-free survival (DFS), while CatB, CatL and StA did not. In lymph node negative patients, high CatB and CatL were also associated with shorter DFS, while u-PA remained the most significant of all these biological markers. In conclusion, this retrospective study showed u-PA to be of better prognostic relevance than the cysteine proteinases, though CatB and CatL were relevant for prognosis in lymph node negative breast cancer patients.
Abstract: Cysteine, serine and metalloproteinases and their respective inhibitors are involved in tumor cell invasion and may have prognostic value for the outcome of malignant disease. The aim of the study was to compare the expression of new potential biological tumor markers, the lysosomal cysteine proteinases and their endogenous inhibitors, with that of the serine proteinases and their inhibitors in breast cancinoma and to relate their levels to the clinicopathological factors of the disease. Enzyme-linked immunosorbent assays (ELISAs) were used to measure cysteine cathepsin B (CatB) and cathepsin L (CatL) and their inhibitors, stefin A (StA) and stefin B (StB), together with urokinase (u-PA) and plasminogen activator inhibitor-1 (PAI-1), in 150 cytosols of primary invasive breast carcinoma. A good correlation was found between the levels of the two cysteine proteinases but only a moderate one between those of the cysteine and serine proteinases. u-PA and PAI-1 levels correlated positively with histological grade and negatively with estrogen receptor (ER) status. PAI-1 correlated with most clinicopathological factors that indicate the progression of the disease, while cathepsins and stefins were independent of these factors. In the total group of patients, high u-PA and PAI-1 and low SIB levels correlated significantly with shorter disease-free survival (DFS), while CatB, CatL and StA did not. In lymph node negative patients, high CatB and CatL were also associated with shorter DFS, while u-PA remained the most significant of all these biological markers. In conclusion, this retrospective study showed u-PA to be of better prognostic relevance than the cysteine proteinases, though CatB and CatL were relevant for prognosis in lymph node negative breast cancer patients. (C) 2002 International Society for Preventive Oncology. Published by Elsevier Science Ltd. All rights reserved.
Abstract: An statistical method for analysis of event data analysis, common in electrophysiological investigations of activities of olfactory receptor neurons, is proposed. The method is based on the analysis of the slopes of cumulative frequency distribution function. During the spontaneous activity the distribution function is linear which indicate that probability density distribution is uniform. During the chemical stimulation, the density of action potentials is either increased (excitation--E) or decreased (suppression--S), causing the observable change of the slope. The slope at each event is evaluated by piecewise linear regression. The type (E or S) and time of change of the slope can be determined, providing information for further statistical analyses. Graphically supported computer program (in S-PLUS) is available for interactive or routine use.
Abstract: An statistical method for analysis of event data analysis, common in electrophysiological investigations of activities of olfactory receptor neurons, is proposed. The method is based on the analysis of the slopes of cumulative frequency distribution function. During the spontaneous activity the distribution function is linear which indicate that probability density distribution is uniform. During the chemical stimulation, the density of action potentials is either increased (excitation - E) or decreased (suppression - S), causing the observable change of the slope. The slope at each event is evaluated by piecewise linear regression. The type (E or S) and time of change of the slope can be determined, providing information for further statistical analyses. Graphically supported computer program (in S-PLUS) is available for interactive or routine use.
Abstract: New prognosticators are needed for breast cancer patients after the initial surgical treatment to make therapeutic decisions that ultimately will. affect their DFS, These consist of specific proteolytic enzymes including lysosomal endopeptidases, In this study, the activity and protein concentrations of cathepsins (Cats) D, B, and I, were measured in 282 invasive breast tumor cytosols. These potential biological prognostic indicators were compared with other histopathological parameters, such as tumor size, lymph node involvement, tumor-node-metastasis stage, histological grade, DNA analysis, and steroid receptors, CatD protein concentration correlated with lymph node involvement. CatB and CatL levels correlated significantly with Scarf-Bloom-Richardson histological grade and were also higher in estrogen-negative tumors, and CatB was;as higher in larger tumors. As prognostic markers, CatB concentration was significant for increased risk for recurrence in the entire patient population and specifically also in lymph node-negative patients as follows: high CatB concentration (above 371 mu g/g) in tumor cytosols was significant (P < 0.00) for high risk of recurrence but was of only borderline prognostic significance (P < 0.06) for overall survival of all patients. In lymph node-negative patients, CatB (above 240 mu g/g, P < 0.003) was highly significant for recurrence-free survival, followed by CatL (above 20 mu g/g, P < 0.049) and CatD (above 45 nmol/g, P < 0.044) concentrations. For overall survival of node-negative patients, only CatB was a significant (P < 0.014) prognosticator, We conclude that CatB is useful as a prognostic indicator in lymph node-negative patients. This suggests that selective adjuvant therapy should be applied in this lower risk group of patients when high levels of CatB are determined.
