Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.     

Distribution and diversity of Russian wheat aphid (Hemiptera: Aphididae) biotypes in South Africa and Lesotho

Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae) was recorded for the first time in South Africa in 1978. In 2005, a second biotype, RWASA2, emerged, and here we report on the emergence of yet another biotype, found for the first time in 2009. The discovery of new Russian wheat aphid biotypes is a significant challenge to the wheat, Triticum aestivum L., industry in South Africa. Russian wheat aphid resistance in wheat, that offered wheat producers a long-term solution to Russian wheat aphid control, may no longer be effective in areas where the new biotypes occur. It is therefore critical to determine the diversity and extent of distribution of biotypes in South Africa to successfully deploy Russian wheat aphid resistance in wheat. Screening of 96 Russian wheat aphid clones resulted in identification of three Russian wheat aphid biotypes. Infestations of RWASA1 caused susceptible damage symptoms only in wheat entries containing the Dn3 gene. Infestations of RWASA2 caused susceptible damage symptoms in wheat entries containing Dn1, Dn2, Dn3, and Dn9 resistant genes. Based on the damage-rating scores for the seven resistance sources, a new biotype, which caused damage rating scores different from those for RWASA1 and RWASA2, was evident among the Russian wheat aphid populations tested. This new biotype is virulent to the same resistance sources as RWASA2 (Dn1, Dn2, Dn3, and Dn9), but it also has added virulence to Dn4, whereas RWASA2 is avirulent to this resistance source.     

The BAR domain protein PICK1 regulates cell recognition and morphogenesis by interacting with Neph proteins

Neph proteins are evolutionarily conserved membrane proteins of the immunoglobulin superfamily that control the formation of specific intercellular contacts. Cell recognition through these proteins is essential in diverse cellular contexts such as patterning of the compound eye in Drosophila melanogaster, neuronal connectivity in Caenorhabditis elegans, and the formation of the kidney filtration barrier in mammals. Here we identify the PDZ and BAR domain protein PICK1 (protein interacting with C-kinase 1) as a Neph-interacting protein. Binding required dimerization of PICK1, was dependent on PDZ domain protein interactions, and mediated stabilization of Neph1 at the plasma membrane. Moreover, protein kinase C (PKCα) activity facilitated the interaction through releasing Neph proteins from their binding to the multidomain scaffolding protein zonula occludens 1 (ZO-1), another PDZ domain protein. In Drosophila, the Neph homologue Roughest is essential for sorting of interommatidial precursor cells and patterning of the compound eye. RNA interference-mediated knockdown of PICK1 in the Drosophila eye imaginal disc caused a Roughest destabilization at the plasma membrane and a phenotype that resembled rst mutation. These data indicate that Neph proteins and PICK1 synergistically regulate cell recognition and contact formation.     

Degradation of azo dyes by laccase and ultrasound treatment

The goal of this work was to investigate the decomposition of azo dyes by oxidative methods, such as laccase and ultrasound treatments. Each of these methods has strong and feeble sides. The laccase treatment showed high decolorization rates but cannot degrade all investigated dyes (reactive dyes), and high anionic strength led to enzyme deactivation. Ultrasound treatment can decolorize all tested dyes after 3 h at a high energy input, and prolonged sonication leads to nontoxic ionic species, which was demonstrated by ion chromatography and toxicity assays. For the first time, it was shown that a combination of laccase and ultrasound treatments can have synergistic effects, which was shown by higher degradation rates. Bulk light absorption and ion-pairing high-performance liquid chromatography (IP-HPLC) were used for process monitoring, while with reversed-phase HPLC, a lower number of intermediates than expected by IP-HPLC was found. Liquid chromatography-mass spectrometry indicated that both acid orange dyes lead to a common end product due to laccase treatment. Acid Orange 52 is demethylated by laccase and ultrasound treatment. Further results confirmed that the main effect of ultrasound is based on *OH attack on the dye molecules.     

