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| Open AccessFEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins
When and how microglia engulf synapses and myelin is still unclear. Here, the authors provide a suite of flow cytometry-based approaches to quantify engulfment, paving the way for high-throughput assessment of microglial function in health and disease.
- Lasse Dissing-Olesen
- , Alec J. Walker
- & Beth Stevens
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Article
| Open AccessA magneto-activated nanoscale cytometry platform for molecular profiling of small extracellular vesicles
Exosomal PD-L1 (exoPD-L1) is a biomarker predicting immunotherapeutic responses. Here the authors report NanoEPIC, a nanoscale cytometry platform that enables phenotypic sorting and exoPD-L1 profiling from blood plasma by using magnetic-activated ranking to differentiate exosomal subpopulations.
- Kangfu Chen
- , Bill T. V. Duong
- & Shana O. Kelley
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Article
| Open AccessSpecific, sensitive and quantitative protein detection by in-gel fluorescence
Recombinant proteins in complex solutions are typically detected with tag-specific antibodies in Western blots. Here, the author describes an antibody-free alternative in which tagged proteins are detected directly in polyacrylamide gels via fluorophore-labelling of the tagged protein using a ligase.
- Adrian C. D. Fuchs
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Article
| Open AccessThe rapid and highly parallel identification of antibodies with defined biological activities by SLISY
The covid pandemic has highlighted the need for rapid antibody development. Here, authors develop an approach called SLISY, which uses NGS with phage display to simultaneously assess millions of clones to rapidly isolate specific antibodies against SARS-CoV-2 and its evolving variants.
- Steve Lu
- , Austin K. Mattox
- & Kenneth W. Kinzler
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Article
| Open AccessA conformation-specific nanobody targeting the nicotinamide mononucleotide-activated state of SARM1
SARM1 is a key player in axon degeneration. Here, the authors generate a nanobody, which specifically recognizes the NMN-bound state of SARM1 and helps resolve the SARM1 structure in an intermediate state of activation.
- Yun Nan Hou
- , Yang Cai
- & Yong Juan Zhao
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Article
| Open AccessA marine sponge-derived lectin reveals hidden pathway for thrombopoietin receptor activation
The mode of cytokine receptor activation is diverse. Here, the authors find that the marine-sponge derived lectin ThC, a bivalent sugar binding protein, activates human cytokine receptor MPL. This mode of action resembles the pathogenic activation of MPL by mutant molecular chaperon calreticulin in hematologic malignancies.
- Hiromi Watari
- , Hiromu Kageyama
- & Ryuichi Sakai
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Article
| Open AccessPriming conditions shape breadth of neutralizing antibody responses to sarbecoviruses
Vaccination and infection history determine the breadth of neutralizing antibody response to SARS-CoV-2 variants and other sarbecoviruses with breakthrough or natural infection combined with vaccination or booster vaccination with mRNA vaccine providing highest neutralization.
- Janice Zhirong Jia
- , Chee Wah Tan
- & Sophie A. Valkenburg
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Article
| Open AccessImproved immunoassay sensitivity and specificity using single-molecule colocalization
A major challenge of enzyme-linked immunosorbent assays is discriminating true signal from non-specific binding. Here the authors present a Single-Molecule Colocalization Assay (SiMCA) which eliminates such effects, enabling reproducible detection of picomolar protein concentrations.
- Amani A. Hariri
- , Sharon S. Newman
- & H. Tom Soh
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Article
| Open AccessA CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples
CRISPR diagnostics are routinely used for the detecting nucleic acids, but rarely for clinically important proteins. Here, by translating a CRISPR-based DNA test into an ultrasensitive assay for antibodies, the authors achieve antibody detection from serum samples at attomolar concentrations.
- Yanan Tang
- , Turun Song
- & Feng Li
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Article
| Open AccessDetection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR
Neutralizing antibodies are critical for conferring immunity against SARS-CoV-2. Here, Dahn et al. report a PCR assay termed SONIA (Split-Oligonucleotide Neighboring Inhibition Assay) for measuring neutralizing antibodies against multiple SARS-CoV-2 strains in fingerprick dried blood spot samples.
- Kenneth Danh
- , Donna Grace Karp
- & Cheng-ting Tsai
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Article
| Open AccessHomogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies
Neutralisation assays are key to understanding immune responses to SARS-CoV-2 infection or vaccination. Here, the authors report a surrogate virus neutralization assay called Neu-SATiN, which measures neutralization directly from sera, and allows easy adaptation to variant-specific testing.
- Sun Jin Kim
- , Zhong Yao
- & Shawn C. Owen
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Poster
| Open AccessImmune monitoring and treatment in immune-mediated inflammatory diseases
Immune monitoring assists in the diagnosis and clinical management of immune-mediated inflammatory diseases.
- Femke van Wijk
- , Marjolein de Bruin
- & Stefan Nierkens
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Article
| Open AccessPICASSO allows ultra-multiplexed fluorescence imaging of spatially overlapping proteins without reference spectra measurements
Ultra-multiplexed fluorescence imaging is currently difficult. Here the authors report PICASSO which enables 15-colour imaging of spatially overlapping proteins in a single-round of imaging; they combine it with cyclic immunofluorescence to achieve 45-colour imaging of the mouse brain in 3 cycles.
- Junyoung Seo
- , Yeonbo Sim
- & Jae-Byum Chang
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Article
| Open AccessEfficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci
Trans-chromosomic (Tc) mice have helped the development of therapeutic antibodies, but chromosome instability limits its application. Here the authors develop a new line of Tc mice with full human Ig heavy and kappa loci integrated into the mouse artificial chromosome for stable passage, and confirm efficient generation of B cell responses and specific antibodies.
- Hiroyuki Satofuka
- , Satoshi Abe
- & Yasuhiro Kazuki
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Article
| Open AccessDevelopment and characterization of functional antibodies targeting NMDA receptors
Selective targeting individual subtypes of N-methyl-D-aspartate receptors (NMDARs) is a desirable therapeutic strategy for neurological disorders. Here, the authors report identification of a functional antibody that specifically targets and allosterically down-regulates ion channel activity of the GluN1—GluN2B NMDAR subtype.
- Nami Tajima
- , Noriko Simorowski
- & Hiro Furukawa
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Article
| Open AccessA pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies
The most potent neutralizing antibodies are typically generated from convalescent patients and immunized animals. Here, the authors show it is possible to generate highly potent human neutralizing antibodies against the SARS-CoV-2 spike protein directly from a semisynthetic naïve antibody library.
- Fortunato Ferrara
- , M. Frank Erasmus
- & Andrew R. M. Bradbury
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Article
| Open AccessA cell-free nanobody engineering platform rapidly generates SARS-CoV-2 neutralizing nanobodies
Faster, higher throughput antibody engineering methods are needed. Here the authors present CeVICA, a cell-free nanobody engineering platform using ribosome display and computational clustering analysis for in vitro selection; they use this to develop nanobodies against the RBD of SARS-CoV-2 spike protein.
- Xun Chen
- , Matteo Gentili
- & Aviv Regev
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Article
| Open AccessDigital immunoassay for biomarker concentration quantification using solid-state nanopores
The concentration of a biomarker in solution can be determined by counting single molecules. Here the authors report a digital immunoassay scheme with solid-state nanopore readout to quantify a target protein and use this to measure thyroid-stimulating hormone from human serum.
- Liqun He
- , Daniel R. Tessier
- & Vincent Tabard-Cossa
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Article
| Open AccessAntibody toolkit reveals N-terminally ubiquitinated substrates of UBE2W
UBE2W catalyzes the ubiquitination of protein N-termini but its substrate spectrum is largely unknown. Here, the authors discover mAbs selective for peptides derived from N-terminally ubiquitinated proteins, solve the structure of a peptide-bound mAb and apply the mAbs to map endogenous UBE2W substrates by proteomics.
- Christopher W. Davies
- , Simon E. Vidal
- & James T. Koerber
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Article
| Open AccessA plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays
Many current immunoassays require multiple washing, incubation and optimization steps. Here the authors present Ratiometric Plug-and-Play Immunodiagnostics (RAPPID), a generic assay platform that uses ratiometric bioluminescent detection to allow sandwich immunoassays to be performed directly in solution.
- Yan Ni
- , Bas J. H. M. Rosier
- & Maarten Merkx
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Article
| Open AccessT cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses
Understanding the immune response to SARS-CoV-2 is dependent on being able to distinguish COVID-19 immune responses from cross-reactive immune responses to other coronaviruses. Here the authors show that choice of antigens and whether an ICS, ELISPOT or T cell proliferation assay is used has a major effect on this discriminatory ability.
- Ane Ogbe
- , Barbara Kronsteiner
- & Susanna Dunachie
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Article
| Open AccessA comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies
Understanding the human antibody response to influenza A virus strains is important for vaccine development. Here, Creanga et al. generate a panel of 55 replication-deficient reporter viruses representing diversity of human H1N1 and H3N2, and pandemic subtypes and characterize the neutralization profile of 24 antibodies and polyclonal sera.
- Adrian Creanga
- , Rebecca A. Gillespie
- & Masaru Kanekiyo
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Article
| Open AccessA conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF
TNF can be inhibited by small molecules that stabilize the TNF trimer in an asymmetric conformation. Here, the authors develop a monoclonal antibody that selectively binds this inactive form of TNF, enabling both target engagement assessment and structural characterization of TNF binding to TNF receptor 1.
- Daniel J. Lightwood
- , Rebecca J. Munro
- & Alastair D. G. Lawson
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Article
| Open AccessAnalysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray
COVID-19 diagnosis is commonly performed by PCR testing, however, serologic methods are more accurate and versatile for monitoring disease burden and epidemiology. Here the authors report a protein microarray with antigens from SARS-CoV-2, SARS-CoV, MERS-CoV as well as common human respiratory viruses.
- Rafael R. de Assis
- , Aarti Jain
- & Saahir Khan
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Article
| Open AccessStandardization of ELISA protocols for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling
Understanding the infection parameters and host responses against SARS-CoV-2 require data from large cohorts using standardized methods. Here, the authors optimize a serum ELISA protocol that has minimal cross-reactivity and flexible sample collection workflow in an attempt to standardize data generation and help inform on COVID-19 pandemic and immunity.
- Carleen Klumpp-Thomas
- , Heather Kalish
- & Kaitlyn Sadtler
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Article
| Open AccessUltrasensitive antibody-aptamer plasmonic biosensor for malaria biomarker detection in whole blood
Reliable plasmonic biosensors with high throughput and ease of use are highly sought after. Here, the authors report a plasmon-enhanced fluorescence antibody-aptamer biosensor based on a gold nanoparticle array, and demonstrate its use for effective specific detection of a malaria marker, at femtomolar level, in whole blood.
- Antonio Minopoli
- , Bartolomeo Della Ventura
- & Raffaele Velotta
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Article
| Open AccessFormat chain exchange (FORCE) for high-throughput generation of bispecific antibodies in combinatorial binder-format matrices
Bispecific antibodies have been generated in many different formats and it is becoming clear that rational design alone cannot create optimal functionalities. Here the authors introduce the high throughput methodology, Format Chain Exchange (FORCE), to enable combinatorial generation of bispecific antibodies.
- Stefan Dengl
- , Klaus Mayer
- & Ulrich Brinkmann
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Article
| Open AccessLight microscopy based approach for mapping connectivity with molecular specificity
Mapping neuroanatomy is a foundational goal of connectomics, and the gold standard method is electron microscopy as light microscopy lacks nanoscale resolution. Here the authors develop a strategy using multicolor genetic labeling (Brainbow) and expansion microscopy to map putative synaptic connections using light microscopy.
- Fred Y. Shen
- , Margaret M. Harrington
- & Dawen Cai
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Article
| Open AccessCross-reactive neutralizing human survivor monoclonal antibody BDBV223 targets the ebolavirus stalk
Human antibodies cross-reactive for several viruses within the Ebolavirus genus have been identified. Here the authors present the crystal structure of such a neutralizing monoclonal antibody (mAb) targeting the stalk of Bundibugyo virus glycoprotein and show that mAb binding may interfere with trimeric bundle assembly and/or the viral membrane.
