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This Protocol Extension details the standardized production of iPS cell-derived macrophages in an intermediate-scale benchtop bioreactor, providing a valuable tool for academic labs focused on human immune cells.

The DNA-dependent protease SPRTN cleaves toxic DNA-protein crosslinks (DPCs). Here, the authors show that SPRTN is activated by DPC-ubiquitylation through an allosteric ubiquitin binding interface. This regulatory mechanism enables precise control of SPRTN activity during DNA repair.

A mode of bidirectional microglia–neuron crosstalk through Hex–GM2–MGL2 and centred around GM2 ganglioside turnover is essential for homeostatic regulation of central nervous system membrane components.

The involvement of cell death pathways in the early stage of pancreatic ductal adenocarcinoma (PDAC) development, especially KRAS-dependent acinar-to-ductal metaplasia (ADM), remains to be investigated. Here, the authors find that TAK1 mediates cell survival during ADM transdifferentiation through suppression of apoptosis and necroptosis, which could be targeted for prevention and treatment of PDAC.

Commonly used organic dyes can photoconvert to blue-shifted fluorescent species, especially under intense illumination. The mechanism of this process and how to avoid unwanted artifacts in super-resolution microscopy are explored here.

A full-body nanobody-based immunolabeling and clearing method that renders mice transparent in 3 weeks, enhancing the signal of fluorescent proteins and allowing their reliable detection and quantification, at cellular resolution, within an entire body.

Withanolides are plant steroids with potent bioactivities found in many medicinal plants including Withania somnifera, but their biosynthetic pathway is largely unknown. Here, the authors report the genome assembly of W. somnifera and reveal a conserved gene cluster in Solanaceae plants for withanolide biosynthesis.

The Candida albicans toxin, candidalysin, is embedded in an unusual conserved precursor peptide sequence (Ece1). The precursor is not required to block premature pore-forming toxicity, but rather to prevent candidalysin auto-aggregation.

The catalytic mechanism of [Fe]-hydrogenases is not well understood. Now a signal-enhanced nuclear magnetic resonance method based on parahydrogen is introduced to study [Fe]-hydrogenase under turnover conditions in situ, revealing intermediates of the catalytic cycle.

Multiomic factor analysis of blood multiomic data, including single-cell transcriptomics, for individuals with either acute or chronic coronary syndrome identifies immune cell signatures that correlate with treatment outcomes.

The study shows that the HIV-1 Nef protein stabilizes actin, thereby preventing R12C release and priming of RIG-I–like receptors. HIV-1 containing a mutant Nef unable to bind the actinmodulating kinase PAK2, triggers enhanced interferon responses.

By combining left- and right-handed DNA-PAINT probes, Unterauer et al. achieve simple, robust, and highly multiplexed super-resolution. They show 13-plex neuronal maps, revealing nanoscale organization of cytoskeleton, organelles, and synapses.

Fatty liver disease exacerbates atherosclerosis, but the underlying mechanisms remain unclear. Here, the authors show that D-dopachrome tautomerase (D-DT/MIF-2) acts as an atypical chemokine, promoting both atherosclerosis and hepatic lipid accumulation.

Federated learning (FL) algorithms have emerged as a promising solution to train models for healthcare imaging across institutions while preserving privacy. Here, the authors describe the Federated Tumor Segmentation (FeTS) challenge for the decentralised benchmarking of FL algorithms and evaluation of Healthcare AI algorithm generalizability in real-world cancer imaging datasets.

A molecular mechanism is revealed through which commensal bacteria modulate intestinal epithelial barrier function.

Coating technologies call for effective methods capable of suppressing the coffee-ring effect for a uniform particle deposition. Rey et al. show homogeneous drying patterns can be achieved via physically adsorbing polymers onto particle surfaces and the method is applicable to a wide range of materials regardless of the shape of the dispersed particles.

Cell-type-resolved spatial proteomics of the skin from patients with toxic epidermal necrolysis reveals that it is driven by JAK/STAT signaling, leading to successful treatment of this potentially fatal condition in patients using JAK inhibitors.

Branched organoids embedded in collagen gels can recapitulate the phenotypic landscape and pronounced molecular and morphological heterogeneity of pancreatic ductal adenocarcinoma.

Olfactory information from Kenyon cells in the mushroom body and reward information from pPAM dopaminergic neurons is required for appetitive olfactory learning and memory. Here, the authors report evidence for a feedback circuit mechanism between Kenyon cells and pPAM neurons for reward memory that involves short neuropeptide F.

The nanoscale organization of the antigen-antibody complexes influences the therapeutic action of monoclonal antibodies. Here, the authors present a multi-target 3D RESI imaging assay for the nanometer spatial analysis of CD20 in complex with therapeutic monoclonal antibodies within intact cells, to analyse the interdependency between the mode of antibody binding and the therapeutic function.

Augmin is essential for microtubule nucleation during cell division. This study reveals how the conserved calponin homology domain of the augmin subunit HAUS6 binds to microtubules to orient augmin for microtubule branching.

Metabolic processes may be different in immune cells between neonates and adults. Here the authors measure metabolic changes in human monocytes from different aged donors and find enhanced oxidative phosphorylation fueling myeloid differentiation in neonates which contrasts glycolytic responses in early childhood and more effective inflammatory response.

Imaging of muscle fibrosis with a handheld device could potentially serve as a biomarker for disease progression and response to therapy in patients with Duchenne muscular dystrophy.

Antibodies targeting the spike protein of coronaviruses are potential candidates for therapeutic development. Here, Bertoglio et al. use phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve universal antibody gene libraries HAL9/10 that block interaction with ACE2 receptor to inhibit infection.

The combination of isotopic tracers with root phenotyping reveals specific patterns in photosynthate allocation. These patterns are reflected in rhizodeposition and result in spatially distinct microbiomes within the plant root system.

Surgical adhesions are organ-joining bands of scar tissue that remain clinically untreatable. Here, the authors show that adhesions are formed through expansive mesothelial membrane bridges, and that blocking these with small molecules prevents formation of adhesions in mice.

MYC drives S-phase progression and immune invasion in pancreatic ductal adenocarcinoma (PDAC), but the underlying mechanisms are not fully understood. Here, the authors show that the transcription elongation complex PAF1c controls the competition of different gene sets for RNA polymerase and elongation factors to regulate these MYC-associated mechanisms in PDAC.

Through a CRISPR-activation screen, Ravid Lustig et al. show that GATA transcription factors activate long-range Xist enhancers to upregulate Xist expression and initiate X chromosome inactivation in early mouse embryos.

The authors provide protocols for rapid and scalable genome engineering in somatic cells of the liver and pancreas through both viral and nonviral delivery of CRISPR components into living mice.

Extracting quantitative information on biomolecular oligomerisation with high resolution remains a significant challenge. Here, the authors propose SPINNA, a framework that compares nearest-neighbour distances from experimental single-protein position data with those obtained from simulations based on a model of protein oligomerisation.

Obesity leads to pathological expansion of white adipose tissue driving vascular dysfunction. Here, the authors utilize single-cell RNA sequencing to elucidate endothelial heterogeneity and demarcate key differences in obesity-associated vascular alterations in subcutaneous and visceral white adipose tissue.

Existing methods for nanoscale visualization of biological targets in thick samples require complex hardware. Here, the authors combine the standard spinning disk confocal (SDC) microscopy with DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) to image proteins, DNA and RNA deep in cells.

Here the authors show that active DNA demethylation and transcription factor occupation at distal regulatory elements is essential for pluripotency maintenance in dormancy conditions.

Innate lymphoid cells (ILC) are involved with different immune responses. Here the authors show that Interferon regulatory factor 1 (IRF1) is important for intestinal ILC3 accumulation during Citrobacter rodentium infection and promotes release of the protective cytokine IL-22 and response to IL-23.

It is unclear how cell compartmentalization emerged in prebiotic conditions. Now it is shown that a temperature gradient in a confined space can bring the core components of a cell together.

Single-molecule localisation microscopy is limited by low labeling and detection efficiencies of the molecular probes. Here the authors report a framework to obtain absolute molecular quantities on a true molecular scale; the data reveal a ternary adhesion complex underlying cell-matrix adhesion.

An assay based on the electrophoresis of whole-cell lysates embedded in agarose plugs separates soluble from immobilized proteins, enabling the purification and the subsequent identification of DNA–protein cross-links.

Self-derived DNA may trigger interferon-driven autoinflammation mediated by the cGAS-STING axis. Here, the authors find that mutations in the GTPase ARF1 cause an interferonopathy by promoting aberrant mitochondrial DNA release and impairing STING recycling.

The authors introduce a single-molecule DNA-barcoding method, resolution enhancement by sequential imaging, that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents.

Using brain organoids models, Prigione and colleagues uncovered the impact of Huntington’s disease on human brain developmental and identified early dysregulation of CHCHD2, which disrupted mitochondria and might serve as a therapeutic target.

Hegazy and colleagues demonstrate that epithelial oncostatin M receptor expression drives gut inflammation and tumor formation.

Tobacco smoking and cold exposure are environmental modulators of human energy metabolism suppressing appetite and increasing energy expenditure, respectively. Here, the authors develop a novel pharmacological strategy in which they simultaneously mimic the metabolic benefits of both phenomena through small-molecule combination therapy, and show that this treatment improves metabolic health of obese mice.

MethyLYZR, an epigenetic classifier of brain tumors, provides clinically relevant cancer classification results within 15 min of sequencing, with potential applications for neurosurgical intraoperative use.

Printed organic and inorganic electronics continue to be of large interest for several applications. Here, the authors propose laser printing as a facile process for fabricating printed electronics with minimum feature sizes below 1 µm and demonstrate functional diodes, memristors, and physically unclonable functions.

Ubiquitin (Ub) and Ub-like modifiers (Ubls) can form chains of various topologies, but preparing defined chains for functional studies remains challenging. Here, the authors develop chemoenzymatic tools to tailormake Ub/Ubl chains and study the involvement of specific Ub/SUMO chains in DNA repair.

Here, using cryo-EM and biochemistry, the authors delineate how the XPD helicase unorthodoxly uses its Arch domain to separate double-stranded DNA upon approaching a DNA lesion, promoting our understanding of NER bubble formation and damage verification.

The risk of developing active tuberculosis (TB) in individuals with latent TB infection is highly variable within and among different risk groups. A personalized risk predictor was developed to better target preventative treatment to individuals at greatest risk, supporting evidence-based clinical decision-making for latent TB.

In DNA-PAINT, transient binding of dye-labeled oligonucleotides to their target strands creates the ‘blinking’ required for stochastic nanoscopy. This protocol describes how to apply DNA-PAINT, from sample preparation to data processing.