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Transfer RNA (tRNA) is an important RNA in cells that decodes messenger RNA (mRNA) codons during protein translation to ensure correct amino acid sequences. The biogenesis of tRNA involves multiple processing steps to produce mature and functional molecules. Pseudouridine (Ψ), a derivative of uridine, is an abundant RNA modification and occurs at multiple positions within tRNAs. These modified sites are highly conserved across organisms. Classical biochemical studies have established that Ψ stabilises RNA-RNA interactions, but structural characterisations and molecular dynamics simulations reveal that Ψ can locally remodel tRNA architecture in ways that are dictated by where it is within tRNAs. Advances in transcriptome-wide Ψ mapping have uncovered additional modified sites beyond those previously described, with several novel sites appearing to be regulated in a cellular context-dependent manner. Furthermore, dysregulated pseudouridylation has been implicated in conditions ranging from cancer to inherited genetic disorders. Together, these developments reframe our understanding of Ψ from a well-established RNA stabiliser to a modification with roles far more dynamic, context-dependent, and clinically relevant than previously appreciated. The present review summarises and discusses the up-to-date developments in the impacts of pseudouridylation on human tRNA biogenesis, tRNA functions, and human health. More mechanistic questions remain open and will require further investigation. As pseudouridylation can also happen in mRNA and rRNA, exploring the interplay between these RNAs will be crucial for fundamental biology and advancing Ψ applications in biotechnology and biomedical uses.

Organelle contact sites are highly dynamic and specialized regions where distinct organelles come into proximity, enabling direct inter-organelle communication. These structures play fundamental roles in cellular homeostasis by coordinating the exchange of lipids, metabolites, and ions, as well as regulating key processes such as organelle dynamics, mitochondrial fission, autophagy, and metabolic integration. Alterations in contact site architecture and function have been increasingly associated with a wide range of human diseases, including neurodegeneration, metabolic disorders, and cancer. Despite their biological relevance, the nanoscale nature and dynamic behaviour of contact sites have historically posed significant challenges for their accurate detection and functional characterization. Here, we provide a comprehensive overview of the methodologies currently available to study organelle contact sites, ranging from classical approaches such as electron microscopy and biochemical fractionation to advanced imaging techniques and genetically encoded reporters. We discuss recent developments in high-resolution and live-cell microscopy that have improved the spatial and temporal resolution of contact site analysis, as well as emerging tools designed to selectively label, quantify, and manipulate these interfaces. Attention is given to the next generation of engineered reporters capable of sensing molecular and ionic exchanges at contact sites, thereby moving beyond structural description toward functional interrogation. By critically evaluating the strengths and limitations of existing approaches, we aim to provide a framework for selecting appropriate tools and to highlight future directions in the field. Ultimately, advancing our ability to monitor and dissect organelle contact sites will be essential for understanding their contribution to cellular physiology and disease.

As the optimal source of nutrition for infants, investigations into the human milk lipidome have been quite extensive. Much of the work, however, has been focused on major lipid components such as triglycerides, possibly undermining its actual complexity. This review focuses on two minor but bioactive lipid classes in human milk: fatty acid esters of hydroxy fatty acids (FAHFAs) and alkyl-diacylglycerols (TG(O)s). FAHFAs are known to exhibit anti-diabetic and anti-inflammatory effects, while TG(O)s are important for the prevention of childhood obesity. With the knowledge that early nutrition and metabolic health influence the risk of metabolic dysfunctions later in life, a comprehensive understanding of FAHFAs and TG(O)s, along with reliable characterisations in human milk, would better allow for the development of accurate human milk fat substitutes. This could have future implications as alternative or preventive treatments for infants with early markers of metabolic dysfunction, including diabetes and obesity. The structural characteristics, pathways for biosynthesis and degradation, bioactivity, dietary sources, and characterisations of FAHFAs and TG(O)s in human milk are discussed. Their statuses as emerging lipid classes, however, is reflected in the incomplete understanding of their biochemical pathways. Characterisations of FAHFAs and TG(O)s in human milk are relatively poor, and contradicting results are reported. This review also addresses the challenges involved in the study of minor lipids in complex biological matrices, and the possible reasons underlying the slower evolution of our understanding of FAHFAs and TG(O)s in human milk and their associations with health outcomes.

Double-stranded RNA (dsRNA) is a universal indicator of viral replication and dysregulated RNA metabolism. Detection of dsRNA triggers some of the most powerful innate immune responses in human cells. Although these molecules differ in origin and structure, viral dsRNAs share the defining geometric and electrostatic features of the A-form helix, enabling their sequence-independent recognition by multiple sensor systems. Cytosolic receptors, like retinoic acid-inducible gene I (RIG-I), melanoma differentiation associated gene 5 (MDA5), and protein kinase R (PKR), as well as the oligoadenylate synthase (OAS)/RNase L pathway, convert dsRNA binding into interferon induction, translational arrest, and widespread RNA decay, while endosomal Toll-like receptor 3 (TLR3) and the inflammasome sensor NLR family pyrin domain containing 1 (NLRP1) expand surveillance to internalised or structurally disruptive RNAs. Counterbalancing these pathways, the RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) marks endogenous dsRNA through A-to-I conversion, preventing inadvertent activation of innate immune response and maintaining self versus non-self discrimination. Although all of these sensors recognise the A-form helix, each extracts distinct structural and chemical information from dsRNA and converts it into a specific response: RIG-I detects short duplexes with 5'-triphosphorylated ends; MDA5 assembles cooperatively along long uninterrupted helices; PKR integrates duplex length with translational control; OAS proteins act as strict reporters of helix regularity; and TLR3 as well as NLRP1 respond to dsRNA in compartment- and context-dependent ways. Epitranscriptomic marks and chemical modifications-including 2'-O-methylation, N6-methyladenosine, pseudouridine, and ADAR1-mediated inosine-further refine sensing by modulating helical stability and end structure, establishing a biochemical 'self-code' that shapes RNA immunogenicity. Together, these pathways form an integrated network that distinguishes between viral and endogenous dsRNA and coordinates antiviral defence with immune tolerance.

Initially identified through chromosomal translocations in lymphomas, the BCL7 protein family, comprising the three paralogues BCL7A, BCL7B, and BCL7C, has recently emerged as a core component of mammalian SWI/SNF ATP-dependent chromatin remodeling complexes. Although their functions remained poorly understood for many years, recent structural and biochemical studies have substantially improved our understanding of their roles. Cryo-electron microscopy studies revealed that BCL7 proteins interact with nucleosomes through a conserved N-terminal arginine anchor motif that binds the nucleosomal acidic patch and stabilize the actin-related protein module through a conserved β-hairpin motif. These findings identify BCL7 proteins as structural elements that contribute to nucleosome engagement and SWI/SNF complex integrity. Comparative analyses further suggest that key structural features of BCL7 proteins are conserved across evolution despite limited sequence similarity. In addition to their roles in chromatin remodeling, increasing evidence links BCL7 proteins to hematological malignancies, solid tumors, and developmental disorders, highlighting their emerging value as biomarkers and potential therapeutic targets. This review summarizes current knowledge on the structure, evolution, and functions of the BCL7 family and outlines future directions for elucidating their contribution to chromatin regulation and disease.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently inactivated tumor suppressors in human cancers, serving as a critical negative regulator of phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Although genetic mutation or deletion commonly underlie functional PTEN loss, accumulating evidence indicates that post-transcriptional and post-translational mechanisms also substantially contribute to PTEN suppression. Phosphatases of regenerating liver (PRLs), comprising PRL1, PRL2, and PRL3, are oncogenic phosphatases frequently overexpressed in both solid and hematological malignancies. Emerging studies reveal that PRLs can downregulate PTEN through a post-translational mechanism by direct dephosphorylation of PTEN at Tyr336, therefore promoting PTEN ubiquitination and proteasomal degradation. PRLs can also reduce PTEN expression through a post-transcriptional mechanism by dephosphorylating the inhibitory Tyr570 in JAK2, thereby activating the JAK2/STAT3-mediated miR-21 expression. These coordinated actions collectively amplify PI3K-AKT signaling, consequently promoting proliferation, survival, and metastasis. In the present review, we synthesize current knowledge of PRL structure, evolution, and functional diversity, evaluate genetic, biochemical, and organismal evidence linking PRLs to PTEN regulation, and discuss insights on PRL oncogenicity derived from experimental models. We further examine context-dependent functions of PRLs, unresolved questions regarding catalytic versus scaffold activities, and the therapeutic potential of targeting the PRL-PTEN axis. Understanding how PRLs modulate PTEN activity may reveal new strategies to restore tumor suppressor function in PTEN-deficient cancers.

Calcium (Ca2+) signaling is a fundamental regulator of virtually all aspects of eukaryotic cell physiology, including gene expression, secretion, metabolism, motility, and cell fate decisions. The spatial and temporal control of cytosolic Ca2+ signals relies on a coordinated interplay between intracellular Ca2+ stores and plasma membrane (PM) Ca2+ channels. A critical advance in this field over the past two decades was the molecular identification of stromal interaction molecule 1 (STIM1) as the long-sought Ca2+ sensor that couples depletion of endoplasmic reticulum Ca2+ stores to Ca2+ influx across the PM. STIM1 has been established as a core component of store-operated Ca2+ entry, acting through direct activation of ORAI Ca2+ channels. However, accumulating evidence now indicates that STIM1 functions extend beyond this canonical role. STIM1 participates in the regulation of multiple classes of ion channels, contributes to the organization of membrane contact sites, and acts as a signaling scaffold influencing cellular processes independently of classical store depletion. This review summarizes the discovery and canonical functions of STIM1 and focuses on its emerging non-canonical roles, highlighting how STIM1 has evolved from an ER Ca2+ sensor into a multifunctional signaling hub.

Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motor neuron disease, characterised by the degeneration of upper and lower motor neurons. The cytoplasmic aggregation of TDP-43 (TAR DNA-binding protein 43), an RNA-binding protein, is considered a hallmark of ALS pathology, found in nearly all postmortem cases of ALS. TDP-43 is normally primarily nuclear, where it has a widespread role in gene regulation. Mutations, extrinsic stressors, and alterations in RNA homeostasis in ALS lead to nuclear depletion of TDP-43 and the formation of cytosolic TDP-43 aggregates. This causes multiple downstream effects on neuronal function and degeneration as well as gene expression. TDP-43 is a promising target as a biomarker, as it is found to be elevated in the biofluids of ALS patients, and its cytoplasmic aggregation can also be observed in peripheral tissues; however, methodological variability and technical limitations currently preclude the establishment of TDP-43 as a standalone biomarker. There are also promising therapeutic strategies in development targeting TDP-43 pathology, but a critical challenge that remains is achieving a balance between eliminating toxic aggregates and preserving the essential functions of TDP-43. In summary, with further research, considering TDP-43 pathology in ALS gives hope for finding future novel diagnostics and therapeutics for ALS.

The continuing development and characterisation of human-induced pluripotent stem cell (hiPSC)-derived cell-types has opened up a virtually endless source of human, physiologically relevant cells, available at scale, for scientific research. The technology's maturation and refinement have allowed additional cell-types and sub-types to become available. The first step in adopting these novel cell-types is to properly characterise these cells and compare how they perform against the longer-established cell-types. Parallel to the progress in iPSC-derived cells has been the great strides in the platforms developed to assess and analyse the characteristics and functions of cells. These improved platforms have greatly increased the range, throughput and quality of the functional data that can be obtained from cell-types, including iPSC-derived cells. Research into cardiomyocytes in particular has been greatly enhanced by these platforms as cardiomyocytes not only have the expected cellular markers, proteomics and transcriptomics but are also electrically active and capable of contracting, opening a wide vista of potential assays. If human iPSC-derived cardiomyocytes are to confidently replace and supplement the existing animal and cellular models of the heart, it has to be demonstrated that they correctly replicate (or even improve) upon the functions and pharmacology of the existing heart models used on these new and improved platforms. Therefore, this review compares the functional and pharmacological differences seen between Axol's human iPSC-derived atrial and ventricular cardiomyocyte cells on a range of established and newer platforms demonstrating the advantages of using chamber-specific human iPSC-derived cardiomyocytes and discussing how their use could supplement these emerging techniques.

For decades, the voltage-dependent anion-selective channel (VDAC), formerly known as the mitochondrial porin, was considered a simple pore enabling nearly free permeability across the outer mitochondrial membrane. This simplified view has been progressively dismantled through the discovery of three mammalian isoforms (VDAC1, VDAC2, and VDAC3) with the gradual attribution, often serendipitous, of diverse cellular roles beyond passive metabolite exchange. Recent advances in cryo-electron microscopy have catalyzed a breakthrough in VDAC research. Three converging lines of evidence are reshaping our understanding: (a) high-resolution structures of VDAC within its native protein complexes; (b) discovery of unexpected functions, including phospholipid scrambling and regulation of outer membrane permeabilization through higher-order oligomeric assemblies; and (c) structural determination of VDAC interactions with macromolecules, as well as small-molecule modulators. Collectively, these insights have strengthened the consideration of VDAC as a multifunctional signaling hub and therapeutic target, with emerging small molecules and peptides designed to modulate gating, oligomerization, and interfering with interacting partners. The aim of this review is to summarize current structural, functional, and pharmacological advances in VDAC biology, emphasizing how oligomerization dynamics and isoform specificity orchestrate mitochondrial behavior and offering perspectives on therapeutic strategies for diseases driven by mitochondrial dysfunction.

