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https://www.readbyqxmd.com/read/28815497/viral-delivery-of-gfp-dependent-recombinases-to-the-mouse-brain
#1
Jonathan C Y Tang, Stephanie Rudolph, Constance L Cepko
Many genetic tools have been developed that use green fluorescent protein (GFP) and its derivatives for labeling specific cell populations in organisms and in cell culture. To extend the use of GFP beyond labeling purposes, we developed methods and reagents that use GFP as a driver of biological activities. We used nanobodies that bind GFP to engineer CRE-DOG and Flp-DOG, recombinases that can induce Cre/lox and Flp/FRT recombination in a GFP-dependent manner, respectively. Here, we present a protocol to deliver CRE-DOG and Flp-DOG into the mouse brain by recombinant AAV infection...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28811312/myosin-ii-is-not-required-for-drosophila-tracheal-branch-elongation-and-cell-intercalation
#2
Amanda Ochoa-Espinosa, Stefan Harmansa, Emmanuel Caussinus, Markus Affolter
The Drosophila tracheal system consists of an interconnected network of monolayered epithelial tubes that ensures oxygen transport in the larval and adult body. During tracheal dorsal branch (DB) development, individual DBs elongate as a cluster of cells, led by tip cells at the front and trailing cells in the rear. Branch elongation is accompanied by extensive cell intercalation and cell lengthening of the trailing stalk cells. Although cell intercalation is governed by Myosin II (MyoII)-dependent forces during tissue elongation in the Drosophila embryo that lead to germ-band extension, it remained unclear whether MyoII plays a similar active role during tracheal branch elongation and intercalation...
August 15, 2017: Development
https://www.readbyqxmd.com/read/28796912/nanobody-mediated-resistance-to-grapevine-fanleaf-virus-in-plants
#3
Caroline Hemmer, Samia Djennane, Léa Ackerer, Kamal Hleibieh, Aurélie Marmonier, Sophie Gersch, Shahinez Garcia, Emmanuelle Vigne, Véronique Komar, Mireille Perrin, Claude Gertz, Lorène Belval, François Berthold, Baptiste Monsion, Corinne Schmitt-Keichinger, Olivier Lemaire, Bernard Lorber, Carlos Gutiérrez, Serge Muyldermans, Gérard Demangeat, Christophe Ritzenthaler
Since their discovery, single-domain antigen-binding fragments of camelid-derived heavy chain-only antibodies, also known as Nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode-transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient...
August 10, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28772027/escherichia-coli-surface-display-for-the-selection-of-nanobodies
#4
REVIEW
Valencio Salema, Luis Ángel Fernández
Nanobodies (Nbs) are the smallest functional antibody fragments known in nature and have multiple applications in biomedicine or environmental monitoring. Nbs are derived from the variable segment of camelid heavy chain-only antibodies, known as VHH. For selection, libraries of VHH gene segments from naïve, immunized animals or of synthetic origin have been traditionally cloned in E. coli phage display or yeast display systems, and clones binding the target antigen recovered, usually from plastic surfaces with the immobilized antigen (phage display) or using fluorescence-activated cell sorting (FACS; yeast display)...
August 3, 2017: Microbial Biotechnology
https://www.readbyqxmd.com/read/28771239/use-of-fluorescence-detected-sedimentation-velocity-to-study-high-affinity-protein-interactions
#5
Sumit K Chaturvedi, Jia Ma, Huaying Zhao, Peter Schuck
Sedimentation velocity (SV) analytical ultracentrifugation (AUC) is a classic technique for the real-time observation of free macromolecular migration in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size distribution, and interactions. Although traditionally limited to determination of the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in marked improvements in detection sensitivity and size resolution during the past decades...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28768817/cxcr4-specific-nanobodies-as-potential-therapeutics-for-whim-syndrome
#6
Raymond H de Wit, Raimond Heukers, Hendrik Brink, Angela Arsova, David Maussang, Pasquale Cutolo, Beatrijs Strubbe, Henry Vischer, Francoise Bachelerie, Martine J Smit
WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: Warts, Hypogammaglobulinemia, Infections and Myelokathexis. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4. Consequently, these CXCR4-WHIM mutants have a gain of function in response to its ligand CXCL12, which results in abnormal leukocyte migration and thereby immune system dysfunctions. Treatment of WHIM patients currently consists of symptom relief, leading to unsatisfactory clinical responses...
