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Article
| Open Access
Single-cell lipidomics enabled by dual-polarity ionization and ion mobility-mass spectrometry imaging
Single-cell analysis provides unique insight into individual cell dynamics and cell-to-cell heterogeneity. Here, the authors utilize trapped ion mobility separation coupled with dual-polarity ionization mass spectrometry imaging to enable high-throughput in situ profiling of single-cell lipidome.
- Hua Zhang
- , Yuan Liu
- & Lingjun Li
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Article
| Open Access
Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system
Transport assays using purified glucose transporters (GLUTs) have proven to be difficult to implement, hampering deeper mechanistic insights. Here the authors have optimized a transport assay in liposomes that will provide insight to study other membrane transport proteins.
- Albert Suades
- , Aziz Qureshi
- & David Drew
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Matters Arising
| Open AccessThe G protein preference of orexin receptors is currently an unresolved issue
- Jyrki P. Kukkonen
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Article
| Open Access
Specific, 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 Access
A cationic motif upstream Engrailed2 homeodomain controls cell internalization through selective interaction with heparan sulfates
Here, the authors show that cell internalization of Engrailed2 not only relies on its homeodomain, but also involves a cationic region which interacts selectively with cell surface heparan sulfates.
- Sébastien Cardon
- , Yadira P. Hervis
- & Sandrine Sagan
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Article
| Open Access
Critical Offset Magnetic PArticle SpectroScopy for rapid and highly sensitive medical point-of-care diagnostics
Sensitive methods for antibody detection tend to be expensive and slow. Here, the authors report a magnetic particle spectroscopy method named COMPASS, as a rapid and low-cost technique which is comparable to ELISA in terms of sensitivity but with a measurement times of seconds.
- Patrick Vogel
- , Martin Andreas Rückert
- & Volker Christian Behr
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Article
| Open Access
Pre-equilibrium biosensors as an approach towards rapid and continuous molecular measurements
Biosensors using ligand-receptor binding tend to operate under equilibrium conditions, but this can make real-time monitoring challenging. Here the authors provide a theoretical foundation for biosensing where ligand concentrations can be continuously measured without needing to reach equilibrium.
- Nicolò Maganzini
- , Ian Thompson
- & Hyongsok Tom Soh
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Article
| Open Access
Demonstration of intracellular real-time molecular quantification via FRET-enhanced optical microcavity
The authors introduce an active-mode optical microcavity sensor with enhanced sensitivity via Förster resonant energy transfer. Changes in lasing wavelengths of both donor and acceptor enable quantitative molecular analysis and real-time monitoring of intracellular molecules.
- Yaping Wang
- , Marion C. Lang
- & Pu Wang
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Article
| Open Access
Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices
Efforts to engineer artificial allosteric systems are ongoing. Here the authors analyse factors influencing development of artificial small molecule:protein complexes that act as chemically induced dimerization (CID) systems; they use one of the CIDs to construct an electrochemical biosensor of methotrexate.
- Zhong Guo
- , Oleh Smutok
- & Kirill Alexandrov
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Article
| Open Access
Measuring expression heterogeneity of single-cell cytoskeletal protein complexes
Existing methods for multimeric protein complex quantification in single cells suffer from limited selectivity and sensitivity. Here the authors report Single-cell protein Interaction Fractionation Through Electrophoresis and immunoassay Readout (SIFTER) and use this to probe the effects of cellular stress.
- Julea Vlassakis
- , Louise L. Hansen
- & Amy E. Herr
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Article
| Open Access
A 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 Access
A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis
There is a clear need for rapid, accurate and scalable Covid-19 diagnostics. Here the authors use PfAgo to detect viral sequences amplified by RT-LAMP in a handheld battery-powered device.
- Guanhua Xun
- , Stephan Thomas Lane
- & Huimin Zhao
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Article
| Open Access
An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing
As the COVID-19 pandemic continues, variants of the virus are emerging. Here the authors present a diagnostic assay that can detect wildtype and known variants using engineered Cas12a.
- Kean Hean Ooi
- , Mengying Mandy Liu
- & Meng How Tan
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Article
| Open Access
Conserved strategies of RNA polymerase I hibernation and activation
RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in eukaryotes. Here, the authors present three cryo-EM structure of S. pombe Pol I in different functional states among them a dimer structure and discuss conserved and organism-specific features of Pol I.
- Florian B. Heiss
- , Julia L. Daiß
- & Christoph Engel
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Article
| Open Access
Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay
Rapid and early detection of SARS-CoV-2 will aid intervention to stop disease spread. Here the authors present a one-pot CRISPR-based rapid detection system with visual readout.
- Xiong Ding
- , Kun Yin
- & Changchun Liu
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Article
| Open Access
Clathrin light chain A drives selective myosin VI recruitment to clathrin-coated pits under membrane tension
Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Here authors find and structurally characterize the selective interaction between CLCa and the actin motor protein myosin VI which act together to generate the force that leads to invagination and fission at the apical surface.
- Matteo Biancospino
- , Gwen R. Buel
- & Simona Polo
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Article
| Open Access
Profiling surface proteins on individual exosomes using a proximity barcoding assay
The use of antibodies to capture and profile exosomes limits the number of target proteins that can be detected. Here the authors develop a proximity-dependent barcoding assay that allows profiling of 38 surface proteins on individual exosomes from heterogeneous samples such as serum and seminal fluid.