Membrane proteins are essential to cellular signaling, transport, and homeostasis, yet their amphipathic nature, dependence on lipids or detergents, and typically difficult expression and purification make them difficult targets for structural methods such as X-ray crystallography. Moreover, most membrane proteins in the human proteome are too small for investigation by single-particle electron cryomicroscopy methods. Microcrystal Electron Diffraction (MicroED) has emerged as a powerful method for overcoming these barriers, allowing structure determination from nanocrystals of membrane proteins embedded in the near-native environment of the lipid membrane. In the present review, we discuss how recent improvements in MicroED, such as focused ion-beam milling and high-throughput data collection approaches, facilitate structure determination and investigation of protein dynamics. We focus on applications involving junction-forming proteins, G protein-coupled receptors, and ion channels, where MicroED has revealed physiologically relevant assemblies, lipid interactions, and transient functional states that were not attainable by other structural biology methods.

The vital role of brain-derived neurotrophic factor (BDNF) in neuronal development, synaptic plasticity, and neuroprotection has been explored for decades. Therefore, the expression, processing, and signalling activities of this neurotrophin, which is reliant upon TrkB and p75NTR receptors, have been well characterised in both health and disease. This review summarises the latest findings on BDNF dysregulation in neuropathologies. Indeed, across diseases of both the central and peripheral nervous systems, BDNF signalling is frequently disrupted, contributing to neuronal dysfunction and degeneration. Consequently, through direct or indirect enhancement of its expression and/or function, BDNF has proved to be a promising therapeutic target across many neurological conditions. However, the complexity of its regulation and interaction with several different receptors underpins the need for further research to deepen our understanding of BDNF disruption in neuropathologies and to achieve its therapeutic potential.

Lipid droplets (LDs) have a multitude of functions ranging from lipid storage to fighting infection and are decorated with a variety of proteins on their surface that determine their functions and behaviours. Mass spectrometric analysis has identified the vast array of LD-localised proteins, which have recently been shown to be dynamic, changing in response to cellular stress, infection, and altered homeostasis. Here, we review the key mechanisms of cytoplasmic protein interactions with the LD, highlighting conventional features like amphipathic helices, atypical sequence-based motifs, protein-protein interactions, and post-translational modifications that confer dynamic targeting of proteins to the surface of the LD. A better understanding of the transient LD proteome and the mechanisms that confer LD protein targeting will allow researchers to develop a more thorough understanding of LD biology, and the role of LDs in cellular homeostasis and disease.

Tunneling nanotubes (TNTs) are thin, actin-based membrane bridges that establish direct cytoplasmic continuity between distant cells, enabling the transfer of diverse cargoes ranging from ions and proteins to organelles such as mitochondria. Since their discovery in 2004, TNTs have been identified in numerous cell types and linked to an expanding range of physiological and pathological functions. Yet, their molecular identity and mechanisms of formation remain elusive. The most defining and least understood step in TNT biogenesis is membrane fusion, the process by which TNTs achieve open-ended continuity between cells, and this represents a critical frontier in the field. This review integrates recent advances in TNT biology, emphasizing the interplay between actin cytoskeletal dynamics, plasma membrane composition, and cell adhesion during TNT formation. It also draws mechanistic parallels with established models of membrane fusion, highlighting fundamental principles and shared regulators across fusion systems, many of which have been implicated in TNT functionality. By combining molecular, biophysical, and imaging perspectives, this review proposes a conceptual framework for TNT formation and fusion, identifies major methodological gaps, and outlines future directions to unravel the mechanisms that underlie intercellular cytoplasmic continuity.

Protein phosphorylation is one of the most common and versatile regulatory mechanisms in cells. Most human proteins are phosphorylated at multiple sites, giving rise to large numbers of possible phosphorylation patterns. Each phosphorylation pattern can lead to a different functional or pathological outcome. Yet, linking defined phosphorylation patterns to specific biological functions remains a major experimental challenge. In this review we describe the main strategies to study phosphorylation patterns at the protein and domain levels and highlight how they complement each other. We first discuss cellular approaches, including phosphomimetics, kinase-based assays, and genetic code expansion, which allow working in a native environment but have their significant drawbacks. We then describe in vitro methods, such as enzymatic phosphorylation and semi-synthetic phosphoproteins generated by ligation, which afford mechanistic insights but result in low yields and are difficult to scale for producing libraries. We focus on synthetic phosphopeptide libraries as tools that offer precise control over the number and position of phosphosites and are uniquely suited for systematic mapping of phosphorylation patterns. This comes at a price of not working at the protein level, but rather at the domain level. Peptide libraries are often used for preliminary identification of key phosphorylations, later studied in detail at the protein level. We conclude that ideally more than one method should be used and that these methods should not be viewed as competing but rather as complementary. A combined use of several of these approaches provides a practical toolbox for dissecting how phosphorylation patterns regulate protein behavior.

Ubiquitination is a versatile post-translational modification process in which the small globular protein ubiquitin is covalently attached to substrate proteins to generate diverse cellular signals. Although originally characterized by its role in proteasome-mediated protein degradation, ubiquitination is now recognized as a central regulator of numerous processes, including signaling, trafficking, and immunity. Canonical ubiquitination is mediated by a cascade of E1 (activating), E2 (conjugating), and E3 (ligase) enzymes that repeatedly conjugate ubiquitin molecules to lysine residues on substrate proteins, leading to the formation of polyubiquitin chains with distinct topologies. The modification is reversed by deubiquitinating enzymes. Notably, components of the ubiquitin system comprise approximately 7% of the human proteome, underscoring its importance in biological regulation. Recent advances have revealed the broad scope of ubiquitination. Ubiquitin was found to conjugate not only to lysine but also to serine, threonine, and cysteine, indicating its unexpected chemical flexibility. Furthermore, ubiquitination can be directed toward other post-translational modifications, particularly glycosylation and ADP-ribosylation, highlighting the extensive crosstalk between modification systems. Strikingly, lipids, sugars, metabolites, nucleic acids, and even synthetic small-molecule compounds have been identified as ubiquitinated substrates. The hypothesis that virtually all classes of molecules are targeted by ubiquitination has become increasingly plausible. Taken together, these findings redefine ubiquitination as a far more general modification process than previously appreciated. In this mini-review, we focus on recent progress in non-proteinaceous ubiquitination research, summarize emerging substrate classes, and discuss key challenges in elucidating the underlying mechanisms and physiological roles of this expanding modification landscape.

Toxins, substances that are produced by living organisms with the potential to cause harm, demonstrate great diversity in their structure, function, and origin. Though some toxins have been repurposed for use as novel therapeutics, research tools, or for application in agriculture, the mechanism of action for many toxins remains uncharacterised. Pooled CRISPR screens offer a high-throughput and unbiased method for rapid annotation of the host cell genome and identification of factors mediating or modifying intoxication. In this review, we provide a brief overview of CRISPR screening before detailing how screens have been used to characterise toxins from various biological kingdoms. We highlight certain cell entry factors and intracellular processes as conserved targets of various toxins. Finally, we highlight limitations in the methods of CRISPR screens used thus far and make recommendations as to how screen design can be modified to more completely characterise toxin activity and elucidate systemic effects of intoxication.

The mismatch repair (MMR) system is an essential DNA repair mechanism that recognizes and corrects single base-base mismatches and unpaired nucleotides that escaped the proofreading exonuclease activity of DNA polymerases or recombination intermediates. This pathway is highly conserved throughout evolution. However, the nature and number of MMR proteins differ between eukaryotes and prokaryotes. Even more, the plant MMR system contains an ancient duplicated MMR protein. In addition, developmental processes vary among eukaryotic organisms. One striking feature is plant genome stability maintenance over multiple generations because embryogenesis and seed development occur after many divisions during plant vegetative growth. Thus, it was of our interest to review the present state of knowledge with respect to the MMR mechanism from eukaryotic organisms, with special comparisons between human, yeast, and plant systems.

During each cell cycle, cells must decide whether to continue to proliferate or to exit the cell cycle into a reversible arrest state, known as quiescence, or G0. This decision must be highly regulated to ensure proper tissue homeostasis. Studies on kinase-driven signalling pathways that regulate this decision point have dominated the field, and the role of phosphatases remains comparatively underexplored, yet the role of phosphatases is vitally important in signal transduction. In the present review, we examine how phosphatases contribute to the regulation of quiescence in mammalian cells across three stages: entry into quiescence, maintenance of the quiescent state, and quiescence exit into the cell cycle. We discuss how phosphatases counteract mitogenic signalling pathways, including MAPK/ERK and PI3K-AKT-mTOR, and maintain low cyclin-dependent kinase (CDK) activity through dephosphorylation of key cell cycle regulators, such as the retinoblastoma family proteins and CDK inhibitors. Finally, we highlight emerging evidence that dynamic regulation of phosphatase activity shapes the transition from quiescence back into proliferation. Understanding how phosphatases regulate the reversible nature of cell cycle arrest is important for understanding how tissues maintain homeostasis and how dysregulation of quiescence contributes to disease, including cancer.