August 2, 2017: Journal of Pharmacology and Experimental Therapeutics
https://www.readbyqxmd.com/read/28754949/cell-permeable-nanobodies-for-targeted-immunolabelling-and-antigen-manipulation-in-living-cells
#7
Henry D Herce, Dominik Schumacher, Anselm F L Schneider, Anne K Ludwig, Florian A Mann, Marion Fillies, Marc-André Kasper, Stefan Reinke, Eberhard Krause, Heinrich Leonhardt, M Cristina Cardoso, Christian P R Hackenberger
Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments...
August 2017: Nature Chemistry
https://www.readbyqxmd.com/read/28741941/efficient-delivery-of-quantum-dots-into-the-cytosol-of-cells-using-cell-penetrating-poly-disulfide-s
#8
Emmanuel Derivery, Eline Bartolami, Stefan Matile, Marcos Gonzalez-Gaitan
Quantum dots (QDs) are extremely bright, photostable, nanometer particles broadly used to investigate single molecule dynamics in vitro. However, the use of QDs in vivo to investigate single molecule dynamics is impaired by the absence of an efficient way to chemically deliver them into the cytosol of cells. Indeed, current methods (using cell-penetrating peptides for instance) provide very low yields: QDs stay at the plasma membrane or are trapped in endosomes. Here, we introduce a technology based on cell-penetrating poly(disulfide)s that solves this problem: we deliver about 70 QDs per cell, and 90% appear to freely diffuse in the cytosol...
August 2, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28731329/generation-and-characterization-of-anti-vglut-nanobodies-acting-as-inhibitors-of-transport
#9
COMPARATIVE STUDY
Stephan Schenck, Laura Kunz, Daniela Sahlender, Els Pardon, Eric R Geertsma, Iaroslav Savtchouk, Toshiharu Suzuki, Yvonne Neldner, Saša Štefanić, Jan Steyaert, Andrea Volterra, Raimund Dutzler
The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library...
August 1, 2017: Biochemistry
https://www.readbyqxmd.com/read/28725224/nanobody-technology-a-versatile-toolkit-for-microscopic-imaging-protein-protein-interaction-analysis-and-protein-function-exploration
#10
REVIEW
Els Beghein, Jan Gettemans
Over the last two decades, nanobodies or single-domain antibodies have found their way in research, diagnostics, and therapy. These antigen-binding fragments, derived from Camelid heavy chain only antibodies, possess remarkable characteristics that favor their use over conventional antibodies or fragments thereof, in selected areas of research. In this review, we assess the current status of nanobodies as research tools in diverse aspects of fundamental research. We discuss the use of nanobodies as detection reagents in fluorescence microscopy and focus on recent advances in super-resolution microscopy...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28720704/cytosolic-interaction-of-type-iii-human-cd38-with-cib1-modulates-cellular-cyclic-adp-ribose-levels
#11
Jun Liu, Yong Juan Zhao, Wan Hua Li, Yun Nan Hou, Ting Li, Zhi Ying Zhao, Cheng Fang, Song Lu Li, Hon Cheung Lee
CD38 catalyzes the synthesis of the Ca(2+) messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca(2+) stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells...
July 18, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28713367/nanobodies-as-tools-to-understand-diagnose-and-treat-african-trypanosomiasis
#12
REVIEW
Benoit Stijlemans, Patrick De Baetselier, Guy Caljon, Jan Van Den Abbeele, Jo A Van Ginderachter, Stefan Magez
African trypanosomes are strictly extracellular protozoan parasites that cause diseases in humans and livestock and significantly affect the economic development of sub-Saharan Africa. Due to an elaborate and efficient (vector)-parasite-host interplay, required to complete their life cycle/transmission, trypanosomes have evolved efficient immune escape mechanisms that manipulate the entire host immune response. So far, not a single field applicable vaccine exists, and chemotherapy is the only strategy available to treat the disease...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28690114/maintenance-of-miranda-localization-in-drosophila-neuroblasts-involves-interaction-with-the-cognate-mrna
#13
Anne Ramat, Matthew Hannaford, Jens Januschke
How cells position their proteins is a key problem in cell biology. Targeting mRNAs to distinct regions of the cytoplasm contributes to protein localization by providing local control over translation. Here, we reveal that an interdependence of a protein and cognate mRNA maintains asymmetric protein distribution in mitotic Drosophila neural stem cells. We tagged endogenous mRNA or protein products of the gene miranda that is required for fate determination with GFP. We find that the mRNA localizes like the protein it encodes in a basal crescent in mitosis...
July 24, 2017: Current Biology: CB
https://www.readbyqxmd.com/read/28687476/actin-nanobodies-uncover-the-mystery-of-actin-filament-dynamics-in-toxoplasma-gondii
#14
Isabelle Tardieux
While the intracellular parasite Toxoplasma relies on a divergent actomyosin motor to support unique speeds in directional movement, the dynamics and architecture of parasite actin filaments remain a much-discussed issue. Using actin chromobodies, Periz et al. started to unveil how networks of dynamic F-actin connect Toxoplasma progeny and expand in the replicative vacuole.
August 2017: Trends in Parasitology
https://www.readbyqxmd.com/read/28675373/dpp-from-the-anterior-stripe-of-cells-is-crucial-for-the-growth-of-the-drosophila-wing-disc
#15
Shinya Matsuda, Markus Affolter
The Dpp morphogen gradient derived from the anterior stripe of cells is thought to control growth and patterning of the Drosophila wing disc. However, the spatial-temporal requirement of dpp for growth and patterning remained largely unknown. Recently, two studies re-addressed this question. By generating a conditional null allele, one study proposed that the dpp stripe is critical for patterning but not for growth. In contrast, using a membrane-anchored nanobody to trap Dpp, the other study proposed that Dpp dispersal from the stripe is required for patterning and also for medial wing disc growth, at least in the posterior compartment...
July 4, 2017: ELife
https://www.readbyqxmd.com/read/28673288/nanobodies-raised-against-monomeric-%C3%A9-synuclein-inhibit-fibril-formation-and-destabilize-toxic-oligomeric-species
#16
Marija Iljina, Liu Hong, Mathew H Horrocks, Marthe H Ludtmann, Minee L Choi, Craig D Hughes, Francesco S Ruggeri, Tim Guilliams, Alexander K Buell, Ji-Eun Lee, Sonia Gandhi, Steven F Lee, Clare E Bryant, Michele Vendruscolo, Tuomas P J Knowles, Christopher M Dobson, Erwin De Genst, David Klenerman
BACKGROUND: The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson's disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS...
July 3, 2017: BMC Biology
https://www.readbyqxmd.com/read/28668496/generation-of-nanobodies-against-slyd-and-development-of-tools-to-eliminate-this-bacterial-contaminant-from-recombinant-proteins
#17
Yaozhong Hu, Ema Romão, Didier Vertommen, Cécile Vincke, Francisco Morales-Yánez, Carlos Gutiérrez, Changxiao Liu, Serge Muyldermans
The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response...
September 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28659622/determination-of-aspergillus-pathogens-in-agricultural-products-by-a-specific-nanobody-polyclonal-antibody-sandwich-elisa
#18
Ting Wang, Peiwu Li, Qi Zhang, Wen Zhang, Zhaowei Zhang, Tong Wang, Ting He
Aspergillus and its poisonous mycotoxins are distributed worldwide throughout the environment and are of particular interest in agriculture and food safety. In order to develop a specific method for rapid detection of Aspergillus flavus to forecast diseases and control aflatoxins, a nanobody, PO8-VHH, highly reactive to A. flavus was isolated from an immunized alpaca nanobody library by phage display. The nanobody was verified to bind to the components of extracellular and intracellular antigen from both A...
June 28, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28656083/in-vivo-immunotherapy-of-lung-cancer-using-cross-species-reactive-vascular-endothelial-growth-factor-nanobodies
#19
Fatemeh Kazemi-Lomedasht, Kamran Pooshang-Bagheri, Mahdi Habibi-Anbouhi, Ensiyeh Hajizadeh-Safar, Delavar Shahbazzadeh, Hasan Mirzahosseini, Mahdi Behdani
OBJECTIVES: Lung cancer is the main leading cause of cancer death worldwide. Angiogenesis is the main step in proliferation and spreading of tumor cells. Targeting vascular endothelial growth factor (VEGF) is an effective approach for inhibition of cancer angiogenesis. Nanobodies (NBs) are a novel class of antibodies derived from the camel. Unique characteristics of Nbs like their small size and good penetration to tumor tissues makes them promising tools in drug development. Development of NBs targeting both human and mouse VEGF is required for understanding their in vivo functions...
May 2017: Iranian Journal of Basic Medical Sciences
https://www.readbyqxmd.com/read/28646705/identification-and-in-vitro-characterization-of-novel-nanobodies-against-human-granulocyte-colony-stimulating-factor-receptor-to-provide-inhibition-of-g-csf-function
#20
Hamid Bakherad, Seyed Latif Mousavi Gargari, Zargham Sepehrizadeh, Hossein Aghamollaei, Ramezan Ali Taheri, Maryam Torshabi, Mojtaba Tabatabaei Yazdi, Walead Ebrahimizadeh, Neda Setayesh
It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH...
September 2017: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
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