- Liam B. King
- , Brandyn R. West
- & Erica Ollmann Saphire
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Article
| Open AccessRationally designed carbohydrate-occluded epitopes elicit HIV-1 Env-specific antibodies
Areas of HIV envelope (Env) that aren’t covered by glycans are potential targets for antibodies. Here, the authors computationally design small protein mimics of four such epitopes and show that they can induce Env binding antibodies in rabbits.
- Cheng Zhu
- , Elena Dukhovlinova
- & Nikolay V. Dokholyan
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Article
| Open AccessCombining discovery and targeted proteomics reveals a prognostic signature in oral cancer
Oral cancer has region-specific histopathological and molecular characteristics, complicating its classification by the standard tumor-node-metastasis system. Here, the authors combine discovery and targeted proteomics with IHC to identify region-specific and saliva biomarkers for oral cancer prognosis.
- Carolina Moretto Carnielli
- , Carolina Carneiro Soares Macedo
- & Adriana Franco Paes Leme
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Article
| Open AccessA quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes
Chromatin-associated protein complexes play a critical role in the regulation of gene expression in health and disease. Here, the authors describe a sensitive mass spectrometry-based method to monitor the dynamic interactions of endogenous chromatin-associated protein complexes in clinical samples.
- Evangelia K. Papachristou
- , Kamal Kishore
- & Jason S. Carroll
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Article
| Open AccessNext generation histology methods for three-dimensional imaging of fresh and archival human brain tissues
Current available tissue clearing techniques are mostly used for rodent tissues. Here, the authors develop OPTIClear solution for fresh and archival human brain tissue clearing and establish associated protocols for three-dimensional histological investigations.
- Hei Ming Lai
- , Alan King Lun Liu
- & Steve M. Gentleman
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Article
| Open AccessRational design of a trispecific antibody targeting the HIV-1 Env with elevated anti-viral activity
Broadly neutralizing antibodies targeting HIV Env could potentially be utilized as therapeutics. Here, Steinhardt et al. engineer a trispecific antibody with specificity for the receptor-binding site, a conserved Env glycan patch and the Env membrane proximal region with nearly pan-isolate neutralization breadth and high potency.
- James J. Steinhardt
- , Javier Guenaga
- & Yuxing Li
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Article
| Open AccessSelenoprotein P-neutralizing antibodies improve insulin secretion and glucose sensitivity in type 2 diabetes mouse models
Selenoprotein P is secreted by the liver and when present in excess it promotes development of type 2 diabetes. Here the authors develop neutralizing antibodies to target human and mouse selenoprotein P, and show that they improve insulin secretion and glucose tolerance in mouse models.
- Yuichiro Mita
- , Kaho Nakayama
- & Yoshiro Saito
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Article
| Open AccessQuantitative analysis of protein-protein interactions and post-translational modifications in rare immune populations
Rare immune or stem cell populations are difficult to quantitatively analyze for protein-protein interactions or post-translational modifications despite advances in proteomics. Here the authors develop proximity ligation imaging cytometry for high-resolution detection at a single cell level.
- Ayelet Avin
- , Maayan Levy
- & Jakub Abramson
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Article
| Open AccessInsufficient antibody validation challenges oestrogen receptor beta research
A large body of work into the role of oestrogen receptor b (ERb) in breast cancer is contradictory, hindering future progress. Here the authors conduct extensive validation of anti-ERb antibodies , and show that normal and cancerous breast tissue do not express ERb, consistent with RNA-seq data.
- Sandra Andersson
- , Mårten Sundberg
- & Anna Asplund
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Article
| Open AccessProfiling protein expression in circulating tumour cells using microfluidic western blotting
Circulating tumour cells (CTCs) are rare cells found in the blood of certain cancer patients. Here, the authors develop a cytometry tool that appends a microfluidic western blot to a CTC isolation workflow and apply it to profile a panel of proteins in single CTCs isolated from ER+ breast cancer patients.
- Elly Sinkala
- , Elodie Sollier-Christen
- & Amy E. Herr
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Article
| Open AccessA bead-based western for high-throughput cellular signal transduction analyses
Dissecting cellular signalling requires the analysis of large numbers of proteins. Here the authors describe DigiWest, a high-throughput protein detection method that combines the concept of western and widely-used bead array systems that allows rapid quantification of hundreds of specific proteins.
