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https://www.readbyqxmd.com/read/29331148/isolation-of-anti-extra-cellular-vesicle-single-domain-antibodies-by-direct-panning-on-vesicle-enriched-fractions
#1
Milica Popovic, Elisa Mazzega, Barbara Toffoletto, Ario de Marco
BACKGROUND: The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis. METHODS: Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents...
January 13, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29328338/binding-affinity-prediction-of-nanobody-protein-complexes-by-scoring-of-molecular-dynamics-trajectories
#2
Miguel A Soler, Sara Fortuna, Ario de Marco, Alessandro Laio
Nanobodies offer a viable alternative to antibodies for engineering high affinity binders. Their small size has an additional advantage: it allows exploiting computational protocols for optimizing their biophysical features, such as the binding affinity. The efficient prediction of this quantity is still considered a daunting task especially for modelled complexes. We show how molecular dynamics can successfully assist in the binding affinity prediction of modelled nanobody-protein complexes. The approximate initial configurations obtained by in silico design must undergo large rearrangements before achieving a stable conformation, in which the binding affinity can be meaningfully estimated...
January 12, 2018: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/29327239/a-small-protein-probe-for-correlated-microscopy-of-endogenous-proteins
#3
Marit A de Beer, Jeroen Kuipers, Paul M P van Bergen En Henegouwen, Ben N G Giepmans
Probes are essential to visualize proteins in their cellular environment, both using light microscopy as well as electron microscopy (EM). Correlated light microscopy and electron microscopy (CLEM) requires probes that can be imaged simultaneously by both optical and electron-dense signals. Existing combinatorial probes often have impaired efficiency, need ectopic expression as a fusion protein, or do not target endogenous proteins. Here, we present FLIPPER-bodies to label endogenous proteins for CLEM. Fluorescent Indicator and Peroxidase for Precipitation with EM Resolution (FLIPPER), the combination of a fluorescent protein and a peroxidase, is fused to a nanobody against a target of interest...
January 11, 2018: Histochemistry and Cell Biology
https://www.readbyqxmd.com/read/29276515/nanobodies-as-novel-agents-for-targeting-angiogenesis-in-solid-cancers
#4
REVIEW
Roghaye Arezumand, Abbas Alibakhshi, Javad Ranjbari, Ali Ramazani, Serge Muyldermans
Solid cancers are dependent on angiogenesis for sustenance. The FDA approval of Bevacizumab in 2004 inspired many scientists to develop more inhibitors of angiogenesis. Although several monoclonal antibodies (mAbs) are being administered to successfully combat various pathologies, the complexity and large size of mAbs seem to narrow the therapeutic applications. To improve the performance of cancer therapeutics, including those blocking tumor angiogenesis, attractive strategies such as miniaturization of the antibodies have been introduced...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/29274374/the-effect-of-subcellular-localization-on-the-efficiency-of-egfr-targeted-vhh-photosensitizer-conjugates
#5
Sanne A M van Lith, Dirk van den Brand, Rike Wallbrecher, Lina Wübbeke, Sander M J van Duijnhoven, Petri I Mäkinen, Janneke S Hoogstad-van Evert, Leon Massuger, Seppo Ylä-Herttuala, Roland Brock, William P J Leenders
Photodynamic therapy (PDT) is an emerging method to treat light-accessible malignancies. To increase specificity and allow dose reduction, conjugates of photosensitizers (PS) with antibodies against tumor-associated antigens have been developed for photoimmunotherapy (PIT). However, so far it is unclear whether cellular internalization of these conjugates after binding affects PIT efficacy. The use of low molecular weight llama single domain antibodies (VHHs, nanobodies) for PIT is preferred above full size antibodies because of better tumor penetration...
December 20, 2017: European Journal of Pharmaceutics and Biopharmaceutics
https://www.readbyqxmd.com/read/29258303/antibody-based-fusion-proteins-allow-ca2-rewiring-to-most-extracellular-ligands
#6
Anam Qudrat, Kevin Truong
The Ca2+ signaling toolkit is the set of proteins used by living systems to generate and respond to Ca2+ signals. The selective expression of these proteins in particular tissues, cell types and subcellular locations allows the Ca2+ signal to regulate a diverse set of cellular processes. Through synthetic biology, the Ca2+ signaling toolkit can be expanded beyond the natural repertoire to potentially allow a non-natural ligand to control downstream cellular processes. To realize this potential, we exploited the ability of an antibody to bind its antigen exclusively in combination with the ability of the cytoplasmic domain of vascular endothelial growth factor receptor 2 (VEGFR2) to generate a Ca2+ signal upon oligomerization...
January 2, 2018: ACS Synthetic Biology
https://www.readbyqxmd.com/read/29246751/generation-and-characterization-of-an-anti-delta-like-ligand-4-nanobody-to-induce-non-productive-angiogenesis
#7
Rasoul Baharlou, Nader Tajik, Mahdi Behdani, Mohammad Ali Shokrgozar, Amir Hassan Zarnani, Fatemeh Shahhosseini, Mahdi Habibi-Anbouhi
Antibody-based targeting of angiogenesis is a key approach for cancer treatment. Delta-like ligand 4 (DLL4) plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. It forecasts the prognosis of human malignancies and blocking its signaling can help to inhibit neovascularization and tumor metastasis. Nanobodies are the smallest antigen-binding domains of heavy chain antibodies in camelidae. The aim of this study was to develop a Nanobody against DLL4 and apply binding and functional approaches to target it...
December 12, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/29241555/inhibiting-the-ca2-influx-induced-by-human-csf
#8
Anna Drews, Suman De, Patrick Flagmeier, David C Wirthensohn, Wei-Hsin Chen, Daniel R Whiten, Margarida Rodrigues, Cécile Vincke, Serge Muyldermans, Ross W Paterson, Catherine F Slattery, Nick C Fox, Jonathan M Schott, Henrik Zetterberg, Christopher M Dobson, Sonia Gandhi, David Klenerman
One potential therapeutic strategy for Alzheimer's disease (AD) is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca2+ influx caused by the CSF of individuals affected with AD and healthy controls, and we have found comparable effects for both types of samples...
December 12, 2017: Cell Reports
https://www.readbyqxmd.com/read/29232933/a-split-luciferase-reporter-recognizing-gfp-and-mcherry-tags-to-facilitate-studies-of-protein-protein-interactions
#9
Mehdi Moustaqil, Akshay Bhumkar, Laura Gonzalez, Lisa Raoul, Dominic J B Hunter, Pascal Carrive, Emma Sierecki, Yann Gambin
The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some protein-protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins...
December 11, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29220018/ablynx-s-nanobody-fragments-go-places-antibodies-cannot
#10
Cormac Sheridan
No abstract text is available yet for this article.
December 8, 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/29216932/nanobodies-targeting-conserved-epitopes-on-the-major-outer-membrane-protein-of-campylobacter-as-potential-tools-for-control-of-campylobacter-colonization
#11
Charlotte Vanmarsenille, Inés Díaz Del Olmo, Jelle Elseviers, Gholamreza Hassanzadeh Ghassabeh, Kristof Moonens, Didier Vertommen, An Martel, Freddy Haesebrouck, Frank Pasmans, Jean-Pierre Hernalsteens, Henri De Greve
Campylobacter infections are among the most prevalent foodborne infections in humans, resulting in a massive disease burden worldwide. Broilers have been identified as the major source of campylobacteriosis and reducing Campylobacter loads in the broiler caeca has been proposed as an effective measure to decrease the number of infections in humans. Failure of current methods to control Campylobacter in broilers stresses the urgency to develop novel mitigation measures. We obtained six nanobodies with a broad specificity, that recognize strains belonging to the two most relevant species, Campylobacter jejuni and Campylobacter coli...
December 8, 2017: Veterinary Research
https://www.readbyqxmd.com/read/29215864/platinum-nanocatalyst-amplification-redefining-the-gold-standard-for-lateral-flow-immunoassays-with-ultra-broad-dynamic-range
#12
Colleen N Loynachan, Michael R Thomas, Eleanor R Gray, Daniel A Richards, Jeongyun Kim, Benjamin S Miller, Jennifer Clare Brookes, Shweta Agarwal, Vijay Chudasama, Rachel A McKendry, Molly M Stevens
Paper-based lateral flow immunoassays (LFIAs) are one of the most widely used point-of-care (PoC) devices, however, their application in early disease diagnostics is often limited due to insufficient sensitivity for the requisite sample sizes and the short timeframes of PoC testing. To address this, we developed a serum-stable, nanoparticle catalyst-labelled LFIA with a sensitivity surpassing that of both current commercial and published sensitivities for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV...
December 7, 2017: ACS Nano
https://www.readbyqxmd.com/read/29213270/nanobodies-and-nanobody-based-human-heavy-chain-antibodies-as-antitumor-therapeutics
#13
REVIEW
Peter Bannas, Julia Hambach, Friedrich Koch-Nolte
Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv)...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/29213077/allosteric-nanobodies-uncover-a-role-of-hippocampal-mglu2-receptor-homodimers-in-contextual-fear-consolidation
#14
Pauline Scholler, Damien Nevoltris, Dimitri de Bundel, Simon Bossi, David Moreno-Delgado, Xavier Rovira, Thor C Møller, Driss El Moustaine, Michaël Mathieu, Emilie Blanc, Heather McLean, Elodie Dupuis, Gérard Mathis, Eric Trinquet, Hervé Daniel, Emmanuel Valjent, Daniel Baty, Patrick Chames, Philippe Rondard, Jean-Philippe Pin
Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways. Here we identify llama nanobodies that specifically recognize mGlu2 receptors, among the eight subtypes of mGluR subunits. Among these nanobodies, DN10 and 13 are positive allosteric modulators (PAM) on homodimeric mGlu2, while DN10 displays also a significant partial agonist activity...
