Enteroendocrine L cells function as chemosensors in the intestine, secreting glucagon-like peptide-1 (GLP-1) to signal nutrient ingestion to the brain. Since GLP-1 secretion is induced by a variety of nutrients, including sugars, amino acids, and fatty acids, GLP-1 receptor-expressing neurons are also activated regardless of the nutrient identity, making it unclear how the brain discriminates between different nutrients. By examining vesicle dynamics and extracellular signals associated with GLP-1, glutamate, and ATP in GLUTag cells using total internal reflection fluorescence (TIRF) microscopy, a technique that selectively visualizes fluorescent molecules near the plasma membrane, we demonstrated that distinct nutrients evoke specific secretory patterns. D-glucose induced the co-secretion of GLP-1, glutamate, and ATP, whereas L-glutamine induced the secretion of GLP-1 and glutamate but not ATP. Butyrate and sucralose, which are also GLP-1 secretagogues, did not induce the secretion of glutamate or ATP. While Ca[2+] acts as the trigger for exocytosis, additional regulatory mechanisms are required to generate these distinct secretory patterns. We therefore investigated the cAMP and cGMP pathways and found that neither their activation nor inhibition detectably affected glutamate and ATP secretion, suggesting the involvement of other nutrient-specific signaling pathways. Collectively, our findings suggest that L cells distinguish and encode nutrient identity as combinatorial secretion patterns of GLP-1 and neurotransmitters.

Genetic and functional studies strongly implicate microglia in the pathology of Alzheimer's disease. The Triggering Receptor Expressed on Myeloid cells 2 (TREM2) pathway is an important functional regulator of microglia, promoting phagocytosis of apoptotic cells, debris and pathogenic proteins. Genome-wide association studies have identified risk-bearing polymorphisms in several members of the pathway including INPP5D, encoding the inositol phosphatase SHIP1. Recent studies utilising in vitro and in vivo disease-relevant models have identified a role for SHIP1 in modulating TREM2 signalling and neurotoxic microglial activation. In this study, we characterised the role of SHIP1 in regulating TREM2 signalling and myeloid functions using human-induced pluripotent stem cell (hiPSC)-derived macrophages. Characterisation of isogenic SHIP1 knockout (SHIP1 KO) macrophages showed decreased expression of the lipopolysaccharide (LPS) coreceptor CD14, which translated to lower secretion of pro-inflammatory cytokines. Additionally, loss of SHIP1 in this macrophage model attenuated TREM2 signalling, which was further reflected in a reduced level of phagocytosis of apoptotic neurons compared to the parental line. The observed effects on TREM2 signalling did not correspond to changes in total protein levels or shedding. Further examination of macrophage scavenger receptor expression revealed reduced cell surface levels of CD163 and CD206 involved in resolution of inflammation, but increased levels of the phagocytic receptor MerTK. Morphological analysis revealed a less amoeboid-activated phenotype and increased adhesion in the SHIP1 KO macrophages. Taken together, our data indicate that SHIP1 plays a key role in modulating TREM2 signalling, inflammation and phagocytosis.

Ageing is characterized by the progressive accumulation of molecular and cellular damage, driven by both genetic and nongenetic factors and ultimately leading to functional decline and an increased risk of disease. Convergent evidence indicates that many of the mechanisms influencing ageing are intimately related to the ability of the organism to cope with intrinsic and extrinsic stress. Key determinants of this capacity are the molecular pathways that coordinate stress responses throughout life. Among these, general and cargo-specific autophagy have emerged as integral components of cellular and systemic adaptation to stress. Here, we focus on mitophagy, a selective form of autophagy targeting mitochondria, and nucleophagy, the selective autophagic recycling of nuclear material. These processes help safeguard the integrity of two organelles that are central to long-term cellular survival and function. Such homeostatic mechanisms are vital in long-lived neurons, where they mediate damage removal during ageing to protect against neurodegeneration. We further discuss how perturbations in these autophagic pathways may contribute to ageing and neurodegenerative disorders. Finally, we outline key open questions and future challenges in the field, including the largely unexplored possibility of functional connections between mitophagy and nucleophagy, which we identify as an important direction for future research.

T cells are central mediators of antitumour immunity and are the cellular basis of many successful cancer immunotherapies, including immune checkpoint blockade, T-cell receptor (TCR)-engineered therapies and chimeric antigen receptor (CAR) T cells. However, clinical responses remain variable, and many tumours evade or resist T-cell-mediated control. Understanding how T cells recognise tumour cells, deliver cytotoxic signals, produce inflammatory cytokines and shape tumour cell fate is therefore essential for improving modern immunotherapies. In vitro tumour T-cell coculture systems provide a powerful experimental framework for dissecting these processes under controlled conditions. They allow investigators to manipulate antigen specificity, HLA/MHC restriction, tumour genotype, T-cell activation state, cytokine exposure and drug-mediated perturbation in ways that are difficult to achieve in vivo. However, these systems require careful design, because assay outcomes are strongly influenced by tumour model, antigen expression, effector-to-target ratio, assay duration and readout. This guide provides a practical framework for studying tumour T-cell interactions in vitro, with a focus on CD8+ T-cell-mediated tumour recognition and killing. It discusses commonly used mouse and human antigen-specific models, how antigen expression and presentation can be experimentally controlled, T-cell activation and coculture conditions, cytotoxicity assays, immune synapse imaging, competition assays, cytokine-mediated bystander killing, transcriptional profiling, CRISPR perturbation strategies, therapeutic testing and patient-derived models. The aim is to help researchers choose the most appropriate model, experimental setup and readout for the biological question being asked and to avoid common interpretive pitfalls when studying tumour immune sensitivity, resistance and therapeutic response.

Obstructive nephropathy is a renal pathology caused by impaired urinary outflow. Although ureteral occlusion in clinical practice is often reversible, the recovery phase after obstruction relief remains incompletely understood and lacks mechanism-based interventions. Caloric restriction (CR) is widely considered a nephroprotective strategy and has shown therapeutic potential in several models of kidney injury. In this study, we investigated whether a 35% CR pretreatment for 4 weeks modulates the early postobstructive renal response in a rat model of reversible unilateral ureteral obstruction (R-UUO). Pretreatment with CR did not enhance early kidney recovery 24 h after restoration of urine flow, as evaluated by serum creatinine, urea, and renal injury markers. During this phase, CR also failed to improve fibrosis and tissue remodeling markers (α-SMA, MMP2, Tgfb1). However, CR was associated with reduced renal Ngal expression and a shift in the inflammatory response, characterized by lower neutrophil infiltration. Meanwhile, macrophage markers CD163, Cd206, and Tlr2 remained elevated. CR altered acetylation-related molecular response, including opposite shifts in acetylation of histone H3 and histone H4, increased SIRT3, and decreased SIRT1, and resulted in selective preservation of oxidative phosphorylation complex I. These data indicate that under severe postobstructive kidney injury, CR induces selective inflammatory and metabolic-epigenetic remodeling without clear early morphofunctional rescue. Mitochondrial function and epigenetic plasticity appear to be CR-responsive elements, while timing and severity of injury appear critical for manifestation of phenotypic nephroprotection. Thus, development of more targeted CR protocols or CR mimetics may be required to translate observed molecular shifts into morphofunctional benefit.

The recent COVID-19 pandemic and the epidemic outbreak of Mpox have demonstrated that viruses, despite their apparently simple structure, are sophisticated microorganisms that have evolved complicated pro-survival mechanisms. Their potential to manipulate cell death pathways can affect and determine the outcome of the infection cycle and the host's immune response. RNA viruses typically display a more acute and aggressive nature, as they often promote cell death. In this way, they proliferate and release their virions to infect adjacent cells and perpetuate their life cycle. Common consequences of RNA virus infection include hyperinflammatory responses in hosts, typically leading in damage to the epithelial barrier. Conversely, DNA viruses modulate these pathways to sustain a balance between the lytic cycle, during which they replicate, and latency, wherein they remain inactive until an appropriate stimulus triggers reactivation. Achieving this balance and transitioning between stages of infection requires the inhibition of cellular death processes. In both instances, the viruses seem to respond to the cellular context, enhancing their adaptability through mutations, so to deceive host defenses and therefore secure their survival. This highlights the necessity to comprehend the processes via which these biological threats induce cell death, thereby facilitating the development of broad-spectrum or targeted antiviral treatments.

