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nELISA miniaturizes the sandwich immunoassay and transforms it into a massively scalable platform for quantitative proteomics.

Using fluorescence lifetime as the readout modality offers more reproducible and quantitative outputs compared to conventional fluorescent barcoding, being independent of sample concentration and measurement methods. Here, the authors design a photo-switchable nanogel exhibiting variable fluorescence lifetime, and demonstrate visual mapping by using fluorescence lifetime imaging microscopy on a sub-cellular scale.

By unifying data from engineered β-barrel nanopores and supported by modelling, it is demonstrated that the lumen charge in a β-barrel nanopore governs rectification and voltage-driven gating, with applications in computing using nanofluidic synapses.

The principal mid-visible light-harvesting system in cyanobacteria is the phycobilisome. Now, using broadband multidimensional spectroscopy, delocalized vibronic excitations and sub-picosecond excitation transfer pathways have been observed in the rods of intact phycobilisomes. An observed kinetic bottleneck in the phycobilisome’s core arises from the intramolecular charge-transfer character of the bilin chromophores, enabling photoregulatory processes to operate on the >10-ps timescale.

The membrane tetraspan protein MS4A4A is expressed on tissue-resident and tumor-associated macrophages. Locati and colleagues show that MS4A4A colocalizes with the β-glucan receptor dectin-1 to enhance cytokine and reactive oxygen species production and to enhance NK cell–mediated control of tumor metastasis.

The development of antimalarials against the human liver and asexual blood stages is one of the top public health challenges. Here, the authors report a single-step biochemical assay for the characterization of prolyl-tRNA synthetase inhibitors, and develop high-affinity inhibitors for the enzyme, including elusive triple-site ligands.

STARSS measures rotational dynamics of large molecules and their complexes.

The human Shu complex promotes homologous recombination by regulating RAD51. Here the authors reveal that the Shu complex proteins, SWSAP1-SWS1, decorate the RAD51 filament on ssDNA and facilitate its strand exchange reaction by stimulating RPA diffusion on ssDNA. Lastly, that SWSAP1-SWS1 knockouts are Olaparib sensitive.

Cell migration in confined environments is initiated by a cytoplasmic pool of anillin and Ect2 that promotes RhoA/myosin II-mediated activation at the poles of migrating cells, in a process dependent on the extracellular environment stiffness.

Studying membrane contact sites at high spatiotemporal resolution is still challenging. Here, authors introduce a series of dynamic chemogenetic reporters to visualize these subcellular regions and study the associated calcium signals.

Multicolor imaging employing genetically-encodable fluorescent proteins permits spatiotemporal live cell imaging of multiple cues. Here, authors use multicolor lifetime imaging to visualize quadruple-labelled human immunodeficiency viruses within cellular contexts.

Room-temperature-solution (20 °C) and low-temperature-solution (80 °C) synthesis procedures are developed for a new class of metal halide perovskite high-entropy semiconductor single crystals.

Cryogenic electron microscopy structures of the Synechocystis phycobilisome—alone and bound with orange carotenoid protein—reveal detailed information regarding the biophysical basis of the control of cyanobacterial light harvesting.

A head-to-head double-ring complex of the human multifunctional DNA repair protein RAD52 mediates protection of stalled replication forks during replication stress, protecting them from reversal by SMARCAL1 motor.

PXo bodies, non-canonical multilamellar organelles, serve as a reservoir for intracellular inorganic phosphate and are a critical regulator of both cytosolic phosphate levels and tissue homeostasis.

The presence of conformational substates of a catalytically competent 'closed' state in the ligand-free form of adenylate kinase is detected. Molecular dynamics simulations indicated that the partially closed conformations were sampled in nanoseconds, and NMR and single-molecule FRET experiments revealed the sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale.

Mass cytometry can be used to scale up multiplexed serology testing, as shown for the simultaneous detection of antibody levels against multiple SARS-CoV-2 proteins in hundreds of serum samples.

Changes in intracellular GTP levels are not considered as a regulatory event in RAC1 activation in live cells since total GTP levels are substantially higher than the RAC1 GTP dissociation constant determined in vitro. Here, the authors demonstrate that the availability of free GTP in live cells controls the activity of RAC1 and cell invasion.

P450 oxidoreductase (POR) selectively activates numerous cytochromes P450 (CYP), crucial for metabolism of drugs, steroids and xenobiotics and natural product biosynthesis. Here, the authors identify ligands that bind POR and bias its specificity towards CYP redox partners, activating distinct metabolic cascades in cells.

Stabilisation of stalled replication forks prevents excessive fork reversal and genome instability. Here authors reveal a RAD52-dependent replication fork protection mechanism.

Single-cell gene expression analysis reveals phenotypic heterogeneity to enable bacterial specialization over the course of plant colonization.

Visualization of endogenous G-quadruplexes (G4s) in living cells by fluorescence microscopy has been hampered by the high concentrations of G4-binding probes required, which can artificially induce additional G4 formation. Now, a G4-specific fluorescent probe (SiR-PyPDS) has been developed that enables single-molecule and real-time detection of individual G4 structures in living cells without perturbing G4 formation and dynamics.

spFRET microscopy analysis reveals how FACT reversibly uncoils DNA from nucleosomes during remodeling, thus modulating DNA accessibility in vitro and in vivo.

The safety and efficacy of ribonucleoproteins for genome editing can be enhanced by formulations of lipid nanoparticles optimized for enhanced stability, delivery efficiency and editing potency of the protein complexes.

NMR and Raman spectroscopies pinpoint the role of the protein droplet surface and RNA in the liquid droplet maturation mechanism of the FUS protein. A crust-like β-sheet structure is formed on the surface of FUS droplets during aging.

A general method for enzymatic synthesis of base-modified RNA was developed using engineered thermostable DNA polymerases enabling introduction of site-specific modifications or synthesis of hypermodified RNA not accessible by in vitro transcription.

The identity of hair cells’ mechanoelectrical transduction (MET) channel-complex components is unknown. Here, the authors used multiple biochemical, genetic, and functional approaches to show that mouse hair cells utilize Piezo1 and Piezo2 isoforms as part of the MET-complex component.

Crystal Mackall and colleagues report that antigen-independent signaling of chimeric antigen receptors (CARs) causes T cell exhaustion and reduced therapeutic efficacy of CAR T cells that can be overcome by incorporating the 4-1BB costimulatory domain into the CAR.

Adaptable, turn-on maturation (ATOM) biosensors use monobody or nanobody targeting to control fluorescent protein maturation for fluorescence in the presence of target biomolecules, enabling bright and specific cellular biosensing.

The vast scale of emerging on-demand chemical collections presents a challenge for efficiently identifying promising drug candidates. Here, the authors develop a bottom-up computational strategy to first explore fragment space and then exploit the most promising scaffolds, successfully identifying diverse and potent BRD4 binders.

Döring, van der Vorst, Yan, Neideck et al. present a non-canonical chemokine pathway involving CCL17 signaling through CCR8, which induces CCL3 expression independent of CCR4 and suppresses the functions of atheroprotective T_reg cells.

Coiled-coil protein origami is a strategy for the de novo design of polypeptide nanostructures based on coiled-coil dimer forming peptides, where a single chain protein folds into a polyhedral cage. Here, the authors design a single-chain triangular bipyramid and also demonstrate that the bipyramid can be self-assembled as a heterodimeric complex, comprising pre-defined subunits.

Singlet oxygen is a highly useful reagent for organic synthesis, disinfection and photodynamic therapy, but its high reactivity calls for systems where its photochemical generation can be switched on and off on demand. Here, the authors report porphyrin-decorated pH-switchable [2]rotaxanes for singlet oxygen photoproduction, finding that molecular folding of the rotaxanes influences the on/off switching in an unforeseen way.

Curaxins are a recently discovered class of anti-cancer agents that disturbs DNA/histone interactions within. Here the authors provide evidence that curaxins affect the spatial genome organization and compromise enhancer-promoter communication necessary for expression of several oncogenes, including MYC.

A combination of bulk and single-molecule fluorescence analysis reveals the choreography of binding and rearrangement of individual DNA-binding domains of RPA during homologous recombination.

Optogenetic approaches to control protein-protein interactions usually require overexpression of the target proteins. Here the authors integrate intrabodies into near-infrared- and blue-light activatable optogenetic tools to control endogenous proteins in mammalian cells.

Signalling of the B cell receptor (BCR) is pivotal for survival and activation of naïve B cells. Here the authors show, using super-resolution microscopy techniques, that BCRs exist primarily as monomers and dimers in resting B cells, and oligomerize only on stimulation, thereby implicating a function of BCR clustering patterns on B cell biology.

Imaging in living Arabidopsis roots reveals that protein complexes can change their conformation in a cell-type-dependent manner to regulate specific gene expression programs leading to precise specification and maintenance of particular cell fates within the root meristem.

An exome wide association study of UK Biobank data revealed 158 variants and 105 genes significantly associated with kidney function traits and disease. The findings are supported by functional evidence for a previously unreported mutation in CLDN10.

Nuclear import of transcriptional regulators and export of mRNA are essential steps for the manufacture of proteins in the cytoplasm. Here, Rajakylä et al.link these two activities by showing that the mRNA export factor Ddx19 promotes the nuclear import of the transcriptional coactivator MKL1.

STIM proteins sense calcium depletion in the endoplasmic reticulum and in response activate calcium influx through Orai1 channels located at the plasma membrane. Here, Miederer et al. identify a novel splice variant of STIM2 that fails to interact with and activate Orai1 and may act to fine-tune cellular calcium homeostasis by negatively regulating calcium influx.

A dogma in the inflammasome field is that NLRP3 activation occurs at dispersed vesicles of the trans-Golgi network. Here, Ricci and colleagues find that these vesicles are of endosomal origin and that endosomes comprise the compartment where NLRP3 is activated.

Determination of interactions between native proteins in cells is important for understanding function. Here the authors report MolBoolean as a method to detect interactions between endogenous proteins in subcellular compartments, using antibody-DNA conjugates for identification and signal amplification.

This Primer offers a practical and rational introduction to macromolecular crystallography, whether to engage directly with or to critically assess results, with a focus on understanding the diffraction data, solving the phase problem, building and refining the atomic model, and interpreting the resulting atomic structure.

Thermally activated delayed fluorescence organic light-emitting diodes (TADF-OLEDs) rely on high triplet energy interlayers to confine excitons, which results in reduced performance. Here, the authors report high-performance blue TADF-OLEDs with below bandgap electroluminescence.

Alzheimer’s disease is often considered a disease of neurons. This study reveals that astrocytes are also impaired by the disease and that these cells contribute more to memory deterioration than previously thought.

Calvo-Rodriguez et al. show elevated calcium levels in neuronal mitochondria in a mouse model of cerebral β-amyloidosis after plaque deposition, which precede rare neuron death events in this model. The mechanism involves toxic extracellular Aβ oligomers and the mitochondrial calcium uniporter.