Abstract: New prognosticators are needed for breast cancer patients after the initial surgical treatment to make therapeutic decisions that ultimately will affect their DFS. These consist of specific proteolytic enzymes including lysosomal endopeptidases. In this study, the activity and protein concentrations of cathepsins (Cats) D, B, and L were measured in 282 invasive breast tumor cytosols. These potential biological prognostic indicators were compared with other histopathological parameters, such as tumor size, lymph node involvement, tumor-node-metastasis stage, histological grade, DNA analysis, and steroid receptors. CatD protein concentration correlated with lymph node involvement. CatB and CatL levels correlated significantly with Scarf-Bloom-Richardson histological grade and were also higher in estrogen-negative tumors, and CatB was higher in larger tumors. As prognostic markers, CatB concentration was significant for increased risk for recurrence in the entire patient population and specifically also in lymph node-negative patients as follows: high CatB concentration (above 371 micrograms/g) in tumor cytosols was significant (P < 0.00) for high risk of recurrence but was of only borderline prognostic significance (P < 0.06) for overall survival of all patients. In lymph node-negative patients, CatB (above 240 micrograms/g, P < 0.003) was highly significant for recurrence-free survival, followed by CatL (above 20 micrograms/g, P < 0.049) and CatD (above 45 nmol/g, P < 0.044) concentrations. For overall survival of node-negative patients, only CatB was a significant (P < 0.014) prognosticator. We conclude that CatB is useful as a prognostic indicator in lymph node-negative patients. This suggests that selective adjuvant therapy should be applied in this lower risk group of patients when high levels of CatB are determined.
Abstract: Olfactory discrimination of amino acids was investigated in brown bullhead catfish (Ameiurus nebulosus). Based on the magnitude of the observed food search activity of catfish conditioned to single amino acids, the tested compounds were classified as being detected by the catfish as equal to, similar to, or different from the conditioned stimulus. L-Proline (L-Pro)-conditioned brown bullhead catfish discriminated all amino acids from L-Pro, but catfish conditioned to L-valine (L-Val) and L-isoleucine (L-Ile) did not discriminate L-Val from L-Ile nor L-Ile from L-Val; however, all other amino acids tested were always discriminated from these two compounds. Catfish conditioned to L-alanine (L-Ala) discriminated basic, acidic and several neutral amino acids with long side-chains (LCNs) from L-Ala; however, they did not always discriminate L-Ala from all neutral amino acids with short side-chains (SCNs). The L-norleucine (L-nLeu)-conditioned fish responded to L-norvaline (L-nVal), L-methionine (L-Met) and L-Ala similarly to L-nLeu, indicating that these amino acids are detected as similar or identical to L-nLeu. L-nLeu was, however, discriminated from L-Ala in L-Ala-conditioned catfish. Interestingly, L-leucine (L-Leu) was discriminated from the conditioned stimuli, L-Ala, L-Ile and L-Val, indicating independent receptors for L-Leu. Although conditioned catfish discriminated other amino acids from L-arginine hydrochloride (L-Arg), in some tests they were unable to discriminate L-Arg from L-lysine hydrochloride (L-Lys). These results imply the existence of independent olfactory receptive pathways for: (i) L-Pro; (ii) basic amino acids (L-Arg and L-Lys); (iii) L-Leu; (iv) other neutral amino acids with branched side-chains (L-Ile and L-Val); (v) neutral amino acids with long linear side-chains (L-nLeu, L-nVal and L-Met); (vi) neutral amino acids with short side-chains; and (vii) amino acids with sulfhydryl groups (L-Cys and L-homoCys).
