The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

Simultaneous determination of natural and synthetic steroid estrogens and their conjugates in aqueous matrices by liquid chromatography / mass spectrometry

An analytical method for the simultaneous determination of nine free and conjugated steroid estrogens was developed with application to environmental aqueous matrices. Solid phase extraction (SPE) was employed for isolation and concentration, with detection by liquid chromatography/mass spectrometry (LC/MS) using electrospray ionisation (ESI) in the negative mode. Method recoveries for various aqueous matrices (wastewater, lake and drinking water) were determined, recoveries proving to be sample dependent. When spiked at 50 ng/l concentrations in sewage influent, recoveries ranged from 62-89 % with relative standard deviations (RSD) < 8.1 %. In comparison, drinking water spiked at the same concentrations had recoveries between 82-100 % with an RSD < 5%. Ion suppression is a known phenomenon when using ESI; hence its impact on method recovery was elucidated for raw sewage. Both ion suppression from matrix interferences and the extraction procedure has bearing on the overall method recovery. Analysis of municipal raw sewage identified several of the analytes of interest at ng/l concentrations, estriol (E3) being the most abundant. Only one conjugate, estrone 3-sulphate (E1-3S) was observe

One Problem, Many Solutions: Simple Statistical Approaches Help Unravel the Complexity of the Immune System in an Ecological Context

The immune system is a complex collection of interrelated and overlapping solutions to the problem of disease. To deal with this complexity, researchers have devised multiple ways to measure immune function and to analyze the resulting data. In this way both organisms and researchers employ many tactics to solve a complex problem. One challenge facing ecological immunologists is the question of how these many dimensions of immune function can be synthesized to facilitate meaningful interpretations and conclusions. We tackle this challenge by employing and comparing several statistical methods, which we used to test assumptions about how multiple aspects of immune function are related at different organizational levels. We analyzed three distinct datasets that characterized 1) species, 2) subspecies, and 3) among- and within-individual level differences in the relationships among multiple immune indices. Specifically, we used common principal components analysis (CPCA) and two simpler approaches, pair-wise correlations and correlation circles. We also provide a simple example of how these techniques could be used to analyze data from multiple studies. Our findings lead to several general conclusions. First, relationships among indices of immune function may be consistent among some organizational groups (e.g. months over the annual cycle) but not others (e.g. species); therefore any assumption of consistency requires testing before further analyses. Second, simple statistical techniques used in conjunction with more complex multivariate methods give a clearer and more robust picture of immune function than using complex statistics alone. Moreover, these simpler approaches have potential for analyzing comparable data from multiple studies, especially as the field of ecological immunology moves towards greater methodological standardization

Spatially structured environmental filtering of collembolan traits in late successional salt marsh vegetation

Both the environment and the spatial configuration of habitat patches are important factors that shape community composition and affect species diversity patterns. Species have traits that allow them to respond to their environment. Our current knowledge on environment to species traits relationships is limited in spite of its potential importance for understanding community assembly and ecosystem function. The aim of our study was to examine the relative roles of environmental and spatial variables for the small-scale variation in Collembola (springtail) communities in a Dutch salt marsh. We used a trait-based approach in combination with spatial statistics and variance partitioning, between environmental and spatial variables, to examine the important ecological factors that drive community composition. Turnover of trait diversity across space was lower than for species diversity. Most of the variation in community composition was explained by small-scale spatial variation in topography, on a scale of 4-6 m, most likely because it determines the effect of inundation, which restricts where habitat generalists can persist. There were only small pure spatial effects on species and trait diversity, indicating that biotic interactions or dispersal limitation probably were less important for structuring the community at this scale. Our results suggest that for springtails, life form (i.e. whether they live in the soil or litter or on the surface/in vegetation) is an important and useful trait to understand community assembly. Hence, using traits in addition to species identity when analysing environment-organism relationships results in a better understanding of the factors affecting community composition

The heterogeneity of wooded-agricultural landscape mosaics influences woodland bird community assemblages

Context Landscape heterogeneity (the composition and configuration of different landcover types) plays a key role in shaping woodland bird assemblages in wooded-agricultural mosaics. Understanding how species respond to landscape factors could contribute to preventing further decline of woodland bird populations. Objective To investigate how woodland birds with different species traits respond to landscape heterogeneity, and to identify whether specific landcover types are important for maintaining diverse populations in wooded-agricultural environments. Methods Birds were sampled from woodlands in 58 2 x 2 km tetrads across southern Britain. Landscape heterogeneity was quantified for each tetrad. Bird assemblage response was determined using redundancy analysis combined with variation partitioning and response trait analyses. Results For woodland bird assemblages, the independent explanatory importance of landscape composition and landscape configuration variables were closely interrelated. When considered simultaneously during variation partitioning, the community response was better represented by compositional variables. Different species responded to different landscape features and this could be explained by traits relating to woodland association, foraging strata and nest location. Ubiquitous, generalist species, many of which were hole-nesters or ground foragers, correlated positively with urban landcover while specialists of broadleaved woodland avoided landscapes containing urban areas. Species typical of coniferous woodland correlated with large conifer plantations. Conclusions At the 2 x 2 km scale, there was evidence that the availability of resources provided by proximate landcover types was highly important for shaping woodland bird assemblages. Further research to disentangle the effects of composition and configuration at different spatial scales is advocated

