Characterisation of an inbred mouse strain with a deletion of the [alpha]-synuclein locus.

mRNA expression profiling was performed on a knock-in mouse model with a mutation in the NMDA receptor subunit NRl (N598R) that affects the receptors' function as coincidence detector. This approach was aimed at identifying downstream effects produced by the Ca2+ influx through NMDA receptors at the level of gene expression. cDNA array technology revealed striking differences only in the mRNA expression level of [alpha]-synuclein, a protein that has been implicated in the pathophysiology of a range of neurodegenerative diseases. However, this was not caused by the NRl mutation, but by a chromosomal deletion of the [alpha]-synuclein gene locus in the C57BL/6J inbred mice that were used for backcrossing the mutant strain. The deletion was shown to be present only in a subpopulation of C57BL/6J mice, now- referred to as C57BL/6JOlaHsd-Del(6)Snca1Slab. In addition to [alpha]-synuclein, other genes may be affected by the deletion that is estimated to be 120-500 kb in size. [alpha]-synuclein-deficient animals appear phenotypically normal. They show no compensatory upregulation of other members of the synuclein family, namely [alpha]-synuclein and [alpha]-synuclein. Similarly, the expression of synphilin-1, a known interacting partner of [alpha]-synuclein was unaffected. The C57BL/6JOlaHsd-Del(6)SncalSlab mouse model should help in the understanding of the physiological function of [alpha]-synuclein and its involvement in synucleinopathies. Also, the findings exemplify unexpected complications that may arise during the study of transgenic models or inbred strains. A Sindbis virus system was developed for the expression of fluorescent [alpha]-synuclein fusion proteins in neurons. A range of recombinant virion preparations was tested in plaque assays and the expression of the recombinant proteins was characterised. Initial analysis of the expression of [alpha]-synuclein-eGFP in organotypic hippocampal neurons suggested that the protein accumulated in presynaptic locations. This approach could be used for the study of the subcellular localisation and of protein interactions of [alpha]-synuclein

Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice

BACKGROUND: The presynaptic protein α-synuclein is involved in a range of neurodegenerative diseases. Here we analyze potential compensatory mechanisms in α-synuclein null mutant mice. Furthermore, the findings reveal problems that may be associated with inbred mouse strains. RESULTS: Expression profiling by cDNA array technology in a transgenic mouse model revealed striking differences only in the expression level of α-synuclein. This was caused by a chromosomal deletion of the α-synuclein locus in the C57BL/6J inbred strain used for backcrossing. However, the deletion is only present in a subpopulation of C57BL/6J mice, namely animals from Harlan. No other genes are known to be affected by the deletion, which is estimated to be smaller than 2 cM. We propose to name this strain C57BL/6S. C57BL/6S animals appear phenotypically normal. They show no upregulation of β-synuclein or γ-synuclein, excluding a compensatory mechanism. Also, the expression of synphilin-1 was unaffected. CONCLUSIONS: The C57BL/6S strain should help in the understanding of the physiological function of α-synuclein and its involvement in synucleinopathies. Also, the findings exemplify unexpected complications that may arise during the study of transgenic models or inbred strains, in particular when combined with genome wide screening techniques

Impact of soil management practices on soil fertility and disease suppressiveness

Soil management practices are targeted to provide adequate crop nutrition and to ensure durable soil fertility and to avoid negative environmental impacts. Soil management also aims to reduce pest and disease pressure on crops. Organic farming is believed to increase soil suppressiveness towards soil-borne diseases as well aerial diseases. In this paper we will discuss the potential of soil manage-ment as a tool to improve disease suppressiveness in practice

Mapping the energy and diffusion landscapes of membrane proteins at the cell surface using high-density single-molecule imaging and Bayesian inference: application to the multi-scale dynamics of glycine receptors in the neuronal membrane

Protein mobility is conventionally analyzed in terms of an effective diffusion. Yet, this description often fails to properly distinguish and evaluate the physical parameters (such as the membrane friction) and the biochemical interactions governing the motion. Here, we present a method combining high-density single-molecule imaging and statistical inference to separately map the diffusion and energy landscapes of membrane proteins across the cell surface at ~100 nm resolution (with acquisition of a few minutes). When applying these analytical tools to glycine neurotransmitter receptors (GlyRs) at inhibitory synapses, we find that gephyrin scaffolds act as shallow energy traps (~3 kBT) for GlyRs, with a depth modulated by the biochemical properties of the receptor-gephyrin interaction loop. In turn, the inferred maps can be used to simulate the dynamics of proteins in the membrane, from the level of individual receptors to that of the population, and thereby, to model the stochastic fluctuations of physiological parameters (such as the number of receptors at synapses). Overall, our approach provides a powerful and comprehensive framework with which to analyze biochemical interactions in living cells and to decipher the multi-scale dynamics of biomolecules in complex cellular environments.Comment: 23 pages, 4 figure

