Divalent Cations Crosslink Vimentin Intermediate Filament Tail Domains to Regulate Network Mechanics

Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca²⁺ and Mg²⁺, showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l_P ≈ 0.5 μm, and Young's modulus, Y ≈ 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells.     

Molecular detection of Babesia canis in Dermacentor reticulatus ticks collected in Slovakia

Dermacentor reticulatus ticks are recognized as the most important vectors of Babesia canis, the aetiological agent of canine babesiosis occurring throughout Europe. Vector competence of D. reticulatus for B. canis is well described and experimentally determined; however, by using molecular analysis it was proven so by one recent study in Russia. Herein, the additional molecular evidence of B. canis infection in D. reticulatus ticks collected in Slovakia is provided. Using PCR followed by sequencing of distinctive amplicons we determined the presence of Babesia canis canis in one of 100 tested adult ticks. Two zoonotic pathogens, Francisella tularensis and Coxiella burnetii, were previously isolated from D. reticulatus ticks in Slovakia. In our samples, we detected only the presence of F. tularensis.     

Role of the leader sequence in tobacco pectin methylesterase secretion

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.     

Chloroplast heat shock protein Cpn60 from Chlamydomonas reinhardtii exhibits a novel function as a group II intron-specific RNA-binding protein

Intron-binding proteins in eukaryotic organelles are mainly encoded by the nuclear genome and are thought to promote the maturation of precursor RNAs. Here, we present a biochemical approach that enable the isolation of a novel nuclear-encoded protein from Chlamydomonas reinhardtii showing specific binding properties to organelle group II intron RNA. Using FPLC chromatography of chloroplast protein extracts, a 61-kDa RNA-binding protein was isolated and then tentatively identified by mass spectrometry as the chloroplast heat shock protein Cpn60. Heterologous Cpn60 protein was used in RNA protein gel mobility shift assays and revealed that the ATPase domains of Cpn60 mediates the specific binding of two group II intron RNAs, derived from the homologous chloroplast psaA gene and the heterologous mitochondrial LSU rRNA gene. The function of Cpn60 as a general organelle splicing factor is discussed.     

Antibody-bound amyloid precursor protein upregulates ornithine decarboxylase expression

Alzheimer's disease is a neurodegenerative disorder characterised by extracellular accumulation of the Abeta peptide, derived from the amyloid precursor protein (APP). The function of APP as a cell surface receptor was examined by ligand-mimicking using an antibody against the APP extracellular domain. Alterations in gene expression evoked by antibody-bound APP were analysed using human pathway-finder gene arrays and the largest change in expression levels was found for ornithine decarboxylase (ODC). These results were confirmed by Western blotting which showed even higher upregulation on the protein level. APP knockdown by RNAi verified that upregulation of ODC was APP-mediated. This APP signalling event did not require gamma-secretase cleavage, as it was independent of the presence of presenilin-1 or -2. The induced ODC expression was rapid and biphasic, resembling growth-factor stimulated signalling events. This study shows that antibody-bound APP leads to altered gene expression that may be relevant to AD.     

Linking anti-predator behaviour and habitat quality: group effect in nest defence of a passerine bird

In habitats where the density of breeding individuals is higher, breeding success has been shown to increase with the number of close conspecific and heterospecific neighbours. However, the mechanisms linking habitat quality, group size of prey individuals and offspring defence are poorly known. In this field study, we examined the relationships between habitat quality and parental nest defence behaviour in the pied flycatcher (Ficedula hypoleuca). We found that mobbing is more intense in unmanaged forests where birds breed in more dense and diverse communities than in heavily managed young forests where heterospecific densities are lower. We also found that the mobbing activities of pied flycatchers breeding in unmanaged mature boreal forests attracted more neighbouring prey individuals than in nearby managed forests. This study shows that habitat quality-mediated effects might be responsible for the decreased group size of mobbing birds in managed forests, which may lead to less effective communal defence.     

High levels of Paleolithic Y-chromosome lineages characterize Serbia

Whether present-day European genetic variation and its distribution patterns can be attributed primarily to the initial peopling of Europe by anatomically modern humans during the Paleolithic, or to latter Near Eastern Neolithic input is still the subject of debate. Southeastern Europe has been a crossroads for several cultures since Paleolithic times and the Balkans, specifically, would have been part of the route used by Neolithic farmers to enter Europe. Given its geographic location in the heart of the Balkan Peninsula at the intersection of Central and Southeastern Europe, Serbia represents a key geographical location that may provide insight to elucidate the interactions between indigenous Paleolithic people and agricultural colonists from the Fertile Crescent. In this study, we examine, for the first time, the Y-chromosome constitution of the general Serbian population. A total of 103 individuals were sampled and their DNA analyzed for 104 Y-chromosome bi-allelic markers and 17 associated STR loci. Our results indicate that approximately 58% of Serbian Y-chromosomes (I1-M253, I2a-P37.2 and R1a1a-M198) belong to lineages believed to be pre-Neolithic. On the other hand, the signature of putative Near Eastern Neolithic lineages, including E1b1b1a1-M78, G2a-P15, J1-M267, J2-M172 and R1b1a2-M269 accounts for 39% of the Y-chromosome. Haplogroup frequency distributions in Western and Eastern Europe reveal a spotted landscape of paleolithic Y chromosomes, undermining continental-wide generalizations. Furthermore, an examination of the distribution of Y-chromosome filiations in Europe indicates extreme levels of Paleolithic lineages in a region encompassing Serbia, Bosnia-Herzegovina and Croatia, possibly the result of Neolithic migrations encroaching on Paleolithic populations against the Adriatic Sea.     

Novel One-step Immunoassays to Quantify α-Synuclein: APPLICATIONS FOR BIOMARKER DEVELOPMENT AND HIGH-THROUGHPUT SCREENING*

Familial Parkinson disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Förster''s resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 h protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120% of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Furthermore, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knockdown increased and two that decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression that may help guide further drug discovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding that will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of compound and genetic libraries, both of which are essential to the development of PD therapies.     

Contribution of STAT4 gene single-nucleotide polymorphism to systemic lupus erythematosus in the Polish population

The STAT4 has been found to be a susceptible gene in the development of systemic lupus erythematosus (SLE) in various populations. There are evident population differences in the context of clinical manifestations of SLE, therefore we investigated the prevalence of the STAT4 G > C (rs7582694) polymorphism in patients with SLE (n = 253) and controls (n = 521) in a sample of the Polish population. We found that patients with the STAT4 C/G and CC genotypes exhibited a 1.583-fold increased risk of SLE incidence (95 % CI = 1.168-2.145, p = 0.003), with OR for the C/C versus C/G and G/G genotypes was 1.967 (95 % CI = 1.152-3.358, p = 0.0119). The OR for the STAT4 C allele frequency showed a 1.539-fold increased risk of SLE (95 % CI = 1.209-1.959, p = 0.0004). We also observed an increased frequency of STAT4 C/C and C/G genotypes in SLE patients with renal symptoms OR = 2.259 (1.365-3.738, p = 0.0014), (p (corr) = 0.0238) and in SLE patients with neurologic manifestations OR = 2.867 (1.467-5.604, p = 0.0016), (p (corr) = 0.0272). Moreover, we found a contribution of STAT4 C/C and C/G genotypes to the presence of the anti-snRNP Ab OR = 3.237 (1.667-6.288, p = 0.0003), (p (corr) = 0.0051) and the presence of the anti-Scl-70 Ab OR = 2.665 (1.380-5.147, p = 0.0028), (p (corr) = 0.0476). Our studies confirmed an association of the STAT4 C (rs7582694) variant with the development of SLE and occurrence of some clinical manifestations of the disease.