Andreev Modes from Phase Winding in a Full-Shell Nanowire-Based Transmon.

We investigate transmon qubits made from semiconductor nanowires with a fully surrounding superconducting shell. In the regime of reentrant superconductivity associated with the destructive Little-Parks effect, numerous coherent transitions are observed in the first reentrant lobe, where the shell carries 2π winding of superconducting phase, and are absent in the zeroth lobe. As junction density was increased by gate voltage, qubit coherence was suppressed then lost in the first lobe. These observations and numerical simulations highlight the role of winding-induced Andreev states in the junction.

Near-infrared scanning cavity ringdown for optical loss characterization of supermirrors.

A cavity ringdown system for probing the spatial variation of optical loss across high-reflectivity mirrors is described. This system is employed to examine substrate-transferred crystalline supermirrors and to quantify the effect of manufacturing process imperfections. Excellent agreement is observed between the ringdown-generated spatial measurements and differential interference contrast microscopy images. A 2-mm diameter ringdown scan in the center of a crystalline supermirror reveals highly uniform coating properties with excess loss variations below 1 ppm.

Introduction of a C-terminal hexa-lysine tag increases thermal stability of the LacDiNac binding adhesin (LabA) exodomain from Helicobacter pylori.

Helicobacter pylori is a pathogenic microorganism infecting approximately 50% of the global population, and establishes life-long colonization despite the hostile stomach environment. H. pylori employs a wide range of outer membrane proteins (adhesins) for epithelial attachment, which specifically bind to glycans or non-carbohydrate structures expressed on the gastric epithelium. A recently described adhesin from H. pylori is LabA, named after its ability to bind to a disaccharide present in gastric mucus (LacdiNAc-specific adhesin). Here, we describe the recombinant expression of LabA from H. pylori strains J99 and 26695 in E. coli. High yields of recombinant LabA were obtained using periplasmic expression. We found that the addition of a C-terminal hexalysine (6K) tag enhanced the thermal stability of LabA without affecting its secondary structure, using differential scanning fluorimetry and circular dichroism spectroscopy. In contrast to our previous report for another H. pylori adhesin (BabA), the 6K tag did not enhance recombinant protein yield or solubility. Both versions of LabA, with or without the 6K tag, were expressed and isolated from the periplasmic space of Escherichia coli, with a surprisingly high yield of at least 40 mg/L for each independent preparation, following a two-step purification protocol. The proteins were analyzed with mass spectrometry (MS). Unlike its reported effect on stability of BabA, the 6K tag did not appear to protect the N-term of recombinant LabA from partial periplasmic degradation.

Post-transcriptional Control of Tumor Cell Autonomous Metastatic Potential by CCR4-NOT Deadenylase CNOT7.

Accumulating evidence supports the role of an aberrant transcriptome as a driver of metastatic potential. Deadenylation is a general regulatory node for post-transcriptional control by microRNAs and other determinants of RNA stability. Previously, we demonstrated that the CCR4-NOT scaffold component Cnot2 is an inherited metastasis susceptibility gene. In this study, using orthotopic metastasis assays and genetically engineered mouse models, we show that one of the enzymatic subunits of the CCR4-NOT complex, Cnot7, is also a metastasis modifying gene. We demonstrate that higher expression of Cnot7 drives tumor cell autonomous metastatic potential, which requires its deadenylase activity. Furthermore, metastasis promotion by CNOT7 is dependent on interaction with CNOT1 and TOB1. CNOT7 ribonucleoprotein-immunoprecipitation (RIP) and integrated transcriptome wide analyses reveal that CNOT7-regulated transcripts are enriched for a tripartite 3'UTR motif bound by RNA-binding proteins known to complex with CNOT7, TOB1, and CNOT1. Collectively, our data support a model of CNOT7, TOB1, CNOT1, and RNA-binding proteins collectively exerting post-transcriptional control on a metastasis suppressive transcriptional program to drive tumor cell metastasis.

The enzyme activities of Caf1 and Ccr4 are both required for deadenylation by the human Ccr4-Not nuclease module.

In eukaryotic cells, the shortening and removal of the poly(A) tail (deadenylation) of cytoplasmic mRNA is a key event in regulated mRNA degradation. A major enzyme involved in deadenylation is the Ccr4-Not deadenylase complex, which can be recruited to its target mRNA by RNA-binding proteins or the miRNA repression complex. In addition to six non-catalytic components, the complex contains two enzymatic subunits with ribonuclease activity: Ccr4 and Caf1 (Pop2). In vertebrates, each deadenylase subunit is encoded by two paralogues: Caf1, which can interact with the anti-proliferative protein BTG2, is encoded by CNOT7 and CNOT8, whereas Ccr4 is encoded by the highly similar genes CNOT6 and CNOT6L. Currently, it is unclear whether the catalytic subunits work co-operatively or whether the nuclease components have unique roles in deadenylation. We therefore developed a method to express and purify a minimal human BTG2-Caf1-Ccr4 nuclease sub-complex from bacterial cells. By using chemical inhibition and well-characterized inactivating amino acid substitutions, we demonstrate that the enzyme activities of Caf1 and Ccr4 are both required for deadenylation in vitro. These results indicate that Caf1 and Ccr4 cooperate in mRNA deadenylation and suggest that the enzyme activities of Caf1 and Ccr4 are regulated via allosteric interactions within the nuclease module.

A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity.

In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in post-transcriptional gene regulation. The ribonuclease activity of deadenylase enzymes is attributed to either a DEDD (Asp-Glu-Asp-Asp) or an endonuclease-exonuclease-phosphatase domain. Both domains require the presence of two Mg2+ ions in the active site. To facilitate the biochemical analysis of deadenylase enzymes, we have developed a fluorescence-based deadenylase assay. The assay is based on end-point measurement, suitable for quantitative analysis and can be adapted for 96- and 384-well microplate formats. We demonstrate the utility of the assay by screening a chemical compound library, resulting in the identification of non-nucleoside inhibitors of the Caf1/CNOT7 enzyme, a catalytic subunit of the Ccr4-Not deadenylase complex. These compounds may be useful tools for the biochemical analysis of the Caf1/CNOT7 deadenylase subunit of the Ccr4-Not complex and indicate the feasibility of developing selective inhibitors of deadenylase enzymes using the fluorescence-based assay.

[Improvement of Psychosomatic Rehabilitation after Prestationary Intervention]. / Verbesserung des Rehabilitationsergebnis durch prästationäre Intervention.

Aim of the study: Improvement of psychosomatic rehabilitation efforts with prestationary intervention. Method: The study is designed as a prospective and randomisized interventon study including 317 in patients. Result: Most of the patients were women (69.4 %), the mean age was 50.2 years. As measured with the BDI-II patients with prestationary intervention improved more than patients without intervention. The motivation has not been changed significantly in both treatment arms. Various independent cofactors like long duration of unemployment, disablement and patients who apply to pension were identified. Conclusion: Finally a prestationary telephon interview improves the results of psychosomatic rehabilitation measured with BDI.

Improved expression and purification of the Helicobacter pylori adhesin BabA through the incorporation of a hexa-lysine tag.

Helicobacter pylori is a pathogenic bacterium that has the remarkable ability to withstand the harsh conditions of the stomach for decades. This is achieved through unique evolutionary adaptations, which include binding Lewis(b) antigens found on the gastric epithelium using the outer membrane protein BabA. We show here the yield of a recombinant form of BabA, comprising its putative extracellular binding domain, can be significantly increased through the addition of a hexa-lysine tag to the C-terminus of the protein. BabA was expressed in the periplasmic space of Escherichia coli and purified using immobilised metal ion affinity and size exclusion chromatography - yielding approximately 1.8 mg of protein per litre of culture. The hexa-lysine tag does not inhibit the binding activity of BabA as the recombinant protein was found to possess affinity towards HSA-Lewis(b) glycoconjugates.

Phosphonium Polymethacrylates for Short Interfering RNA Delivery: Effect of Polymer and RNA Structural Parameters on Polyplex Assembly and Gene Knockdown.

Synthetic polymers containing quaternary phosphonium salts are an emerging class of materials for the delivery of oligo/polynucleotides. In this work, cationic phosphonium salt-containing polymethacrylates and their corresponding ammonium analogues were synthesized by reversible addition-fragmentation chain transfer polymerization. Both the nature of the charged heteroatom (N vs P) and the length of the spacer separating the cationic units along the polymer backbone (oxyethylene vs trioxyethylene) were systematically varied. Polymers efficiently bound short interfering RNA (siRNA) at N(+)/P(-) or P(+)/P(-) ratios of 2 and above. At a 20:1 ratio, small polyplexes (Rh: 4-15 nm) suitable for cellular uptake were formed that displayed low cytotoxicity. While siRNA polyplexes from both ammonium and phosphonium polymers were efficiently internalized by green fluorescent protein (GFP)-expressing 3T3 cells, no knockdown of GFP expression was observed. However, 65% Survivin gene knockdown was observed when siRNA was replaced with novel, multimerized long interfering RNA in HeLa cells, demonstrating the importance of RNA macromolecular architecture on RNA-mediated gene silencing.

Discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as inhibitors of the human poly(A)-selective ribonuclease Caf1.

Eukaryotic mRNA contains a 3' poly(A) tail, which plays important roles in the regulation of mRNA stability and translation. Well-characterized enzymes involved in the shortening of the poly(A) tail include the multi-subunit Ccr4-Not deadenylase, which contains the Caf1 (Pop2) and Ccr4 catalytic components, and poly(A)-specific ribonuclease (PARN). Two Mg(2+) ions present in the active sites of these ribonucleases are required for RNA cleavage. Here, we report the discovery, synthesis and biochemical profiling of purine-2,6-dione derivatives as (sub)micromolar inhibitors of Caf1.

Comprehensive study on the light shielding potential of thermotropic layers for the development of new materials.

In recent years thermotropic overheating protection glazings have been the focus for both solar thermal collector technology and architecture. A thermotropic glazing changes its light transmittance from highly transparent to light diffusing upon reaching a certain threshold temperature autonomously and reversibly. In thermotropic systems with fixed domains (TSFD) the scattering domains are embedded in a polymer matrix, which exhibits a sudden change of the refractive index upon reaching a threshold temperature. The aim of the present study was to comprehensively investigate the light shielding characteristics and potential of TSFD materials by applying simulation of light scattering in particle-filled layers. In random walk simulations a variety of parameters were varied systematically, and the effect on the light transmission behavior of TSFD was studied. The calculation steps of the simulation process are shown in detail. The simulations demonstrate that there is great potential for the production of functional materials with high overheating protection efficiency.

RNA decay machines: deadenylation by the Ccr4-not and Pan2-Pan3 complexes.

Shortening and removal of the 3' poly(A) tail of mature mRNA by poly(A)-specific 3' exonucleases (deadenylases) is the initial and often rate-limiting step in mRNA degradation. The majority of cytoplasmic deadenylase activity is associated with the Ccr4-Not and Pan2-Pan3 complexes. Two distinct catalytic subunits, Caf1/Pop2 and Ccr4, are associated with the Ccr4-Not complex, whereas the Pan2 enzymatic subunit forms a stable complex with Pan3. In this review, we discuss the composition and activity of these two deadenylases. In addition, we comment on generic and specific mechanisms of recruitment of Ccr4-Not and Pan2-Pan3 to mRNAs. Finally, we discuss specialised and redundant functions of the deadenylases and review the importance of Ccr4-Not subunits in the regulation of physiological processes. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells.

BACKGROUND: Post-translational modifications (PTMs) of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7. METHODS: Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR. RESULTS: Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2, and was prevented by siRNA targeting of SUV420H2. CONCLUSION: In summary, although garcinol and curcumin can both inhibit histone acetyltransferase activities, our results show that these compounds have differential effects on cancer cells in culture. Garcinol treatment alters expression of chromatin modifying enzymes in MCF7 cells, resulting in reprogramming of key histone and p53 PTMs and growth arrest, underscoring its potential as a cancer chemopreventive agent.

Disruption of the Mammalian Ccr4-Not Complex Contributes to Transcription-Mediated Genome Instability.

The mammalian Ccr4-Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4-Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4-Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S.

Deadenylation of cytoplasmic mRNA by the mammalian Ccr4-Not complex.

The Ccr4-Not complex is one of the major deadenylase factors present in eukaryotic cells. This multi-subunit protein complex is composed of at least seven stably associated subunits in mammalian cells including two enzymatic deadenylase subunits: one DEDD (Asp-Glu-Asp-Asp)-type deadenylase (either CNOT7/human Caf1/Caf1a or CNOT8/human Pop2/Caf1b/Calif) and one EEP (endonuclease-exonuclease-phosphatase)-type enzyme (either CNOT6/human Ccr4/Ccr4a or CNOT6L/human Ccr4-like/Ccr4b). Here, the role of the human Ccr4-Not complex in cytoplasmic deadenylation of mRNA is discussed, including the mechanism of its recruitment to mRNA and the role of the BTG/Tob proteins.

Anisotropic interface magnetoresistance in Pt/Co/Pt sandwiches.

We report on an effect of reduced dimensionality on the magnetotransport in cobalt layers sandwiched by platinum. In a current in-plane geometry it is found that the resistivity depends on the magnetization orientation within the plane perpendicular to the current direction. The resistivity shows a symmetry adapted cos(2) dependence on the angle to the surface normal, with the maximum along the surface normal. The Co thickness dependence of the effect in Pt/Co/Pt sandwiches clearly points out that the mechanism behind this effect originates at the Co/Pt interfaces and is disparate to the texture induced geometrical size effect.

Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori.

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcß1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 â€‹Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20-49%), LabA shares an L-shaped fold with SabA and BabA. The 'head' region contains a 4 â€‹+ â€‹3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain ('crown' region), the 'J99-type' (insertion ~31 â€‹amino acids), and the H. pylori '26695 type' (insertion ~46 â€‹amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcß1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding.

Mid-infrared interference coatings with excess optical loss below 10 ppm.

We present high-reflectivity substrate-transferred single-crystal GaAs/AlGaAs interference coatings at a center wavelength of 4.54 µm with record-low excess optical loss below 10 parts per million. These high-performance mirrors are realized via a novel microfabrication process that differs significantly from the production of amorphous multilayers generated via physical vapor deposition processes. This new process enables reduced scatter loss due to the low surface and interfacial roughness, while low background doping in epitaxial growth ensures strongly reduced absorption. We report on a suite of optical measurements, including cavity ring-down, transmittance spectroscopy, and direct absorption tests to reveal the optical losses for a set of prototype mirrors. In the course of these measurements, we observe a unique polarization-orientation-dependent loss mechanism which we attribute to elastic anisotropy of these strained epitaxial multilayers. A future increase in layer count and a corresponding reduction of transmittance will enable optical resonators with a finesse in excess of 100 000 in the mid-infrared spectral region, allowing for advances in high resolution spectroscopy, narrow-linewidth laser stabilization, and ultrasensitive measurements of various light-matter interactions.

The mammalian anti-proliferative BTG/Tob protein family.

The mammalian BTG/Tob family comprises six proteins (BTG1, BTG2/PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob and Tob2), which regulate cell cycle progression in a variety of cell types. They are characterised by the conserved N-terminal domain spanning 104-106 amino acids. Recent biochemical and structural data indicate that the conserved BTG domain is a protein-protein interaction module, which is capable of binding to DNA-binding transcription factors as well as the paralogues CNOT7 (human Caf1/Caf1a) and CNOT8 (human Pop2/Calif/Caf1b), two deadenylase subunits of the Ccr4-Not complex. Consistent with this finding, several members of the BTG/Tob family are shown to be implicated in transcription in the nucleus and cytoplasmic mRNA deadenylation and turnover. The C-terminal regions are less conserved and appear to mediate protein-protein interactions that are unique to each family member. The human and mouse BTG/Tob proteins will be the focus of this review and structural aspects of BTG/Tob interactions with components of the Ccr4-Not complex, and the role of the BTG/Tob proteins in the regulation of gene expression, tumourigenesis and cancer will be discussed.

Presence of cyclophilin A in synovial fluids of patients with rheumatoid arthritis.

Cyclophilins have been suggested to act as leukocyte chemotactic factors produced in the course of inflammation. Therefore we looked for the presence of cyclophilins in the synovial fluids (SF) from patients with rheumatoid arthritis (RA). Peptidyl prolyl cis-trans isomerase activity (PPIase) was measured in SF from knee punctures of 26 patients with RA and five patients with knee osteoarthritis (OA). PPIase was detected in SF from RA patients, but not in samples from OA patients. Enzyme activity was sensitive to inhibition by cyclosporin A (IC50 = 28-50 nM). Estimated concentrations of the SF-derived cyclophilin based on the enzyme activity were in the range of 11 to 705 nM. The presence of cyclophilin in the SF showed disease correlation; its concentration correlated with the number of cells in the SF (r = 0.91, P < 0.0001) and with the percentage of neutrophils in the cellular infiltrate and was higher in more acute cases of joint swelling. In immunoblots of partially purified preparations of SF from RA patients, an approximately 18-kD protein band reacted with polyclonal antibodies that recognize cyclophilin A and B, but not with antibodies specific for cyclophilin B. Sequencing of this protein revealed identity of the NH2-terminal amino acids with those of human cyclophilin A. The finding is unexpected since cyclophilin B rather than A is generally regarded as the secreted isoform, the presence of cyclophilin A being confined to the cytoplasm. Our data support the hypothesis that cyclophilins may contribute to the pathogenesis of inflammatory diseases, possibly by acting as cytokines. This may offer a possible explanation of the effectiveness of cyclosporin A in RA, in addition to the known immunosuppressive effects of the drug.