Impaired Recruitment of Grk6 and β-Arrestin2 Causes Delayed Internalization and Desensitization of a WHIM Syndrome-Associated CXCR4 Mutant Receptor

WHIM (warts, hypogammaglobulinemia, infections, and myelokatexis) syndrome is a rare immunodeficiency syndrome linked to heterozygous mutations of the chemokine receptor CXCR4 resulting in truncations of its cytoplasmic tail. Leukocytes from patients with WHIM syndrome display impaired CXCR4 internalization and enhanced chemotaxis in response to its unique ligand SDF-1/CXCL12, which likely contribute to the clinical manifestations. Here, we investigated the biochemical mechanisms underlying CXCR4 deficiency in WHIM syndrome. We report that after ligand activation, WHIM-associated mutant CXCR4 receptors lacking the carboxy-terminal 19 residues internalize and activate Erk 1/2 slower than wild-type (WT) receptors, while utilizing the same trafficking endocytic pathway. Recruitment of β-Arrestin 2, but not β-Arrestin 1, to the active WHIM-mutant receptor is delayed compared to the WT CXCR4 receptor. In addition, while both kinases Grk3 and Grk6 bind to WT CXCR4 and are critical to its trafficking to the lysosomes, Grk6 fails to associate with the WHIM-mutant receptor whereas Grk3 associates normally. Since β-Arrestins and Grks play critical roles in phosphorylation and internalization of agonist-activated G protein-coupled receptors, these results provide a molecular basis for CXCR4 dysfunction in WHIM syndrome

Site‐specific encoding of photoactivity in antibodies enables light‐mediated antibody‐antigen binding on live cells

Antibodies have found applications in several fields, including, medicine, diagnostics, and nanotechnology, yet methods to modulate antibody‐antigen binding using an external agent remain limited. Here, we have developed photoactive antibody fragments by genetic site‐specific replacement of single tyrosine residues with photocaged tyrosine, in an antibody fragment, 7D12. A simple and robust assay is adopted to evaluate the light‐mediated binding of 7D12 mutants to its target, epidermal growth factor receptor (EGFR), on the surface of cancer cells. Presence of photocaged tyrosine reduces 7D12‐EGFR binding affinity by over 20‐fold in two out of three 7D12 mutants studied, and binding is restored upon exposure to 365 nm light. Molecular dynamics simulations explain the difference in effect of photocaging on 7D12‐EGFR interaction among the mutants. Finally, we demonstrate the application of photoactive antibodies in delivering fluorophores to EGFR‐positive live cancer cells in a light‐dependent manner

NCS-1 associates with adenosine A2A receptors and modulates receptor function

Modulation of G protein-coupled receptor (GPCR) signaling by local changes in intracellular calcium concentration is an established function of Calmodulin (CaM) which is known to interact with many GPCRs. Less is known about the functional role of the closely related neuronal EF-hand Ca2+-sensor proteins that frequently associate with CaM targets with different functional outcome. In the present study we aimed to investigate if a target of CaM—the A2A adenosine receptor is able to associate with two other neuronal calcium binding proteins (nCaBPs), namely NCS-1 and caldendrin. Using bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments we show the existence of A2A—NCS-1 complexes in living cells whereas caldendrin did not associate with A2A receptors under the conditions tested. Interestingly, NCS-1 binding modulated downstream A2A receptor intracellular signaling in a Ca2+-dependent manner. Taken together this study provides further evidence that neuronal Ca2+-sensor proteins play an important role in modulation of GPCR signaling

Cyclotides Isolated from an Ipecac Root Extract Antagonize the Corticotropin Releasing Factor Type 1 Receptor

Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants’ uterotonic properties and further establishing cyclotides as valuable source for GPCR ligand design. In this study we screened a cyclotide extract derived from the root powder of the South American medicinal plant ipecac (Carapichea ipecacuanha) for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRF1R). We identified and characterized seven novel cyclotides. One cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRF1R. A nanomolar concentration of this cyclotide (260 nM) reduced CRF potency by ∼4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V2 receptor, suggesting a CRF1R-specific mode-of-action. These results in conjunction with our previous findings establish cyclotides as modulators of both classes A and B GPCRs. Given the diversity of cyclotides, our data point to other cyclotide-GPCR interactions as potentially important sources of drug-like molecules

Ovarian Cancers Harbour Defects in Non-Homologous End Joining Resulting in Resistance to Rucaparib

Abstract Purpose: DNA damage defects are common in ovarian cancer and can be used to stratify treatment. Although most work has focused on homologous recombination (HR), DNA double-strand breaks are repaired primarily by nonhomologous end joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic instability and have been associated with the development of chemoresistance. Experimental Design: NHEJ was assessed in a panel of ovarian cancer cell lines and 47 primary ascetic-derived ovarian cancer cultures, by measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers. mRNA and protein expression of components of NHEJ was determined using RT-qPCR and Western blotting. Cytotoxicities of cisplatin and the PARP inhibitor rucaparib were assessed using sulforhodamine B (SRB) assays. HR function was assessed using γH2AX/RAD51 foci assay. Results: NHEJ was defective (D) in four of six cell lines and 20 of 47 primary cultures. NHEJ function was independent of HR competence (C). NHEJD cultures were resistant to rucaparib (P = 0.0022). When HR and NHEJ functions were taken into account, only NHEJC/HRD cultures were sensitive to rucaparib (compared with NHEJC/HRC P = 0.034, NHEJD/HRC P = 0.0002, and NHEJD/HRD P = 0.0045). The DNA-PK inhibitor, NU7441, induced resistance to rucaparib (P = 0.014) and HR function recovery in a BRCA1-defective cell line. Conclusions: This study has shown that NHEJ is defective in 40% of ovarian cancers, which is independent of HR function and associated with resistance to PARP inhibitors in ex vivo primary cultures. Clin Cancer Res; 23(8); 2050–60. ©2016 AACR.</jats:p

Orexin-Corticotropin-Releasing Factor Receptor Heteromers in the Ventral Tegmental Area as Targets for Cocaine

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking

Computational steering of a multi-objective evolutionary algorithm for engineering design

The execution process of an evolutionary algorithm typically involves some trial and error. This is due to the difficulty in setting the initial parameters of the algorithm—especially when little is known about the problem domain. This problem is magnified when applied to many-objective optimisation, as care is needed to ensure that the final population of candidate solutions is representative of the trade-off surface. We propose a computational steering system that allows the engineer to interact with the optimisation routine during execution. This interaction can be as simple as monitoring the values of some parameters during the execution process, or could involve altering those parameters to influence the quality of the solutions produced by the optimisation process. The implementation of this steering system should provide the ability to tailor the client to the hardware available, for example providing a lightweight steering and visualisation client for use on a PDA

Nasa's Launch Communications Ground Segment for the 21st Century Florida Spaceport

The National Aeronautics and Space Administration (NASA) Near Earth Network (NEN) Project is implementing a new launch communications ground segment to provide services for the next generation of human and robotic space exploration systems. It will deliver unique and advanced capabilities to accelerate the transformation of Kennedy Space Center into a multi-user spaceport in cooperation with the United States Air Force (USAF). The project has leveraged commercial technologies and remote operations concepts matured in NASAs orbiting satellite ground systems to achieve dramatic lifecycle cost efficiencies as compared to the space shuttle-era ground segment. The purpose of this paper is to discuss the development history, capabilities and anticipated use cases of the NEN Launch Communications Segment (NEN LCS).The NASA Kennedy Space Center is co-located with the USAF Eastern Launch Range at Cape Canaveral, Florida. The USAF operates two launch communications ground stations, but they are not designed to transmit voice, commands or other data to the launch vehicle or astronauts. The bi-directional uplink-downlink communications responsibility for human missions has historically resided with the Goddard Space Flight Center in Greenbelt, Maryland. Several market analyses and feasibility studies investigating concepts to provide NASAs next generation launch communications services were performed during the Constellation Program prior to its cancellation in 2009, and as part of the Kennedy Space Centers follow-on efforts to transform itself into a 21st century multi-user spaceport. In 2012, the Kennedy Space Center and the USAF 45th Space Wing jointly led a study to analyze the market needs of current and future launch systems and assess the operational deficiencies of the Eastern Range infrastructure. The study team issued several recommendations, two of which ultimately became driving operational capability requirements for the NEN LCS: increased telemetry data rates of at least 20 Mbps, and S-band uplink capability. Additional capabilities identified in the requirements development process include spread spectrum modulation support, LDPC 12 and 78 error correction codes, support for IRIG-106 and CCSDS data formats, automated best source selection, and Space Link Extension (SLE) services for data distribution. The NEN LCS is comprised of two permanent ground stations, the new Kennedy Uplink Station (KUS) and refurbished Ponce de Leon (PDL) station. Both stations are remotely operated from the Global Monitor and Control Center at Wallops Flight Facility. This core architecture is extensible through host-tenant arrangements with the U.S. Air Force and deployable assets, enabling agile, tailored and robust solutions to meet the needs of civil, commercial or military customers. The NEN LCS has three use cases:1.To provide agile, tailored and robust launch communications solutions to Florida spaceport customers2.To provide orbital communications services to near-earth customers 3.To provide an experimental proving ground for Space Mobile Network concepts and technologies The NEN LCS driving mission is to support the bi-directional link with the Orion crew capsule and two 20 Mbps telemetry links from the Space Launch System core stage on Exploration Mission-1, the first integrated flight of NASAs flagship human exploration systems

Long-lasting impairment of mGluR5-activated intracellular pathways in the striatum after withdrawal of cocaine self-administration

Background: Cocaine addiction continues to be a major heath concern, and despite public health intervention there is a lack of efficient pharmacological treatment options. A newly identified potential target are the group I metabotropic glutamate receptors (mGluR1/5), with allosteric modulators showing particular promise. Methods: We evaluated the capacity of mGluR1/5 receptors to induce functional responses in ex vivo striatal slices from rats with 1) acute cocaine self-administration (CSA), 2) chronic CSA and 3) 60 days CSA withdrawal by westernblot and extracellular recordings of synaptic transmission. Results: We found that striatal mGluR5 are the principal mediator of the mGluR1/5 agonist DHPG-induced CREB phosphorylation. Both acute and chronic CSA blunted mGluR1/5 effects on CREB phosphorylation in the striatum, which correlated with the capacity to induce long-term depression, an effect which was maintained 60 days after chronic CSA withdrawal. In the nucleus accumbens, the principal brain region mediating the rewarding effects of drugs, chronic CSA blunted mGluR1/5 stimulation of ERK1/2 and CREB. Interestingly, the mGluR5 antagonist/inverse-agonist, MPEP, lead to a specific increase in CREB phosphorylation after chronic CSA specifically in the nucleus accumbens, but not in the striatum. Conclusions: Prolonged CSA, through withdrawal, leads to a blunting of mGluR1/5 responses in the striatum. In addition, specifically in the accumbens, mGluR5 signaling to CREB shifts from an agonist-induced to an antagonist-induced CREB phosphorylation

Sexuality and Affection among Elderly German Men and Women in Long-Term Relationships: Results of a Prospective Population-Based Study

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The study was funded by the German Federal Ministry for Families, Senior Citizens, Women and Youth (AZ 314-1722-102/16; AZ 301-1720-295/2), the Ministry for Science, Research and Art Baden-Württemberg, and the University of Rostock (FORUN 989020; 889048)