Abstract: Olfactory discrimination of amino acids was investigated in brown bullhead catfish (Ameiurus nebulosus). Based on the magnitude of the observed food search activity of catfish conditioned to single amino acids, the tested compounds were classified as being detected by the catfish as equal to, similar to, or different from the conditioned stimulus. L-Proline (L-Pro)-conditioned brown bullhead catfish discriminated all amino acids from L-Pro, but catfish conditioned to L-valine (L-Val) and L-isoleucine (L-Ile) did not discriminate L-Val from L-Ile nor L-Ile from L-Val; however, all other amino acids tested were always discriminated from these two compounds. Catfish conditioned to L-alanine (L-Ala) discriminated basic, acidic and several neutral amino acids with long side-chains (LCNs) from L-Ala; however, they did not always discriminate L-Ala from all neutral amino acids with short side-chains (SCNs). The L-norleucine (L-nLeu)-conditioned fish responded to L-norvaline (L-nVal), L-methionine (L-Met) and L-Ala similarly to L-nLeu, indicating that these amino acids are detected as similar or identical to L-nLeu. L-nLeu was, however, discriminated from L-Ala in L-Ala-conditioned catfish. Interestingly, L-leucine (L-Leu) was discriminated from the conditioned stimuli, L-Ala, L-Ile and L-Val, indicating independent receptors for L-Leu. Although conditioned catfish discriminated other amino acids from L-arginine hydrochloride (L-Arg), in some tests they were unable to discriminate L-Acg from L-lysine hydrochloride (L-Lys). These results imply the existence of independent olfactory receptive pathways for: (i) L-Pro; (ii) basic amino acids (L-Arg and L-Lys); (iii) L-Leu; (iv) other neutral amino acids with branched sidechains (L-Ile and L-Val); (v) neutral amino acids with long linear side-chains (L-nLeu, L-nVal and L-Met), (vi) neutral amino acids with short side-chains; and (vii) amino acids with sulfhydryl groups (L-Cys and L-homoCys).
Abstract: We tested the hypothesis that the respiratory system of hypogean spiders is subject to regressive evolution by examining representatives of the family Dysderidae. This comparison included the epigean species Dysdera ninnii Canestrini 1868, and Harpactea lepida (C.L. Koch 1838), and the hypogean species Stalita taenaria Schiodte 1847, and Parastalita stygia (Joseph 1882). Both tube tracheae and book lungs of these species were measured and compared using 10 indices. Both the tracheal system and hook lungs of the hypogean species were less developed than those of the epigean ones. We suggest that the cause is reduction of the respiratory system as a part of general structural reductions in the troglobites. This is consistent with the lower respiratory rates that characterize many troglobites.
Abstract: A new, troglomorphic subspecies of Asellus aquaticus, A. a. infernus, from Romania, is described and illustrated. The general architecture of the copulatory appendages, studied with SEM in Romanian and Slovenian hypogean and epigean populations, is compared and discussed. A difference in brood size was determined between Romanian hypogean and epigean populations. Extensive analysis of the data using multivariate discriminant function analysis (DFA) allowed the elucidation of racial differentiation patterns in A. aquaticus from Romania and Slovenia. The number of the initially measured morphometric characters was greatly reduced as the contribution of each character to each discriminant function was assessed. DFA divided the sample complex into distinct epigean-A. a. aquaticus, and hypogean forms-A. a. infernus (Romania) and A. a. cavernicolus (Slovenia). The size of pleopod V and its area as well as the isometric (short distal rami) uropod represent the most important discriminant characters. Distinct separation of Romanian and Slovenian cave populations, i.e., the low degree of convergent evolution toward troglomorphism, might be at least to some extent explained by a different origin of populations and by the considerable ecological differences between both habitats.
