Toward the blood-borne miRNome of human diseases

In a multicenter study, we determined the expression profiles of 863 microRNAs by array analysis of 454 blood samples from human individuals with different cancers or noncancer diseases, and validated this 'miRNome' by quantitative real-time PCR. We detected consistently deregulated profiles for all tested diseases; pathway analysis confirmed disease association of the respective microRNAs. We observed significant correlations (P = 0.004) between the genomic location of disease-associated genetic variants and deregulated microRNAs.     

Prescribed burning as an alternative management in grasslands of temperate Europe: The impact on seeds

Fire is not an integral part of terrestrial ecosystems in temperate Europe, nevertheless prescribed burning is proposed to be an alternative to traditional management applied to grasslands. Thus, anthropogenic fire represents a serious challenge to plant species, and there is no information on how the recruitment of species responds to fire. The aim of our study was to assess the effect of fire on seed germination of 16 herbaceous grassland and ruderal species belonging to four common families. We first assessed the fuel load in open habitats in early spring and measured soil temperatures during experimental fires. After that we performed a controlled pot experiment involving heat treatments and experimental fire applied to seeds and followed their germination. Our measurements showed that maximum temperatures are between 73 °C and 264 °C on the ground surface and fire passage is fast, with short residence times of high temperatures. In deeper soil layers (1 cm and 5 cm), temperature increase is negligible. Seed germination of half of the species was decreased by the passage of fire, and a heat shock of 100 °C for 5 min had an even stronger adverse effect. Seeds of three Fabaceae species were stimulated by heat or fire, while negative effects prevailed among species belonging to other families. Anthropogenic fire in grasslands of temperate Europe might reduce recruitment by seed, particularly in species of the Asteraceae and Poaceae, two very important families with a large representation in temperate grasslands. Our results indicate that prescribed burning should be carefully applied in order not to endanger the local persistence of grassland species whose seedling emergence is negatively affected by fire.     

Characterization of a high field Orbitrap mass spectrometer for proteome analysis

The field of proteomics continues to be driven by improvements in analytical technology, notably in peptide separation, quantitative MS, and informatics. In this study, we have characterized a hybrid linear ion trap high field Orbitrap mass spectrometer (Orbitrap Elite) for proteomic applications. The very high resolution available on this instrument allows 95% of all peptide masses to be measured with sub‐ppm accuracy that in turn improves protein identification by database searching. We further confirm again that mass accuracy in tandem mass spectra is a valuable parameter for improving the success of protein identification. The new CID rapid scan type of the Orbitrap Elite achieves similar performance as higher energy collision induced dissociation fragmentation and both allow the identification of hundreds of proteins from as little as 0.1 ng of protein digest on column. The new instrument outperforms its predecessor the Orbitrap Velos by a considerable margin on each metric assessed that makes it a valuable and versatile tool for MS‐based proteomics.     

Optic Neuritis Is Associated with Inner Nuclear Layer Thickening and Microcystic Macular Edema Independently of Multiple Sclerosis

Background

Microcystic macular edema (MME) and inner nuclear layer thickening (INL) were described in multiple sclerosis (MS) and neuromyelitis optica (NMO) patients using optical coherence tomography (OCT). The cause of these findings is currently unknown and a relation to inflammatory or degenerative processes in the optic nerve is discussed.

Objective

The aim of our study was to investigate whether INL thickening and MME are related to optic neuritis (ON) in various neuro-inflammatory disorders causingON: MS, NMO and chronic inflammatory optic neuropathy.

Methods

We retrospectively analyzed data from 216 MS patients, 39 patients with a clinically isolated syndrome, 20 NMO spectrum disorder patients, 9 patients with chronic inflammatory optic neuropathy and 121 healthy subjects. Intra-retinal layer segmentation was performed for the eyes of patients with unilateral ON. Scanning laser ophthalmoscopy (SLO) images were reviewed for characteristic ocular fundus changes.

Results

Intra-retinal layer segmentation showed that eyes with a history of ON displayed MME independent INL thickening compared to contralateral eyes without previous ON. MME was detected in 22 eyes from 15 patients (5.3% of all screened patients), including 7 patients with bilateral edema. Of these, 21 had a prior history of ON (95%). The SLO images of all 22 MME-affected eyes showed crescent-shaped texture changes which were visible in the perifoveal region. A second grader who was blinded to the results of the OCT classified all SLO images for the presence of these characteristic fundus changes. All MME eyes were correctly classified (sensitivity = 100%) with high specificity (95.2%).

Conclusion

This study shows that both MME and INL thickening occur in various neuro-inflammatory disorders associated with ON. We also demonstrate that detection and analysis of MME by OCT is not limited to B-scans, but also possible using SLO images.     

Myasthenia Gravis: Analysis of Serum Autoantibody Reactivities to 1827 Potential Human Autoantigens by Protein Macroarrays

Background

Myasthenia gravis is a disorder of neuromuscular transmission associated with autoantibodies against the nicotinic acetylcholine receptor. We have previously developed a customized protein macroarray comprising 1827 potential human autoantigens, which permitted to discriminate sera of patients with different cancers from sera of healthy controls, but has not yet been evaluated in antibody-mediated autoimmune diseases.

Objective

To determine whether autoantibody signatures obtained by protein macroarray separate sera of patients with myasthenia gravis from healthy controls.

Methods

Sera of patients with acetylcholine receptor antibody-positive myasthenia gravis (n = 25) and healthy controls (n = 32) were analyzed by protein macroarrays comprising 1827 peptide clones.

Results

Autoantibody signatures did not separate patients with myasthenia gravis from controls with sufficient sensitivity, specificity, and accuracy. Intensity values of one antigen (poly A binding protein cytoplasmic 1, p = 0.0045) were higher in patients with myasthenia gravis, but the relevance of this and two further antigens, 40S ribosomal protein S13 (20.8% vs. 0%, p = 0.011) and proteasome subunit alpha type 1 (25% vs. 3.1%, p = 0.035), which were detected more frequently by myasthenia gravis than by control sera, currently remains uncertain.

Conclusion

Seroreactivity profiles of patients with myasthenia gravis detected by a customized protein macroarray did not allow discrimination from healthy controls, compatible with the notion that the autoantibody response in myasthenia gravis is highly focussed against the acetylcholine receptor.     

Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster

Succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate oxidoreductase (QFR) from Escherichia coli are members of the complex II family of enzymes. SQR and QFR catalyze similar reactions with quinones; however, SQR preferentially reacts with higher potential ubiquinones, and QFR preferentially reacts with lower potential naphthoquinones. Both enzymes have a single functional quinone-binding site proximal to a [3Fe-4S] iron-sulfur cluster. A difference between SQR and QFR is that the redox potential of the [3Fe-4S] cluster in SQR is 140 mV higher than that found in QFR. This may reflect the character of the different quinones with which the two enzymes preferentially react. To investigate how the environment around the [3Fe-4S] cluster affects its redox properties and catalysis with quinones, a conserved amino acid proximal to the cluster was mutated in both enzymes. It was found that substitution of SdhB His-207 by threonine (as found in QFR) resulted in a 70-mV lowering of the redox potential of the cluster as measured by EPR. The converse substitution in QFR raised the redox potential of the cluster. X-ray structural analysis suggests that placing a charged residue near the [3Fe-4S] cluster is a primary reason for the alteration in redox potential with the hydrogen bonding environment having a lesser effect. Steady state enzyme kinetic characterization of the mutant enzymes shows that the redox properties of the [3Fe-4S] cluster have only a minor effect on catalysis.     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

High genetic diversity declines towards the geographic range periphery of Adonis vernalis,a Eurasian dry grassland plant

Genetic diversity is important for species' fitness and evolutionary processes but our knowledge on how it varies across a species' distribution range is limited. The abundant centre hypothesis (ACH) predicts that populations become smaller and more isolated towards the geographic range periphery – a pattern that in turn should be associated with decreasing genetic diversity and increasing genetic differentiation. We tested this hypothesis in Adonis vernalis, a dry grassland plant with an extensive Eurasian distribution. Its life‐history traits and distribution characteristics suggest a low genetic diversity that decreases and a high genetic differentiation that increases towards the range edge. We analysed AFLP fingerprints in 28 populations along a 4698‐km transect from the geographic range core in Russia to the western range periphery in Central and Western Europe. Contrary to our expectation, our analysis revealed high genetic diversity (range of proportion of polymorphic bands = 56–81%, He = 0.168–0.238) and low genetic differentiation across populations (Φ_ST = 0.18). However, in congruence with the genetic predictions of the ACH, genetic diversity decreased and genetic differentiation increased towards the range periphery. Spanish populations were genetically distinct, suggesting a divergent post‐glacial history in this region. The high genetic diversity and low genetic differentiation in the remaining A. vernalis populations is surprising given the species' life‐history traits and points to the possibility that the species has been widely distributed in the studied region or that it has migrated from a diverse source in an East–West direction, in the past.