Urinary extracellular vesicles (uEVs) are an attractive source of biomarkers as their cargo of proteins, nucleic acids and lipids reflects cell pathophysiology. The physical properties of uEVs such as their electrical nature measured by the zeta potential (charge at the interface of vesicle and liquid) are seldom explored although their understanding may lead to translational applications of EVs in clinical settings. In diabetes and diabetic kidney disease (DKD), post-translational modifications resulting from loss of glycocalyx (diabetes) and gain of extracellular matrix components (tissue fibrosis) could potentially affect the electrical charge of pathologically relevant EVs. DKD cohorts obtained by adhering to industry-standard reproducible criteria were stratified into stable and progressive disease based on estimated glomerular filtration rate (eGFR) loss and compared to nonkidney disease diabetic and healthy controls. Surface charge analysis revealed a significant difference in the zeta potential of large uEVs between the diabetic and healthy cohorts, as well as between the diabetic and DKD-progressive cohorts. These differences are mechanistically consistent with pathological alterations observed in heparan sulfate (HS) content. Accordingly, uEVs from DKD patients exhibited higher HS levels than those from diabetic individuals, as assessed by SP-IRIS/ExoView using HS-binding capture. Comparative proteomic analysis further demonstrated an increased abundance of the sulfated glycosaminoglycan lumican in DKD uEVs relative to diabetes, consistent with its reported upregulation in early fibrotic stages of DKD. Notably, our findings indicate for the first time that the zeta potential of large uEVs may be predictive of the progression of diabetes into DKD.

This Viewpoint synthesises 25 years of work from a single laboratory on activity-based protease sensing and offers a critical appraisal of where each of the three resulting platforms stands today. The activity of proteolytic enzymes-rather than their abundance-determines the outcome of inflammation, tumour progression, viral entry and barrier dysfunction. Measuring it in clinical samples is demanding: closely related proteases share substrate preferences, and biological matrices are crowded with endogenous inhibitors and competing hydrolases. Over the past 25 years, our group at the University of Gdansk has developed a chemical toolkit along three complementary axes, assembled here, as a probe ladder running from solution chemistry to solid-state interface. First, internally quenched chromogenic and Förster resonance energy transfer (FRET) peptide substrates, designed by iterative deconvolution of combinatorial libraries spanning non-primed and primed subsites, provide selective readouts for serine, cysteine, metallo- and viral proteases. Second, peptide-quantum dot conjugates built on non-toxic ZnO nanocrystals improved detection limits 20-30-fold over the parent solution substrate in two published demonstrators. Third, peptide-modified gold electrodes translate protease cleavage and molecular recognition into a label-free electrochemical signal-exemplified by an electrode that reads 20S proteasome activity and by an antimicrobial-peptide interface for bacterial endotoxin. We applied the resulting probes in exploratory studies of bladder cancer, type-2 diabetes and diabetic nephropathy, and positioned them within the broader landscape of activity-based protease sensing-the activity-based probes of the Bogyo and Cravatt laboratories, the PS-SCL and HyCoSuL combinatorial approaches, and the Weissleder near-infrared imaging program.

Successful tissue repair depends on the precise orchestration of phase-specific cellular responses alongside sustained activity of multifunctional immune regulators. Mast cells, which have primarily been long regarded as effector cells of allergic reactions, have emerged as pivotal modulators of wound repair. Importantly, mast cell activity exhibits a dual nature: while transient activation supports efficient repair and granulation tissue formation, sustained or excessive activation is associated with pathological fibrosis, hypertrophic scarring and keloid formation. Although many experimental studies and clinical evidences implicate a critical role for mast cells in wound repair, several key questions remain. Specifically, the contributions of distinct mast cell subtypes and the precise temporal dynamics of their mediator release, as well as the intricate molecular crosstalk between mast cells, stromal components, and other infiltrating immune cells remans a subject of active investigation. This review highlights current literature on the multifaceted roles of mast cells across the successive phases of wound-healing, and the specific mechanisms by which mast cells support or impair tissue repair, outlining unresolved questions in the field. Through this discussion, we aim to provide a framework for developing new therapeutic strategies to improve healing outcomes and facilitate better scar management.

Mitochondrial DNA (mtDNA) copy number determines the functional state of mitochondria and thus, influences cellular energy production, growth, metabolism, and stress response. Variation in mtDNA copy number has been observed across many cancer types and has been linked to changes in gene expression. However, whether mtDNA copy number has any link to mutation accumulation in cancer is unknown. Further, how mtDNA copy number variation alters expression of cancer-associated pathways and thereby influences cancer progression remains unclear. Here, through an analysis of whole genome data in multiple cancer types, we show that mtDNA copy number increases with an increase in mutational load in cancer samples and the increase in mtDNA copy number bears a signature of compensation for mitochondrial function. We also show that the samples with low-mtDNA generally have increased expression of cancer-promoting genes, including genes involved in cell migration and epithelial-mesenchymal transition (EMT). Taken together, these results reveal a close association of mtDNA copy number with mutational load and disease progression in cancer. These findings provide a way forward toward a better understanding of the role of mtDNA copy number and mitochondrial functional state in cancer progression.

