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https://www.readbyqxmd.com/read/28731329/generation-and-characterization-of-anti-vglut-nanobodies-acting-as-inhibitors-of-transport
#1
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...
July 21, 2017: Biochemistry
https://www.readbyqxmd.com/read/28725224/nanobody-technology-a-versatile-toolkit-for-microscopic-imaging-protein-protein-interaction-analysis-and-protein-function-exploration
#2
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
#3
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
#4
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
#5
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 5, 2017: Current Biology: CB
https://www.readbyqxmd.com/read/28687476/actin-nanobodies-uncover-the-mystery-of-actin-filament-dynamics-in-toxoplasma-gondii
#6
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.
July 4, 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
#7
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
#8
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
#9
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...
June 28, 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
#10
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
#11
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
#12
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...
June 21, 2017: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
https://www.readbyqxmd.com/read/28642127/exploiting-sequence-and-stability-information-for-directing-nanobody-stability-engineering
#13
Patrick Kunz, Tilman Flock, Nicolas Soler, Moritz Zaiss, Cécile Vincke, Yann Sterckx, Damjana Kastelic, Serge Muyldermans, Jörg D Hoheisel
BACKGROUND: Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate effects on conformational stability, protease resistance and aggregation propensity of the protein. METHODS: We analyzed the sequences and thermostabilities of 78 purified nanobody binders...
June 20, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28636463/optimization-of-an-anti-her2-nanobody-expression-using-the-taguchi-method
#14
Alireza Farasat, Fatemeh Rahbarizadeh, Davoud Ahmadvand, Fatemeh Yazdian
Despite being widely used in immunotherapy of cancer, whole antibodies are limited by several disadvantages. This has led to the advent of novel biomolecules such as nanobodies. Taguchi method is a statistical experimental design to study the effect of multiple variables in biological processes. In an effort to overexpress a recombinant anti-HER2 nanobody, we carried out a detailed study to find optimal condition of temperature, induction, culture media, vector and host strain, using Taguchi methodology. A total of 16 various experiments were designed...
June 21, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28635419/multimodal-imaging-gd-nanoparticles-functionalized-with-pittsburgh-compound-b-or-a-nanobody-for-amyloid-plaques-targeting
#15
Jonathan Pansieri, Marie Plissonneau, Nathalie Stransky-Heilkron, Mireille Dumoulin, Laurence Heinrich-Balard, Pascaline Rivory, Jean-François Morfin, Eva Toth, Maria Joao Saraiva, Eric Allémann, Olivier Tillement, Vincent Forge, François Lux, Christel Marquette
AIM: Gadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses. MATERIALS & METHODS: The ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy...
June 21, 2017: Nanomedicine
https://www.readbyqxmd.com/read/28619826/a-toolkit-for-gfp-mediated-tissue-specific-protein-degradation-in-c-elegans
#16
Shaohe Wang, Ngang Heok Tang, Pablo Lara-Gonzalez, Zhiling Zhao, Dhanya K Cheerambathur, Bram Prevo, Andrew D Chisholm, Arshad Desai, Karen Oegema
Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation...
July 15, 2017: Development
https://www.readbyqxmd.com/read/28611730/exploiting-nanobodies-in-the-detection-and-quantification-of-human-growth-hormone-via-phage-sandwich-enzyme-linked-immunosorbent-assay
#17
Hossam Murad, Jana Mir Assaad, Rasha Al-Shemali, Abdul Qader Abbady
BACKGROUND: Monitoring blood levels of human growth hormone (hGH) in most children with short stature deficiencies is crucial for taking a decision of treatment with extended course of daily and expensive doses of recombinant hGH (rhGH or Somatropin(®)). Besides, misusing of rhGH by sportsmen is banned by the World Anti-Doping Agency and thus sensitive GH-detecting methods are highly welcome in this field. Nanobodies are the tiniest antigen-binding entity derived from camel heavy chain antibodies...
2017: Frontiers in Endocrinology
https://www.readbyqxmd.com/read/28611361/discovery-of-a-novel-conformational-equilibrium-in-urokinase-type-plasminogen-activator
#18
Tobias Kromann-Hansen, Eva Louise Lange, Hans Peter Sørensen, Gholamreza Hassanzadeh-Ghassabeh, Mingdong Huang, Jan K Jensen, Serge Muyldermans, Paul J Declerck, Elizabeth A Komives, Peter A Andreasen
Although trypsin-like serine proteases have flexible surface-exposed loops and are known to adopt higher and lower activity conformations, structural determinants for the different conformations have remained largely obscure. The trypsin-like serine protease, urokinase-type plasminogen activator (uPA), is central in tissue remodeling processes and also strongly implicated in tumor metastasis. We solved five X-ray crystal structures of murine uPA (muPA) in the absence and presence of allosteric molecules and/or substrate-like molecules...
June 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28605435/aav9-delivered-bispecific-nanobody-attenuates-amyloid-burden-in-the-gelsolin-amyloidosis-mouse-model
#19
Adriaan Verhelle, Nisha Nair, Inge Everaert, Wouter Van Overbeke, Lynn Supply, Olivier Zwaenepoel, Cindy Peleman, Jo Van Dorpe, Tony Lahoutte, Nick Devoogdt, Wim Derave, Marinee K Chuah, Thierry VandenDriessche, Jan Gettemans
No abstract text is available yet for this article.
June 9, 2017: Human Molecular Genetics
https://www.readbyqxmd.com/read/28591513/unravelling-the-molecular-basis-of-high-affinity-nanobodies-against-hiv-p24-in-vitro-functional-structural-and-in-silico-insights
#20
Eleanor R Gray, Jennifer C Brookes, Christophe Caillat, Valérian Turbé, Benjamin L J Webb, Luke A Granger, Benjamin S Miller, Laura E McCoy, Mohamed El Khattabi, C Theo Verrips, Robin A Weiss, Dorothy M Duffy, Winfried Weissenhorn, Rachel A McKendry
Preventing the spread of infectious diseases remains an urgent priority worldwide, and this is driving the development of advanced nanotechnology to diagnose infections at the point of care. Herein, we report the creation of a library of novel nanobody capture ligands to detect p24, one of the earliest markers of HIV infection. We demonstrate that these nanobodies, one tenth the size of conventional antibodies, exhibit high sensitivity and broad specificity to global HIV-1 subtypes. Biophysical characterization indicates strong 690 pM binding constants and fast kinetic on-rates, 1 to 2 orders of magnitude better than monoclonal antibody comparators...
July 14, 2017: ACS Infectious Diseases
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