- Fridolin Treindl
- , Benjamin Ruprecht
- & Markus F. Templin
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Article
| Open AccessA broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve
A major goal of vaccine design is to protect against a broad range of pathogen strains. Here the authors isolate a new broadly neutralizing antibody against influenza haemagglutinin from human memory B cells, and identify mutations that increase and broaden the neutralization towards H5 HA subtype.
- Ying Fu
- , Zhen Zhang
- & Wayne A. Marasco
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Article
| Open AccessEfficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment
Generating antibodies specific for the peptide–MHCII complexes has been challenging, with only a handful made to date. Here, the authors develop a more efficient approach to generate these antibodies, and demonstrate their potential in research and therapeutic applications.
- Justin A. Spanier
- , Daniel R. Frederick
- & Brian T. Fife
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Article
| Open AccessReactivation of IgG-switched memory B cells by BCR-intrinsic signal amplification promotes IgG antibody production
Antigen receptors on memory B cells enhance their signaling strength by recruiting the cytosolic Grb2 adaptor to their ITT phosphorylation motifs. Here the authors show that inactivating the ITT motif of mouse mIgG1 impairs IgG1 production and T-cell independent reactivation of memory B cells.
- Johannes Lutz
- , Kai Dittmann
- & Niklas Engels
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Article
| Open AccessDigital microfluidic immunocytochemistry in single cells
The ability to measure signalling responses in single cells following short pulses of stimulus would shed insight into temporal thresholds for cell activation. Here the authors introduce a microfluidic platform that allows downstream phosphorylation cascades to be observed following as little as one second of stimulus exposure.
- Alphonsus H. C. Ng
- , M. Dean Chamberlain
- & Aaron R. Wheeler
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Article
| Open AccessA highly potent human antibody neutralizes dengue virus serotype 3 by binding across three surface proteins
There is no licensed vaccine or therapeutic for dengue virus (DENV) infection. Here, the authors show that a highly potent human monoclonal antibody binds to DENV particles in an unusual and very effective way by interacting with three viral envelope proteins.
- Guntur Fibriansah
- , Joanne L. Tan
- & Shee-Mei Lok
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Article
| Open AccessExploiting light chains for the scalable generation and platform purification of native human bispecific IgG
Bispecific antibodies allow for novel therapeutic approaches but industrial-scale production and immunogenicity represent significant challenges. Here Fischer et al.describe a unique human bispecific antibody format that exploits differing light chains to overcome these obstacles.
- Nicolas Fischer
- , Greg Elson
- & Marie Kosco-Vilbois
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Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling
Protein SUMOylation plays an important role in regulation of many cellular processes. Lamoliatte et al.use a monoclonal antibody specific for SUMO3 tryptic peptides to enhance proteomic identification of SUMOylated proteins, and dramatically expand the number of sites known to be modified by SUMO3.
- Frédéric Lamoliatte
- , Danielle Caron
- & Pierre Thibault
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Article |
PAR-CLIP analysis uncovers AUF1 impact on target RNA fate and genome integrity
AUF1 is an RNA-binding protein believed to function mostly by regulating the decay of its target transcripts. Here, Yoon et al.systematically identify the targets of AUF1 and provide insights into how AUF1 functions to regulate various cellular processes by enhancing the decay, stability or translation of specific RNAs.
- Je-Hyun Yoon
- , Supriyo De
- & Myriam Gorospe
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Article
| Open AccessCreation of a gated antibody as a conditionally functional synthetic protein
The ability to control antibody binding could have important medical implications. Here, the authors present a method to engineer phosphatase-controllable antibodies that bind to a specific recognition site in the presence of two biomarker inputs.
- Smita B. Gunnoo
- , Helene M. Finney
- & Benjamin G. Davis
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Optofluidic laser for dual-mode sensitive biomolecular detection with a large dynamic range
Enzyme-linked immunosorbent assays are common biological analyses, but they can be hindered by non-specific binding and background interference. Here, the authors present an optofluidic laser-based ELISA technique with low detection limits and in the presence of significant non-specific binding.
- Xiang Wu
- , Maung Kyaw Khaing Oo
- & Xudong Fan