- Di Wu
- , Junhong Yan
- & Masood Kamali-Moghaddam
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Article
| Open Access
Band-collision gel electrophoresis
Electrophoretic mobility shift assays are widely used in gel electrophoresis to study binding interactions between different molecular species, but these assays access only a subset of reaction possibilities. Here, the authors develop a band-collision gel electrophoresis (BCGE) approach that demonstrates a much wider variety of reaction types.
- Dimitri A. Bikos
- & Thomas G. Mason
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Article
| Open Access
Parallel analysis of tri-molecular biosynthesis with cell identity and function in single cells
Simultaneous quantification of DNA, RNA and protein at the single cell level has not yet been possible. Here the authors introduce a molecular labelling and detection strategy to quantify synthesis of these biomolecules and couple it to transient cell states through parallel quantification of state-dependent biomolecules.
- Samuel C. Kimmey
- , Luciene Borges
- & Sean C. Bendall
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Article
| Open Access
ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling
The GTPase dynamin catalyzes membrane fission but activity of the dynamin-related ATPase EHD1 is unknown. Here, using in vitro reconstitution assays and molecular dynamics simulations, the authors report that EHD1 hydrolyzes ATP to remodel, causing fission of membrane tubes and that this is necessary for endocytic recycling.
- Raunaq Deo
- , Manish S. Kushwah
- & Thomas J. Pucadyil
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Article
| Open Access
A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection
Isothermal DNA amplification techniques are useful for diagnostic applications in place of traditional PCR. Here the authors describe CRISDA, which combines CRISPR–Cas9 with strand displacement amplification and exhibits attomolar sensitivity and single-nucleotide specificity in DNA detection.
- Wenhua Zhou
- , Li Hu
- & Xue-Feng Yu
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Article
| Open Access
An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules
Ubiquitination and ubiquitin-like modifications of proteins regulate multiple cellular processes but identifying substrates of specific E2 and E3 enzymes remains challenging. Here, the authors conjugate E2 enzymes with enrichable ubiquitin derivatives to identify substrates of specific E2/E3 pairs by mass spectrometry.
- Gábor Bakos
- , Lu Yu
- & Jörg Mansfeld
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Article
| Open Access
Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals
The specific glycosylation patterns of biological drugs often impact the efficacy and safety of the therapeutic product. Here the authors describe a native mass spectrometry approach that allows the resolution of highly complex glycosylation patterns on large proteins, which they apply to the therapeutic Fc-fusion protein Etanercept.
- Therese Wohlschlager
- , Kai Scheffler
- & Christian G. Huber
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Article
| Open Access
A switch point in the molecular chaperone Hsp90 responding to client interaction
The heat shock protein 90 (Hsp90) chaperone undergoes large conformational changes during its functional cycle. Here the authors combine in vivo, biochemical, biophysical and computational approaches and provide insights into the allosteric regulation of Hsp90 by identifying and characterizing a switch point in the Hsp90 middle domain.
- Daniel Andreas Rutz
- , Qi Luo
- & Johannes Buchner
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Article
| Open Access
Pixelated spatial gene expression analysis from tissue
Spatial localization of genetic information is important for tissue heterogeneity but difficult to capture with current analytical techniques. Here the authors present “Pixelated RT-LAMP”, an approach that uses parallel on-chip reactions to provide the distribution of target sequences directly from tissue.
- A. Ganguli
- , A. Ornob
- & R. Bashir
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Article
| Open Access
Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting
Current methods to measure post-translational modifications of receptor tyrosine kinases provide ensemble results. Here the authors develop a single-molecule blotting (SiMBlot) assay that detects site-specific phosphorylation patterns at the single-molecule level.
- Kyung Lock Kim
- , Daehyung Kim
- & Sung Ho Ryu
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Article
| Open Access
A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma
Levels of the enzyme myeloperoxidase in the blood are considered a biomarker for the severity of cardiovascular disease. Here the authors report a rapid and inexpensive method for measuring myeloperoxidase activity in human plasma by luminescence, after adsorption of the enzyme to a polymer surface.
- Reece J. Goiffon
- , Sara C. Martinez
- & David Piwnica-Worms
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Article |
One-step pipetting and assembly of encoded chemical-laden microparticles for high-throughput multiplexed bioassays
High-throughput screening allows for the rapid assessment of biochemical compounds and processes, but with increasing scale comes increasing costs. Here, the authors use an array of lithographically encoded hydrogel microparticles as a more accessible screening technique.
- Su Eun Chung
- , Jiyun Kim
- & Sunghoon Kwon
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Article
| Open Access
Multiplexed volumetric bar-chart chip for point-of-care diagnostics
Diagnostic microfluidic devices often require complicated optical systems and computers to quantify results. Here, Qin and colleagues link enzymatic biomarker detection with the displacement of ink, resulting in a device that displays quantitative results as bar graphs directly on the device.
- Yujun Song
- , Yuanqing Zhang
- & Lidong Qin
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Article
| Open Access
Novel sialic acid derivatives lock open the 150-loop of an influenza A virus group-1 sialidase
The influenza virus life cycle relies on sialidases, which are classified as group-1 or group-2, depending on the flexibility of the '150-loop'. In this study, chemical compounds are developed, which lock open the '150-loop', selectively inhibiting the activity of group-1 sialidases.
- Santosh Rudrawar
- , Jeffrey C. Dyason
- & Mark von Itzstein
Deciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction