Spherical shield Patent

Flexible bellows joint shielding sleeve for propellant transfer pipeline

Cross-spectral analysis of physiological tremor and muscle activity. I. Theory and application to unsynchronized EMG

We investigate the relationship between the extensor electromyogram (EMG) and tremor time series in physiological hand tremor by cross-spectral analysis. Special attention is directed to the phase spectrum and the effects of observational noise. We calculate the theoretical phase spectrum for a second order linear stochastic process and compare the results to measured tremor data recorded from subjects who did not show a synchronized EMG activity in the corresponding extensor muscle. The results show that physiological tremor is well described by the proposed model and that the measured EMG represents a Newtonian force by which the muscle acts on the hand.Comment: 9 pages, 6 figures, to appear in Biological Cybernetic

Characterization of three vasopressin receptor 2 variants: an apparent polymorphism (V266A) and two loss-of-function mutations (R181C and M311V).

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown

Computational redesign of acyl-ACP thioesterase with improved selectivity towards medium chain fatty acids at high production levels

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Our computational procedure was demonstrated by tuning substrate binding of the highly-active ‘TesA thioesterase in Escherichia coli in favor of medium-chain (C6-C12) lengths. Specifically, the Iterative Protein Redesign & Optimization procedure (IPRO) was used to design ‘TesA variants with enhanced C12- or C8specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8), the fatty acid products of thioesterase-mediated catalysis. The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A similar sized library created through random mutagenesis failed to identify medium-chain specific, highly-active variants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. In this work, we not only successfully modified ‘TesA substrate preference but in doing so, we identified the third most C12-specific and tenth most C8-specific thioesterase characterized to date. These results are significant because medium-chain fatty acids are limited in natural abundance relative to long-chain fatty acids. This limited supply leads to high costs of C6-C10 oleochemicals such as fatty alcohols, amines, and esters. We expect that the new thioesterase variants will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals. Furthermore, we anticipate that the lessons learned from both successful and failed computational designs can guide algorithmic advancements aiding with future enzyme engineering endeavors

Increased Expression of Cell-Cell Signaling Genes by Stimulated Mononuclear Leukocytes in Patients with Previous Atherothrombotic Stroke A Whole Genome Expression Profile Study

Background/Aims: Inflammation plays an important role in atherosclerosis and stroke. Acute infections are recognized as trigger factors for ischemic stroke. Methods: In this whole genome expression profile study of 15 patients and 15 control subjects, we tested the hypothesis that patients with a history of atherothrombotic stroke show enhanced transcription of inflammatory genes in circulating leukocytes. RNA from unstimulated or lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) was analyzed with Affymetrix U133A GeneChips using a pooling design. Expression of single genes and functional groups of genes was analyzed by global statistical tests. Results: A total of 10,197 probe sets showed positive calls. After correction for multiple testing no single probe set revealed significant differences either without or with LPS stimulation. However, significant global expression differences were found upon LPS stimulation for the group of genes that are involved in cell-cell signaling. Conclusion: LPS stimulation of PBMCs, a condition mimicking bacterial infection, induces differential expression of a group of cell-cell signaling genes in patients with previous atherothrombotic stroke. This finding can be caused by genetic differences between both groups, but acquired risk factors, medication and technical factors may also have contributed to the result. Copyright (C) 2009 S. Karger AG, Base

CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes

© 2020 The Authors G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels

Die Spektren der Protonen aus (d,p)-Reaktionen an schweren Kernenbei Deuteronenenergien unter 12 MeV.

The proton spectra of the (d,p)-reactions on heavy nuclei show, in addition to the low energy continuum and the gross structure at higher energies, a pronounced proton group. The relative intensity of the proton group increases with decreasing energy of the primary deuterons. The dependence of the spectra on the atomic number of the target and the energy dependence and angular distribution for gold have been measured. The results are explained in terms of the properties of the nuclear surface