p3d – Python module for structural bioinformatics

<p>Abstract</p> <p>Background</p> <p>High-throughput bioinformatic analysis tools are needed to mine the large amount of structural data via knowledge based approaches. The development of such tools requires a robust interface to access the structural data in an easy way. For this the Python scripting language is the optimal choice since its philosophy is to write an understandable source code.</p> <p>Results</p> <p>p3d is an object oriented Python module that adds a simple yet powerful interface to the Python interpreter to process and analyse three dimensional protein structure files (PDB files). p3d's strength arises from the combination of a) very fast spatial access to the structural data due to the implementation of a binary space partitioning (BSP) tree, b) set theory and c) functions that allow to combine a and b and that use human readable language in the search queries rather than complex computer language. All these factors combined facilitate the rapid development of bioinformatic tools that can perform quick and complex analyses of protein structures.</p> <p>Conclusion</p> <p>p3d is the perfect tool to quickly develop tools for structural bioinformatics using the Python scripting language.</p

Вітання Президента України В.Ф. Януковича з нагоди 50-річчя обрання академіка Б.Є. Патона президентом Національної академії наук України

Phthalates which are widely used, are ubiquitous in the environment and in some human tissues. It is generally accepted that phthalates exert their toxic action by inhibiting Leydig cell synthesis of testosterone, but in vitro studies have also shown anti-androgenic effects at the receptor level. Some cross-sectional studies have shown inverse associations between urinary levels of phthalates and reproductive hormones, but results are conflicting and the evidence base is limited. The aim of this study was to investigate if levels of di-2-ethylhexyl phthalate (DEHP) and diisononyl phthalate (DiNP) metabolites in serum are associated with serum concentrations of male reproductive hormones and semen quality. A secondary aim was to investigate metabolic pathways of DEHP and DiNP on semen quality and reproductive hormones. A cross-sectional sample of 589 spouses of pregnant women from Greenland, Poland and Ukraine were enrolled between 2002 and 2004. The men gave semen and blood samples and were interviewed. Six phthalate metabolites of DEHP and DiNP were measured by liquid chromatography tandem mass spectrometry in serum. The metabolites were summed according to their molar weight. We observed significant inverse associations between serum levels of the metabolites, the proxies and serum testosterone. Negative associations were also discovered between some metabolites and sex hormone-binding globulin, semen volume and total sperm count Findings are compatible with a weak anti-androgenic action of DEHP metabolites, but less so for DiNP metabolites. Metabolic pathways differed significantly between the three study sites, but without major effect on semen quality or reproductive hormones. (C) 2014 Elsevier Ltd. All rights reserved

Ki67 proliferation in core biopsies versus surgical samples - a model for neo-adjuvant breast cancer studies

Background: An increasing number of neo-adjuvant breast cancer studies are being conducted and a novel model for tumor biological studies, the "window-of-opportunity" model, has revealed several advantages. Change in tumor cell proliferation, estimated by Ki67-expression in pre-therapeutic core biopsies versus post-therapeutic surgical samples is often the primary end-point. The aim of the present study was to investigate potential differences in proliferation scores between core biopsies and surgical samples when patients have not received any intervening anti-cancer treatment. Also, a lack of consensus concerning Ki67 assessment may raise problems in the comparison of neo-adjuvant studies. Thus, the secondary aim was to present a novel model for Ki67 assessment. Methods: Fifty consecutive breast cancer cases with both a core biopsy and a surgical sample available, without intervening neo-adjuvant therapy, were collected and tumor proliferation (Ki67, MIB1 antibody) was assessed immunohistochemically. A theoretical model for the assessment of Ki67 was constructed based on sequential testing of the null hypothesis 20% Ki67-positive cells versus the two-sided alternative more or less than 20% positive cells.. Results: Assessment of Ki67 in 200 tumor cells showed an absolute average proliferation difference of 3.9% between core biopsies and surgical samples (p = 0.046, paired t-test) with the core biopsies being the more proliferative sample type. A corresponding analysis on the log-scale showed the average relative decrease from the biopsy to the surgical specimen to be 19% (p = 0.063, paired t-test on the log-scale). The difference was significant when using the more robust Wilcoxon matched-pairs signed-ranks test (p = 0.029). After dichotomization at 20%, 12 of the 50 sample pairs had discrepant proliferation status, 10 showed high Ki67 in the core biopsy compared to two in the surgical specimen (p = 0.039, McNemar's test). None of the corresponding results for 1000 tumor cells were significant - average absolute difference 2.2% and geometric mean of the ratios 0.85 (p = 0.19 and p = 0.18, respectively, paired t-tests, p = 0.057, Wilcoxon's test) and an equal number of discordant cases after dichotomization. Comparing proliferation values for the initial 200 versus the final 800 cancer cells showed significant absolute differences for both core biopsies and surgical samples 5.3% and 3.2%, respectively (p < 0.0001, paired t-test). Conclusions: A significant difference between core biopsy and surgical sample proliferation values was observed despite no intervening therapy. Future neo-adjuvant breast cancer studies may have to take this into consideration

Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution