A Molecular Mechanism for Synapse Elimination: Novel Inhibition of Locally Generated Thrombin Delays Synapse Loss in Neonatal Mouse Muscle

AbstractActivity-dependent, polyneuronal synapse elimination (ADPSE) is a programmed, regressive event in the development of the nervous system and readily studied at the neuromuscular junction, where it is complete 15–20 days after birth. Local excess, or imbalanced, protease activity is one of several possible underlying mechanisms. In this regard, thrombin mediates activity-dependent synapse loss in anin vitromodel of ADPSE. To test the involvement of thrombinin vivo,we locally applied the leech thrombin-specific inhibitor, hirudin. We monitored neuromuscular behavior, correlated with acetylcholinesterase and silver nitrate histochemistry at endplates, for changes in the timecourse ofin vivosynapse elimination and assayed both thrombin activity and prothrombin expression in developing muscle. Hirudin retarded elimination, without altering motor performance, uniquely at Postnatal Day 5 (P5) and maximally at P9. Reverse transcription–polymerase chain reaction (PCR) showed that neonatal muscle was a source of local prothrombin, with peak expression during the first week after birth. A specific chromogenic assay revealed that local thrombin, activated from muscle-derived prothrombin, peaked during maximal synapse remodeling

Far-field probing of leaky topological states in all-dielectric metasurfaces

© 2018 The Author(s). Topological phase transitions in condensed matter systems give rise to exotic states of matter such as topological insulators, superconductors, and superfluids. Photonic topological systems open a whole new realm of research and technological opportunities, exhibiting a number of important distinctions from their condensed matter counterparts. Photonic modes can leak into free space, which makes it possible to probe topological photonic phases by spectroscopic means via Fano resonances. Based on this idea, we develop a technique to retrieve the topological properties of all-dielectric metasurfaces from the measured far-field scattering characteristics. Collected angle-resolved spectra provide the momentum-dependent frequencies and lifetimes of the photonic modes that enable the retrieval of the effective Hamiltonian and extraction of the topological invariant. Our results demonstrate how the topological states of open non-Hermitian systems can be explored via far-field measurements, thus paving a way to the design of metasurfaces with unique scattering characteristics controlled via topological effects

Enhanced light–matter interactions in dielectric nanostructures via machine-learning approach

A key concept underlying the specific functionalities of metasurfaces is the use of constituent components to shape the wavefront of the light on demand. Metasurfaces are versatile, novel platforms for manipulating the scattering, color, phase, or intensity of light. Currently, one of the typical approaches for designing a metasurface is to optimize one or two variables among a vast number of fixed parameters, such as various materials’ properties and coupling effects, as well as the geometrical parameters. Ideally, this would require multidimensional space optimization through direct numerical simulations. Recently, an alternative, popular approach allows for reducing the computational cost significantly based on a deep-learning-assisted method. We utilize a deep-learning approach for obtaining high-quality factor (high-Q) resonances with desired characteristics, such as linewidth, amplitude, and spectral position. We exploit such high-Q resonances for enhanced light–matter interaction in nonlinear optical metasurfaces and optomechanical vibrations, simultaneously. We demonstrate that optimized metasurfaces achieve up to 400-fold enhancement of the third-harmonic generation; at the same time, they also contribute to 100-fold enhancement of the amplitude of optomechanical vibrations. This approach can be further used to realize structures with unconventional scattering responses

V(D)J Recombination Excision Circles of B- and T-cells as Prognostic Marker in B-Cell Chronic Lymphocytic Leukemia

Background & Aims. T-cell receptor excision circles (TREC) and κ-deleting recombination excision circles (KREC) are extrachromosomal DNA segments generated during V(D)J re combination process that characterize the diversity of the antigen repertoire of T- and B-cells. The aim of our study is to identify the prognostic value of the excision circles in the chronic lymphocytic leukemia (CLL) setting. Methods. The excision circles’ levels were assessed by means of real time PCR in 109 patients with high-risk CLL and 16 matched healthy individuals. Results. KREC levels were signifi cantly (p < 0.001) lower in CLL patients vs. the reference group. TREC levels were lower in groups with unmutated status of immunoglobulin heavy chain variable region genes (p < 0.05) and 11q deletions (p < 0.1). Moreover, the KREC levels were higher in NOTCH1 mutation carriers than in noncarriers (p < 0.05). The comparison of treatment outcomes demonstrated a correlation between a high TREC level and achievement of complete remission. The prognostic value of the biomarker was confirmed by ROC-analysis: AUCTREC = 0.713 (p = 0.001) Conclusion. Association between excision circles’ levels and clinical/laboratory CLL prognostic factors, as well as complete remission achievement, makes possible the implementation of the test for early prediction of the treatment outcome

Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF

CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200

Postsynthesis modification of a cellulose acetate ultrafiltration membrane for applications in water and wastewater treatment

A technique for postsynthesis modification of a cellulose acetate ultrafiltration membrane with possible application in water and wastewater treatment is studied. The technique used an oxidizing agent (persulfate) to develop free radicals on the membrane surface, and that was expected to promote grafting of hydrophilic macromolecules (polyethylene glycol). A chain-transfer agent (2-mercaptoethanol) was tested to control the grafting process, avoiding the formation of long chains that usually lead to high permeability losses in other graft techniques. The modifications aimed at the decrease of the fouling susceptibility of the membrane studied. The possibility of an increase in rejection was also investigated. The membrane was characterized before and after modification, by attenuated total reflectance-Fourier transform-infrared spectroscopy, scanning electron microscopy, atomic force microscopy, and in terms of the rejection of neutral reference solutes. The information given by the different techniques of characterization provided strong evidences of the occurrence of modification, although permeation of (real) foulants was the decisive test. To obtain information about the fouling tendency of the nonmodified and modified membranes, two different kinds of foulants were used: a humic acid (usually found in surface waters) and textile auxiliaries (representing one of the most important industries in Portugal). The results showed an increase in the rejections of the humic acid, and significant improvements in the performance of the membrane with respect to fouling tendency in the case of the textile auxiliarie

Specificity of the E. coli LysR-Type Transcriptional Regulators

Families of paralogous oligomeric proteins are common in biology. How the specificity of assembly evolves is a fundamental question of biology. The LysR-Type Transcriptional Regulators (LTTR) form perhaps the largest family of transcriptional regulators in bacteria. Because genomes often encode many LTTR family members, it is assumed that many distinct homooligomers are formed simultaneously in the same cell without interfering with each other's activities, suggesting specificity in the interactions. However, this assumption has not been systematically tested.A negative-dominant assay with λcI repressor fusions was used to evaluate the assembly of the LTTRs in E. coli K-12. Thioredoxin (Trx)-LTTR fusions were used to challenge the homooligomeric interactions of λcI-LTTR fusions. Eight cI-LTTR fusions were challenged with twenty-eight Trx fusions. LTTRs could be divided into three classes based on their interactions with other LTTRs.Multimerization of LTTRs in E. coli K-12 is mostly specific. However, under the conditions of the assay, many LTTRs interact with more than one noncognate partner. The physiological significance and physical basis for these interactions are not known

Concerted loop motion triggers induced fit of FepA to ferric enterobactin

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles

Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG.

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features

Measurement and Interpretation of Fermion-Pair Production at LEP energies above the Z Resonance

This paper presents DELPHI measurements and interpretations of cross-sections, forward-backward asymmetries, and angular distributions, for the e+e- -> ffbar process for centre-of-mass energies above the Z resonance, from sqrt(s) ~ 130 - 207 GeV at the LEP collider. The measurements are consistent with the predictions of the Standard Model and are used to study a variety of models including the S-Matrix ansatz for e+e- -> ffbar scattering and several models which include physics beyond the Standard Model: the exchange of Z' bosons, contact interactions between fermions, the exchange of gravitons in large extra dimensions and the exchange of sneutrino in R-parity violating supersymmetry.Comment: 79 pages, 16 figures, Accepted by Eur. Phys. J.