December 6, 2017: Nature Communications
https://www.readbyqxmd.com/read/29209322/camelid-single-domain-antibodies-historical-perspective-and-future-outlook
#15
Mehdi Arbabi-Ghahroudi
Tremendous effort has been expended over the past two and a half decades to understand many aspects of camelid heavy chain antibodies, from their biology, evolution, and immunogenetics to their potential applications in various fields of research and medicine. In this article, I present a historical perspective on the development of camelid single-domain antibodies (sdAbs or VHHs, also widely known as nanobodies) since their discovery and discuss the advantages and disadvantages of these unique molecules in various areas of research, industry, and medicine...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/29207143/screening-and-antitumor-effect-of-an-anti%C3%A2-ctla%C3%A2-4-nanobody
#16
Ruirong Wan, Aiqun Liu, Xiaoqiong Hou, Zongqiang Lai, Jieping Li, Nuo Yang, Juntao Tan, Fengzhen Mo, Zixi Hu, Xiaomei Yang, Yongxiang Zhao, Xiaoling Lu
Cytotoxic T‑lymphocyte antigen‑4 (CTLA‑4) is a critical negative regulator of immune responses. CTLA‑4 is rapidly upregulated following T‑cell activation, and then binds to B7 molecules with a higher affinity than CD28. CTLA‑4 may abolish the initiation of the responses of T cells by raising the threshold of signals required for full activation of T cells, and it also may terminate ongoing T-cell responses. This regulatory role has led to the development of monoclonal antibodies (mAbs) designed to block CTLA‑4 activity for enhancing immune responses against cancer...
December 4, 2017: Oncology Reports
https://www.readbyqxmd.com/read/29177623/expression-of-single-domain-antibody-in-different-systems
#17
REVIEW
Yongkang Liu, He Huang
Camelid single-domain antibodies (sdAbs, VHHs, or Nanobodies®) are types of antibody fragments that are composed of the heavy-chain variable domain only. These VHHs possess unique structural and functional features, as they are small in size and exhibit thermal stability and high solubility. Compared to conventional antibodies, VHHs can be manufactured in microorganisms to significantly save on cost, labor, and time since VHHs lack the Fc domain with its N-linked oligosaccharide. Until now, VHHs have been expressed in several kinds of production systems, ranging from prokaryotic cells, yeasts, fungi, insect cells, and mammalian cell lines, to plants...
November 25, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29176642/insight-into-partial-agonism-by-observing-multiple-equilibria-for-ligand-bound-and-gs-mimetic-nanobody-bound-%C3%AE-1-adrenergic-receptor
#18
Andras S Solt, Mark J Bostock, Binesh Shrestha, Prashant Kumar, Tony Warne, Christopher G Tate, Daniel Nietlispach
A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., Gs and β-arrestin. Using 13C methyl methionine NMR for the β1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with Gs-mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody-receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism...
November 27, 2017: Nature Communications
https://www.readbyqxmd.com/read/29163515/nanobody-based-delivery-systems-for-diagnosis-and-targeted-tumor-therapy
#19
REVIEW
Yaozhong Hu, Changxiao Liu, Serge Muyldermans
The development of innovative targeted therapeutic approaches are expected to surpass the efficacy of current forms of treatments and cause less damage to healthy cells surrounding the tumor site. Since the first development of targeting agents from hybridoma's, monoclonal antibodies (mAbs) have been employed to inhibit tumor growth and proliferation directly or to deliver effector molecules to tumor cells. However, the full potential of such a delivery strategy is hampered by the size of mAbs, which will obstruct the targeted delivery system to access the tumor tissue...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/29160309/structure-of-pink1-in-complex-with-its-substrate-ubiquitin
#20
Alexander F Schubert, Christina Gladkova, Els Pardon, Jane L Wagstaff, Stefan M V Freund, Jan Steyaert, Sarah L Maslen, David Komander
Autosomal recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular in the E3 ubiquitin ligase Parkin (PARK2), and in its upstream protein kinase PINK1 (PARK6). PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like domain on structurally protected Ser65 to trigger mitophagy. We here report a crystal structure of a nanobody-stabilised complex between Pediculus humanus corporis (Ph)PINK1 with ubiquitin in the 'C-terminally retracted' (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the PINK1 N-lobe binds ubiquitin via a unique insertion...
October 30, 2017: Nature
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