Ovarian cancer (OC) is typically associated with poor prognosis and high mortality. Chimeric antigen receptor (CAR) T-cell therapy offers a promising strategy, yet its application in solid tumours remains constrained by limited efficacy and on-target/off-tumour toxicities. We previously developed K101CAR T cells targeting CA125, the cleaved extracellular domain of MUC16, and demonstrated potent antitumour activity against OC. However, because MUC16 exhibits low-level expression in certain normal tissues, safety concerns remain regarding potential off-tumour effects. We designed a combinatorial CAR composed of two single-chain variable fragments (scFvs): (i) TAG-72 targeting the Sialyl-Tn antigen highly expressed on malignant cells, and (ii) the K101 scFv. The first unit is linked to a CD3ζ signalling domain, whereas the second is linked to a 4-1BB costimulatory domain, leading to a combinatorial CAR fully reactive against double-positive targets, Tz-KBBCAR. We demonstrate that Tz-KBBCAR T cells efficiently eliminate MUC16[+]/TAG-72[+]-, while sparing MUC16[+]/TAG-72[-]- cells in vitro and markedly suppress tumour progression in vivo. We also observed that compared to K101CAR T cells, Tz-KBBCAR T cells exhibit enhanced expansion, sustained viability, and a reduced exhaustion phenotype. Overall, our study presents an efficient combinatorial CAR T-cell strategy predicted to be safer than single CAR T-cell therapy for OC.

The apolipoprotein E ε4 (APOE4) allele is the strongest common genetic risk factor for late-onset Alzheimer's disease. However, its incomplete penetrance supports the view that it acts as a modifiable systems-level perturbation of brain homeostasis rather than a deterministic driver. Increasing evidence further positions APOE4 protein within interconnected lipid metabolic pathways, with converging genetic, epidemiological, and translational studies implicating disrupted cholesterol transport, lipoprotein metabolism, and lipid oxidation as upstream mechanisms that promote neurodegeneration. Here, we synthesize currently developing therapeutic strategies targeting APOE4 across domains: small molecules, endogenous protectors, emerging biologics, and systemic lipid modulators. Small molecules include structural correctors and natural compounds, while endogenous protection arises from APOE2, the APOE3-Christchurch variant, and modifiers such as CASP7, KLOTHO, the VHL-HIF axis, fibronectin, and NHE6-mediated endosomal regulation. Biologic platforms enable allele-selective and pathway-specific interventions via siRNA, antisense oligonucleotides, and antibodies targeting pathological APOE conformers. In parallel, cholesteryl ester transfer protein (CETP) inhibition has emerged as a systems-level strategy that restores lipid and redox homeostasis, with early clinical evidence showing reductions in neurodegenerative biomarkers, particularly in APOE4 carriers. These advances support an integrative therapeutic framework to restore brain lipid homeostasis, stress resilience and neural-vascular coupling, positioning APOE4 as a modifiable node within a preventable lipid-vascular-neurodegenerative axis.

PARP proteins are enzymes catalyzing ADP-ribosylation, a well-conserved post-translational modification. In addition to the catalytic domain present in all PARPs, these proteins have a wide variety of other domains possessing different functions. Additional domains present in human PARP15 are macrodomains, capable of binding ADP-ribose. PARP15 is the least studied of the macrodomain-containing human PARPs, and it is linked to different diseases from infections to cancer. We show that the full-length canonical isoform 1 of PARP15 auto-ADP-ribosylates robustly on glutamate and aspartate residues, from which the modification can be hydrolyzed by known ADP-ribosylhydrolases. We have been able to locate several modification sites in the region of tandem macrodomains. Consistent with previous findings that the catalytic domain dimerization is necessary for enzymatic activity, we confirm this in the full-length context with the recombinant isoform 1 of PARP15. We show that dimerization is required for the efficient substrate NAD[+] binding, and we provide structural basis for this requirement with the help of a co-crystal structure of the catalytic domain dimer with an unhydrolyzable substrate analog.