Distinct modes of vertebrate hypaxial muscle formation contribute to the teleost body wall musculature

The formation of the body wall musculature in vertebrates is assumed to be initiated by direct ventral extension of the somites/myotomes. This contrasts to the formation of limb muscles and muscles involved in feeding or respiration/ventilation, which are founded by migratory muscle precursors (MMPs) distant to the somites. Here, we present evidence from morphology and expression of molecular markers proposing that the formation of the two muscle layers of the teleost body wall involves both of the above mechanisms: (1) MMPs from somites 5 and 6 found an independent muscle primordium–the so-called posterior hypaxial muscle (PHM)–which subsequently gives rise to the most anterior two segments of the medial obliquus inferioris (OI) muscle. (2) Direct epithelial extension of the hypaxial myotomes generates the OI segments from somite 7 caudalward and the entire lateral obliquus superioris (OS) muscle. The findings are discussed in relation to the evolution of hypaxial myogenic patterning including functional considerations. We hypothesise that the potential of the most anterior somites to generate migratory muscle precursors is a general vertebrate feature that has been differently utilised in the evolution in vertebrate groups.     

A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.     

Haptic Search for Hard and Soft Spheres

In this study the saliency of hardness and softness were investigated in an active haptic search task. Two experiments were performed to explore these properties in different contexts. In Experiment 1, blindfolded participants had to grasp a bundle of spheres and determine the presence of a hard target among soft distractors or vice versa. If the difference in compliance between target and distractors was small, reaction times increased with the number of items for both features; a serial strategy was found to be used. When the difference in compliance was large, the reaction times were independent of the number of items, indicating a parallel strategy. In Experiment 2, blindfolded participants pressed their hand on a display filled with hard and soft items. In the search for a soft target, increasing reaction times with the number of items were found, but the location of target and distractors appeared to have a large influence on the search difficulty. In the search for a hard target, reaction times did not depend on the number of items. In sum, this showed that both hardness and softness are salient features.     

Hypochlorite-modified high density lipoprotein,a high affinity ligand to scavenger receptor class B,type I,impairs high density lipoprotein-dependent selective lipid uptake and reverse cholesterol transport

Hypochlorous acid/hypochlorite (HOCl/OCl(-)), a potent oxidant generated in vivo by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, alters the physiological properties of high density lipoprotein (HDL) by generating a proatherogenic lipoprotein particle. On endothelial cells lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) and scavenger receptor class B, type I (SR-BI), act in concert by mediating the holoparticle of and selective cholesteryl ester uptake from HOCl-HDL. We therefore investigated the ligand specificity of HOCl-HDL to SR-BI-overexpressing Chinese hamster ovary cells. Binding of HOCl-HDL was saturable, and the degree of HOCl modification was the determining factor for increased binding affinity to SR-BI. Competition experiments further confirmed that HOCl-HDL binds with increased affinity to the same or overlapping domain(s) of SR-BI as does native HDL. Furthermore, SR-BI-mediated selective HDL-cholesteryl ester association as well as time- and concentration-dependent cholesterol efflux from SR-BI overexpressing Chinese hamster ovary cells were, depending on the degree of HOCl modification of HDL, markedly impaired. The most significant findings of this study were that the presence of very low concentrations of HOCl-HDL severely impaired SR-BI-mediated bidirectional cholesterol flux mediated by native HDL. The colocalization of immunoreactive HOCl-modified epitopes with apolipoprotein A-I along with deposits of lipids in serial sections of human atheroma shown here indicates that the myeloperoxidase-H(2)O(2)-halide system contributes to oxidative damage of HDL in vivo.     

Fluorescence in situ hybridization test for detection of endometrial carcinoma cells by non-invasive vaginal swab

Endometrial cancer (EC) is the most common gynaecological malignancy with increasing incidence in developed countries. As gold standard, hysteroscopy confirms only 30% of suspected ECs. The detection of EC cells in the vagina by fluorescence in situ hybridization (FISH) after a smear test could reduce invasive procedures in the future. Using array-based comparative genome hybridization (aCGH) on 65 endometrial carcinomas, most frequently imbalanced regions of the tumour genome were identified. Bacterial artificial chromosomes were used to generate FISH-probes homologue to these human regions. The FISH test was hybridized on swabs specimens collected from the vaginal cavity. Samples from six patients without EC were selected as a negative control and on 13 patients with known EC as a positive control. To distinguish between benign and EC cases, the cut-off value has been defined. A first validation of this EC-FISH Test was performed with swabs from 41 patients with suspected EC. The most common genomic imbalances in EC are around the CTNNB1, FBXW7 and APC genes. The cut-off is defined at 32% of analysed cells without diploid signal pattern. This differs significantly between the positive and negative controls (p < 0.001). In a first validation cohort of 41 patients with suspected EC, the EC-FISH Test distinguishes patients with and without EC with a sensitivity of 91% and a specificity of 83%. The negative predictive value is 96%. This is the first report of a non-invasive EC-FISH Test to predict EC in women with suspected EC.