Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations

BACKGROUND: The amplification of RNA with the T7-System is a widely used technique for obtaining increased amounts of RNA starting from limited material. The amplified RNA (aRNA) can subsequently be used for microarray hybridizations, warranting sufficient signal for image analysis. We describe here an amplification-time dependent degradation of aRNA in prolonged standard T7 amplification protocols, that results in lower average size aRNA and decreased yields. RESULTS: A time-dependent degradation of amplified RNA (aRNA) could be observed when using the classical "Eberwine" T7-Amplification method. When the amplification was conducted for more than 4 hours, the resulting aRNA showed a significantly smaller size distribution on gel electrophoresis and a concomitant reduction of aRNA yield. The degradation of aRNA could be correlated to the presence of the T7 RNA Polymerase in the amplification cocktail. The aRNA degradation resulted in a strong bias in microarray hybridizations with a high coefficient of variation and a significant reduction of signals of certain transcripts, that seem to be susceptible to this RNA degrading activity. The time-dependent degradation of these transcripts was verified by a real-time PCR approach. CONCLUSIONS: It is important to perform amplifications not longer than 4 hours as there is a characteristic 'quality vs. yield' situation for longer amplification times. When conducting microarray hybridizations it is important not to compare results obtained with aRNA from different amplification times

Cross‐reactive carbohydrate determinants in atopic and healthy dogs and their influence on allergy test specificity

Background The selection of allergens for immunotherapy in atopic dogs is often based on serum allergy testing. Cross-reactive carbohydrate determinants (CCDs) are common structures in plant and insect allergens that reportedly induce polysensitisation, reduce agreement between intradermal and serum tests and complicate allergen selection. Methods Thirty-four dogs with diagnosed atopic dermatitis and 10 healthy dogs were included in the study. An intradermal test was conducted in atopic dogs, and serum samples from allergic and healthy dogs were analysed for allergen-specific immunoglobulin E (IgE) before and after inhibition of detectable anti-CCD-IgE antibodies. Results Anti-CCD-IgE antibodies were not found in any of the healthy dogs and no polysensitisation to plant and insect allergens was detected. The agreement between intradermal and serum allergy test results in the atopic dogs with anti-CCD-IgE antibodies improved from slight to fair after blocking the anti-CCD-IgE antibodies. In addition, blocking clearly reduced polysensitisation to plant allergens but not to acarid allergens. Limitations Only a limited number of healthy dogs were tested in this study. A gold standard for determining the clinical relevance of IgE sensitisation does not exist. Conclusion Inhibition of anti-CCD-IgE antibodies seems to be of importance to improve serum test specificity for allergen-specific IgE in atopic dogs in relation to intradermal allergy testing

From cave to spring: Understanding transport of suspended sediment particles in a fully phreatic karst conduit using particle analysis and geochemical methods

Karst aquifers are vulnerable to contamination, especially in the context of heavy rainfall events. Contamination is often associated with turbidity that can originate from the soil zone, infiltrating surface waters or resuspension of previously deposited sediments within the aquifer. While turbidity events can be well monitored at karst springs, related information about the sediment origin and the spatiotemporal input function usually remain unknown. Thus, the mobility and attenuation of the particulate matter and associated pollutants can hardly be determined quantitatively. A tracer test with suspended cave sediments and solute tracers for comparison has hence been performed in a karst aquifer at the Blue Spring (Blautopf) in Southern Germany. The tracers were injected in the cave system, at the beginning of a fully phreatic karst conduit, and monitored at the spring after a travel distance of 1250 m. The particle-size distribution was monitored using a particle counter and sediment samples were filtered with 0.45-μm cellulose acetate filters. Particles on the filter were analysed for major and trace elements as well as rare earth elements (REE) by ICP-MS after acid digestion. Results show that (1) sediment particles were transported faster than solutes, which was interpreted as a transport in the main flow path of the conduit, whereas conservative tracers tend to diffuse into smaller fissures as well. (2) All measured particles sizes were transported at similar flow velocities. (3) A transport associated to sediment particles could be shown for all measured elements. This study presents a methodological improvement of comparative sediment tracer tests as well as deeper insights into particle and element transport processes in karst aquifers, originating from previously deposited cave sediment. Results provide deeper knowledge into transport processes of sediment-associated contaminants, such as heavy metals which may strongly be affected by the particle size. This knowledge contributes to a better management of karst water resources in the context of turbidity events

No substantial changes in estrogen receptor and estrogen-related receptor orthologue gene transcription in Marisa cornuarietis exposed to estrogenic chemicals

This article is made available through the Brunel Open Access Publishing Fund. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.Estrogen receptor orthologues in molluscs may be targets for endocrine disruptors, although mechanistic evidence is lacking. Molluscs are reported to be highly susceptible to effects caused by very low concentrations of environmental estrogens which, if substantiated, would have a major impact on the risk assessment of many chemicals. The present paper describes the most thorough evaluation to-date of the susceptibility of Marisa cornuarietis ER and ERR gene transcription to modulation by vertebrate estrogens in vivo and in vitro. We investigated the effects of estradiol-17β and 4-tert-Octylphenol exposure on in vivo estrogen receptor (ER) and estrogen-related receptor (ERR) gene transcription in the reproductive and neural tissues of the gastropod snail M. cornuarietis over a 12-week period. There was no significant effect (p > 0.05) of treatment on gene transcription levels between exposed and non-exposed snails. Absence of a direct interaction of estradiol-17β and 4-tert-Octylphenol with mollusc ER and ERR protein was also supported by in vitro studies in transfected HEK-293 cells. Additional in vitro studies with a selection of other potential ligands (including methyl-testosterone, 17α-ethinylestradiol, 4-hydroxytamoxifen, diethylstilbestrol, cyproterone acetate and ICI182780) showed no interaction when tested using this assay. In repeated in vitro tests, however, genistein (with mcER-like) and bisphenol-A (with mcERR) increased reporter gene expression at high concentrations only (>10−6 M for Gen and >10−5 M for BPA, respectively). Like vertebrate estrogen receptors, the mollusc ER protein bound to the consensus vertebrate estrogen-response element (ERE). Together, these data provide no substantial evidence that mcER-like and mcERR activation and transcript levels in tissues are modulated by the vertebrate estrogen estradiol-17β or 4-tert-Octylphenol in vivo, or that other ligands of vertebrate ERs and ERRs (with the possible exception of genistein and bisphenol A, respectively) would do otherwise.BBSR

