ClutrFree: cluster tree visualization and interpretation

ClutrFree facilitates the visualization and interpretation of clusters or patterns computed from microarray data through a graphical user interface that displays patterns, membership information of the genes and annotation statistics simultaneously. ClutrFree creates a tree linking the patterns based on similarity, permitting the navigation among patterns identified by different algorithms or by the same algorithm with different parameters, and aids the inferring of conclusions from a microarray experiment. AVAILABILITY: The ClutrFree Java source code and compiled bytecode are available as a package under the GNU General Public License at http://bioinformatics.fccc.edu     

How morphological constraints affect axonal polarity in mouse neurons

Neuronal differentiation is under the tight control of both biochemical and physical information arising from neighboring cells and micro-environment. Here we wished to assay how external geometrical constraints applied to the cell body and/or the neurites of hippocampal neurons may modulate axonal polarization in vitro. Through the use of a panel of non-specific poly-L-lysine micropatterns, we manipulated the neuronal shape. By applying geometrical constraints on the cell body we provided evidence that centrosome location was not predictive of axonal polarization but rather follows axonal fate. When the geometrical constraints were applied to the neurites trajectories we demonstrated that axonal specification was inhibited by curved lines. Altogether these results indicated that intrinsic mechanical tensions occur during neuritic growth and that maximal tension was developed by the axon and expressed on straight trajectories. The strong inhibitory effect of curved lines on axon specification was further demonstrated by their ability to prevent formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provided evidence that microtubules were involved in the tension-mediated axonal polarization, acting as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations.     

Coxiella burnetii vaginal shedding and antibody responses in dairy goat herds in a context of clinical Q fever outbreaks

This study, carried out in three goat herds, was aimed at describing individual responses to Q fever infection in an abortive context, focusing on both antibody and shedding levels. Seroprevalence of Coxiella burnetii (Cb) infection and vaginal shedding of 1083 goats were investigated using ELISA and realtime qPCR assays, respectively. At the end of the outbreaks, a seroprevalence of 45.0% was found, and vaginal shedding appeared massive with levels above 10(4) Cb per swab in 42.3% of the whole population and above 10(6) Cb per swab for 90.9% of aborted goats. Susceptible animals (i.e. seronegative nonshedders) were unfrequent (31.2%), most of them being kids (94.7%). Seronegative females were predominant among nonshedders and conversely seropositive ones, predominant among high shedders (above 10(6) Cb per swab). Nevertheless, at least 43.3% of seronegative goats shed bacteria confirming the need of interpreting serology on a herd scale. The subsequent farrowing period was characterized by a significant reduction in the number of clinical cases. Females that had already aborted were more often involved than others. Shedding quantities remained high, particularly for primiparous does, mainly when facing infection for the first time. Thus, Q fever control must be based on both preventive measures directed to the preherd and environmental precautions.     

Mechanisms of DNA methylation and demethylation in mammals

Cytosine methylation is an epigenetically propagated DNA modification that can modify how the DNA molecule is recognized and expressed. DNA methylation undergoes extensive reprogramming during mammalian embryogenesis and is directly linked to the regulation of pluripotency and cellular identity. Studying its regulation is also important for a better understanding of the many diseases that show epigenetic deregulations, in particular, cancer. In the recent years, a lot of progress has been made to characterize the profiles of DNA methylation at the genome level, which revealed that patterns of DNA methylation are highly dynamic between cell types. Here, we discuss the importance of DNA methylation for genome regulation and the mechanisms that remodel the DNA methylome during mammalian development, in particular the involvement of the rediscovered modified base 5-hydroxymethylcytosine.     

Heterogeneity of serum gelatinases MMP‐2 and MMP‐9 isoforms and charge variants

The matrix metalloproteinases (MMPs) gelatinase A (MMP‐2) and gelatinase B (MMP‐9) are mediators of brain injury in multiple sclerosis (MS) and valuable biomarkers of disease activity. We applied bidimensional zymography (2‐DZ) as an extension of classic monodimensional zymography (1‐DZ) to analyse the complete pattern of isoforms and post‐translational modifications of both MMP‐9 and MMP‐2 present in the sera of MS patients. The enzymes were separated on the basis of their isoelectric points (pI) and apparent molecular weights (Mw) and identified both by comparison with standard enzyme preparations and by Western blot analysis. Two MMP‐2 isoforms, and at least three different isoforms and two different states of organization of MMP‐9 (the multimeric MMP‐9 and the N‐GAL‐MMP‐9 complex) were observed. In addition, 2‐DZ revealed for the first time that all MMP‐9 and MMP‐2 isoforms actually exist in the form of charge variants: four or five variants in the N‐GAL complex, more charge variants in the case of MMP‐9; and five to seven charge variants for MMP‐2. Charge variants were also observed in recombinant enzymes and, after concentration, also in sera from healthy individuals. Sialylation (MMP‐9) and phosphorylation (MMP‐2) contributed to molecular heterogeneity. The detection of charge variants of MMP‐9 and MMP‐2 in MS serum samples illustrates the power of 2‐DZ and demonstrates that in previous studies MMP mixtures, rather than single molecules, were analysed. These observations open perspectives for better diagnosis and prognosis of many diseases and need to be critically interpreted when applying other methods for MS and other diseases.     

Embedding of human vertebral bodies leads to higher ultimate load and altered damage localisation under axial compression

Computer tomography (CT)-based finite element (FE) models of vertebral bodies assess fracture load in vitro better than dual energy X-ray absorptiometry, but boundary conditions affect stress distribution under the endplates that may influence ultimate load and damage localisation under post-yield strains. Therefore, HRpQCT-based homogenised FE models of 12 vertebral bodies were subjected to axial compression with two distinct boundary conditions: embedding in polymethylmethalcrylate (PMMA) and bonding to a healthy intervertebral disc (IVD) with distinct hyperelastic properties for nucleus and annulus. Bone volume fraction and fabric assessed from HRpQCT data were used to determine the elastic, plastic and damage behaviour of bone. Ultimate forces obtained with PMMA were 22% higher than with IVD but correlated highly (R² = 0.99). At ultimate force, distinct fractions of damage were computed in the endplates (PMMA: 6%, IVD: 70%), cortex and trabecular sub-regions, which confirms previous observations that in contrast to PMMA embedding, failure initiated underneath the nuclei in healthy IVDs. In conclusion, axial loading of vertebral bodies via PMMA embedding versus healthy IVD overestimates ultimate load and leads to distinct damage localisation and failure pattern.     

Interference with Glycosaminoglycan-Chemokine Interactions with a Probe to Alter Leukocyte Recruitment and Inflammation In Vivo

In vivo leukocyte recruitment is not fully understood and may result from interactions of chemokines with glycosaminoglycans/GAGs. We previously showed that chlorite-oxidized oxyamylose/COAM binds the neutrophil chemokine GCP-2/CXCL6. Here, mouse chemokine binding by COAM was studied systematically and binding affinities of chemokines to COAM versus GAGs were compared. COAM and heparan sulphate bound the mouse CXC chemokines KC/CXCL1, MIP-2/CXCL2, IP-10/CXCL10 and I-TAC/CXCL11 and the CC chemokine RANTES/CCL5 with affinities in the nanomolar range, whereas no binding interactions were observed for mouse MCP-1/CCL2, MIP-1α/CCL3 and MIP-1β/CCL4. The affinities of COAM-interacting chemokines were similar to or higher than those observed for heparan sulphate. Although COAM did not display chemotactic activity by itself, its co-administration with mouse GCP-2/CXCL6 and MIP-2/CXCL2 or its binding of endogenous chemokines resulted in fast and cooperative peritoneal neutrophil recruitment and in extravasation into the cremaster muscle in vivo. These local GAG mimetic features by COAM within tissues superseded systemic effects and were sufficient and applicable to reduce LPS-induced liver-specific neutrophil recruitment and activation. COAM mimics glycosaminoglycans and is a nontoxic probe for the study of leukocyte recruitment and inflammation in vivo.