Abstract: Proteolytic enzymes have been proposed as new biological prognostic indicators to facilitate decisions about treatment of breast cancer patients following surgery. We reported earlier that the activities of cysteine proteinases (CP), cathepsin (Cat) B and cathepsin (Cat) L and the expression of stefin A might be associated with breast tumor progression and prognosis. Here, the protein concentrations of Cats D, B and L and stefin A have been measured in a series of 60 matched pairs of breast tumours and control adjacent tissues, using ELISAs developed in our laboratory. Median tumor concentrations of Cat D (47 pm/mg), Cat B (222 ng/mg) and Cat L (88 ng/mg) were significantly (p<0.0005) increased by 7 fold, 27 fold and 6 fold, respectively. Much greater increases in the activities of Cat B (63 fold) and of Cat L (274 fold) were found, indicating activation of proCat B and proCat L and/or to a decrease in specific endogenous cystatins. However, the 1.6-fold decreased (p<0.0001) levels of inhibition by cystatins could not be entirely responsible for more than 100-fold increased ratio of CP:cystatins activity. Moreover, stefin A was either increased or decreased in tumor samples, resulting in a 1.4-fold median increase in tumors. Comparing the biological parameters with the established histo-pathological prognosticators, we found that the increased protein concentration of Cat B was associated with lymph node involvement (p<0.009) and higher stage (p<0.003), and both Cat B and Cat L activities were more increased in high grade tumours (p<0.05). Survival analysis revealed that stefin A was the most significant prognostic factor for disease-free (p<0.008) and overall survival (p<0.02), followed by increased Cat B activity and protein concentration. Cat L was of borderline significance while Cat D was not significant for prognosis. We conclude that enhanced activation of CP, due partially to an imbalance between cysteine proteinases and inhibitors is linked to the progression of breast cancer. Larger sample size is needed to confirm the prognostic significance of stefin A, Cat B and Cat L.
Abstract: Diurnal vertical migration (DVM) of Daphnia hyalina in Lake Bled was most intense during summer stratification. The extent of DVM varied with the size of the animal and its reproductive state. Migration distances were shortest in immature specimens and longest in ovigerous females. During daytime, ovigerous females stayed deeper in the water column than females without ova or immatures. The daytime temperature of water at the median depth of the ovigerous females did not exceed 10-degrees-C, even in the warmest season. At night they migrated upward to an environment which was warmer by as much as 9-degrees-C. Laboratory observations indicate that specimen’s size and water temperature determine the velocity of passive sinking, such that morning descent of the different groups of Daphnia can be explained by passive sinking alone. Our hypothesis is that the distribution of different groups of D. hyalina in Lake Bled is influenced by two types of predators: fish (Perca fluviatilis L. and Rutilus rutilus (L.)) and larvae of Chaoborus flavicans (Meig.), the latter appearing in the epilimnion during the night. Fish predation has a key-role at the beginning of thermal stratification. Supposing that in spring the gene pool of Daphnia consists of a mix of different genotypes, distributed at different depths during the day, fish predation combined with a presence of fish chemicals favored genotypes with a lower day-depth during the spring/summer period.
Abstract: The macrophytes of Lake Bled were studied from 1987 to 1990. Three main factors influencea the decline of the aquatic vegetation in the lake during that period: (1) reduced light in the littoral zone due to an increase in phytoplankton (2) grazing by herbivorous fish and waterfowl, and (3) direct human impact.
Abstract: Different kinds of data are used in teaching statistics. In applied statistics courses we usually use
real life data related to the main subject matter of our students. Such data are interesting for
students and motivate final interpretation of statistical results. For demonstration of statistical
concepts, computer simulated data with known statistical properties can be used. The advantage
of such data is that results of analysis can be compared with known and pre-defined properties of
data. Many important statistical concepts and procedures can be obviously shown with computer
simulations and dynamic graphics. Such simulations can sometimes be more convincing than
proofs and are appreciated by students.
Abstract: Real life data are usually used in statistics teaching. For illustration of statistical
methods, simulated data with known statistical properties are more suitable.
Computer simulations and examples on simulated data can sometimes be used
instead of proofs. When the methods are tested and familiarized on data with
known properties, they can be effectively used on real life data with unknown
Structure and properties.