Β-(I→3)-D-Glucan Modulates Dna Binding of Nuclear Factors κB, at and IL-6 Leading to an Anti-Inflammatory Shift of the IL-I β/IL-I Receptor Antagonist Ratio

Background: β-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1). Results: Despite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate. Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h: 5000 pg/ ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter. An increase in IL-1 receptor antagonist(RA) production (peak at 24 h: 12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels. Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments. When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA. Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-Iβ and IL-6. As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype. Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2. Conclusion: Thus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction. Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan

β-(1→3)-D-glucan modulates DNA binding of nuclear factors κB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1β/IL-1 receptor antagonist ratio

BACKGROUND: β-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1). RESULTS: Despite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate. Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h: 5000 pg/ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter. An increase in IL-1receptor antagonist(RA) production (peak at 24 h: 12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels. Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments. When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA. Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1β and IL-6. As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype. Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2. CONCLUSION: Thus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction. Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan

Where do Chip and Dale come from? Origins of invasive populations of the Siberian chipmunk in Europe

Among invasive squirrels in Europe, the Siberian chipmunk Eutamias sibiricus, native to North-East Asia, shows the highest number of free-ranging populations in European countries, due to the intense pet trade it underwent between the 1960s and the 1980s. We describe 628-bp cytochrome b sequences from the Netherlands (N = 3), Belgium (N = 4) and Switzerland (N = 1), and through phylogenetic analysis show that they belong to the Korean subspecies. This confirms previous findings that the Korean subspecies (Eutamias sibiricus barberi) has been the most commonly introduced to Europe. Another subspecies (the Northern subspecies, Eutamias sibiricus sibiricus) has only been reported previously in an Italian free-living population. If the splitting of the Korean taxon as a proper species (Eutamias barberi) will be confirmed by analysis of nuclear markers, a revision of the European Regulation 1143/2014 should be conducted to explicitly ban the trade of newly identified taxa.Support to MM for this research (grant LCF/BQ/DR20/11790020) was provided by ‘La Caixa’ Foundation (ID 100010434)

Modest Attenuation of HIV-1 Vpu Alleles Derived from Elite Controller Plasma

In the absence of antiretroviral therapy, infection with human immunodeficiency virus type 1 (HIV-1) can typically not be controlled by the infected host and results in the development of acquired immunodeficiency. In rare cases, however, patients spontaneously control HIV-1 replication. Mechanisms by which such elite controllers (ECs) achieve control of HIV-1 replication include particularly efficient immune responses as well as reduced fitness of the specific virus strains. To address whether polymorphisms in the accessory HIV-1 protein Vpu are associated with EC status we functionally analyzed a panel of plasma-derived vpu alleles from 15 EC and 16 chronic progressor (CP) patients. Antagonism of the HIV particle release restriction by the intrinsic immunity factor CD317/tetherin was well conserved among EC and CP Vpu alleles, underscoring the selective advantage of this Vpu function in HIV-1 infected individuals. In contrast, interference with CD317/tetherin induced NF-κB activation was little conserved in both groups. EC Vpus more frequently displayed reduced ability to downregulate cell surface levels of CD4 and MHC class I (MHC-I) molecules as well as of the NK cell ligand NTB-A. Polymorphisms potentially associated with high affinity interactions of the inhibitory killer immunoglobulin-like receptor (KIR) KIR2DL2 were significantly enriched among EC Vpus but did not account for these functional differences. Together these results suggest that in a subgroup of EC patients, some Vpu functions are modestly reduced, possibly as a result of host selection

An exploratory investigation of brain collateral circulation plasticity after cerebral ischemia in two experimental C57BL/6 mouse models

Brain collateral circulation is an essential compensatory mechanism in response to acute brain ischemia. To study the temporal evolution of brain macro and microcollateral recruitment and their reciprocal interactions in response to different ischemic conditions, we applied a combination of complementary techniques (T2 weighted magnetic resonance imaging [MRI], time of flight [TOF] angiography [MRA], cerebral blood flow [CBF] imaging and histology) in two different mouse models. Hypoperfusion was either induced by permanent bilateral common carotid artery stenosis (BCCAS) or 60 minute transient unilateral middle cerebral artery occlusion (MCAO). In both models, collateralization is a very dynamic phenomenon with a global effect affecting both hemispheres. Patency of ipsilateral posterior communicating artery (PcomA) represents the main variable survival mechanism and the main determinant of stroke lesion volume and recovery in MCAO whereas the promptness of external carotid artery retrograde flow recruitment together with PcomA patency, critically influence survival, brain ischemic lesion volume and retinopathy in BCCAS mice. Finally, different ischemic gradients shape microcollateral density and size