A Holistic Landscape Description Reveals That Landscape Configuration Changes More over Time than Composition: Implications for Landscape Ecology Studies

International audienceBackground: Space-for-time substitution—that is, the assumption that spatial variations of a system can explain and predict the effect of temporal variations—is widely used in ecology. However, it is questionable whether it can validly be used to explain changes in biodiversity over time in response to land-cover changes.Hypothesis: ere, we hypothesize that different temporal vs spatial trajectories of landscape composition and configuration may limit space-for-time substitution in landscape ecology. Land-cover conversion changes not just the surface areas given over to particular types of land cover, but also affects isolation, patch size and heterogeneity. This means that a small change in land cover over time may have only minor repercussions on landscape composition but potentially major consequences for landscape configuration.Methods: sing land-cover maps of the Paris region for 1982 and 2003, we made a holistic description of the landscape disentangling landscape composition from configuration. After controlling for spatial variations, we analyzed and compared the amplitudes of changes in landscape composition and configuration over time.Results: For comparable spatial variations, landscape configuration varied more than twice as much as composition over time. Temporal changes in composition and configuration were not always spatially matched.Significance: The fact that landscape composition and configuration do not vary equally in space and time calls into question the use of space-for-time substitution in landscape ecology studies. The instability of landscapes over time appears to be attributable to configurational changes in the main. This may go some way to explaining why the landscape variables that account for changes over time in biodiversity are not the same ones that account for the spatial distribution of biodiversity

Up-regulation of brain-derived neurotrophic factor in primary afferent pathway regulates colon-to-bladder cross-sensitization in rat

Background In humans, inflammation of either the urinary bladder or the distal colon often results in sensory cross-sensitization between these organs. Limited information is known about the mechanisms underlying this clinical syndrome. Studies with animal models have demonstrated that activation of primary afferent pathways may have a role in mediating viscero-visceral cross-organ sensitization. Methods Colonic inflammation was induced by a single dose of tri-nitrobenzene sulfonic acid (TNBS) instilled intracolonically. The histology of the colon and the urinary bladder was examined by hematoxylin and eosin (H&E) stain. The protein expression of transient receptor potential (TRP) ion channel of the vanilloid type 1 (TRPV1) and brain-derived neurotrophic factor (BDNF) were examined by immunohistochemistry and/or western blot. The inter-micturition intervals and the quantity of urine voided were obtained from analysis of cystometrograms. Results At 3 days post TNBS treatment, the protein level of TRPV1 was increased by 2-fold (p \u3c 0.05) in the inflamed distal colon when examined with western blot. TRPV1 was mainly expressed in the axonal terminals in submucosal area of the distal colon, and was co-localized with the neural marker PGP9.5. In sensory neurons in the dorsal root ganglia (DRG), BDNF expression was augmented by colonic inflammation examined in the L1 DRG, and was expressed in TRPV1 positive neurons. The elevated level of BDNF in L1 DRG by colonic inflammation was blunted by prolonged pre-treatment of the animals with the neurotoxin resiniferatoxin (RTX). Colonic inflammation did not alter either the morphology of the urinary bladder or the expression level of TRPV1 in this viscus. However, colonic inflammation decreased the inter-micturition intervals and decreased the quantities of urine voided. The increased bladder activity by colonic inflammation was attenuated by prolonged intraluminal treatment with RTX or treatment with intrathecal BDNF neutralizing antibody. Conclusion Acute colonic inflammation increases bladder activity without affecting bladder morphology. Primary afferent-mediated BDNF up-regulation in the sensory neurons regulates, at least in part, the bladder activity during colonic inflammation

Intrauterine Growth Restriction Is a Direct Consequence of Localized Maternal Uropathogenic Escherichia coli Cystitis

Despite the continually increasing rates of adverse perinatal outcomes across the globe, the molecular mechanisms that underlie adverse perinatal outcomes are not completely understood. Clinical studies report that 10% of pregnant women will experience a urinary tract infection (UTI) and there is an association of UTIs with adverse perinatal outcomes. We introduced bacterial cystitis into successfully outbred female mice at gestational day 14 to follow pregnancy outcomes and immunological responses to determine the mechanisms that underlie UTI-mediated adverse outcomes. Outbred fetuses from mothers experiencing localized cystitis displayed intrauterine growth restriction (20–80%) as early as 48 hours post-infection and throughout the remainder of normal gestation. Robust infiltration of cellular innate immune effectors was observed in the uteroplacental tissue following introduction of UTI despite absence of viable bacteria. The magnitude of serum proinflammatory cytokines is elevated in the maternal serum during UTI. This study demonstrates that a localized infection can dramatically impact the immunological status as well as the function of non-infected distal organs and tissues. This model can be used as a platform to determine the mechanism(s) by which proinflammatory changes occur between non-contiguous genitourinary organ