The adult hippocampus generates new neurons in the dentate gyrus (DG), where neural stem cells (NSCs) in the subgranular zone differentiate into neuronal and astroglial lineages. Accurate quantification of adult NSCs is critical for assessing neurogenic potential, yet most current methods rely on manual, density-based measurements in limited regions of the DG. These approaches may introduce bias due to morphological variability among tissue sections. To overcome these challenges, we developed the Dentate Gyrus Image-based Spatial quantification Tool (DGIST), an ImageJ/Fiji-based workflow that integrates BioVoxxel and MorphoLibJ plug-ins for cell-based quantification throughout the entire DG. Applied to in vivo datasets, DGIST quantifies age-dependent changes in marker-defined NSC and astroglial populations. Our results demonstrate that DGIST provides a reproducible method for quantifying marker-defined populations in the adult neurogenic niche.
Three-dimensional genome architecture (3DGA) is an active, self-reinforcing system in which chromatin biochemical state and spatial organization are reciprocally coupled. Thus, 3DGA provides a physical substrate through which past regulatory events are encoded, maintained, and re-established, as a form of cellular memory. Understanding how 3DGA arises and persists requires the integration of three complementary approaches, each illuminating aspects of 3DGA that the others cannot fully capture. Chromosome conformation capture and its derivatives, culminating in Hi-C, have generated genome-wide maps of chromatin contacts revealing compartments, topologically associating domains, and loops, but report ensemble-averaged contact frequencies that obscure the variability and dynamics realized in individual nuclei. Super-resolution microscopy and chromatin tracing directly resolve the spatial geometry of chromatin in single cells, revealing that the structural features inferred from Hi-C are probabilistic and heterogeneous rather than fixed, and capturing the transient, stochastic nature of enhancer-promoter contacts. Biochemical reconstitution together with single-molecule approaches define the causal mechanisms underlying the organizational features of 3DGA, but operate on material removed from its native cellular context. We discuss how the integration of these three approaches has shifted our view of 3DGA from a passive correlate of gene expression to a dynamic memory machine.
Solid tumors comprise cancer cells, stromal cells, and extracellular matrix components that together form the tumor microenvironment (TME), a key regulator of tumor progression. Abnormal tumor vasculature results in hypoxia and nutrient limitation, which drive malignant phenotypes through gene expression changes associated with proliferation, invasion, angiogenesis, metastasis, and chemoresistance. Stromal cells, including fibroblasts, endothelial cells, and immune cells, further influence tumor behavior through dynamic interactions with cancer cells. Lysophosphatidic acid (LPA) is a bioactive lipid that signals through G protein-coupled receptors (LPA1-LPA6) to regulate diverse cellular functions. Increasing evidence indicates that LPA receptor-mediated signaling contributes to cancer progression by modulating both cancer cells and stromal components within the TME. Importantly, the biological consequences of LPA receptor-mediated signaling are highly dependent on receptor subtype, cellular context, and microenvironmental conditions, including hypoxia, nutrient deprivation, and stromal-cell interactions. These effects vary depending on cellular context. Furthermore, LPA receptor signaling participates in bidirectional communication among cancer cells, fibroblasts, vascular and lymphatic endothelial cells, and immune cells, thereby shaping the adaptive responses of tumors to environmental stress. This review outlines the roles of LPA receptor-mediated signaling in the TME, with emphasis on hypoxia-related responses and stromal-cancer cell interactions, and discusses how receptor-subtype-specific signaling contributes to malignant progression, therapy resistance, and the potential development of precision-targeted therapeutic strategies.
Due to accumulation of many genetic changes in cancer oncogenes and tumor suppressor genes can generate an immune response by presenting antigenic peptides on the cell surface. On the other hand, in many cases immune response is muted and unable to effectively fight tumors. Recent studies have shown that T-cell responses are dependent on cytotoxic T cells, these responses can be inhibited by the interaction receptors on the surface of immune cells such as PD-1 with the molecules expressed in the surface of tumors, such as PD-L1. Thus, immune therapies were developed to block this interaction thereby triggering the cytotoxic activity of these activated T cells. In this short article we summarize the role of microRNAs, in particularly miR-155 in avoidance of immune mediated tumor rejection by malignant cells.
TP53 is frequently mutated in various cancers, including pancreatic, hepatocellular, and biliary cancers. These mutations are often associated with poor patient survival and resistance to therapy. In this review, we focus on the progress made in elucidating the effects of TP53 on these cancers. Mutations in these three cancer types were chosen to review as they are related in their origins and to components of the hepatobiliary and pancreatic systems.
NLRP3 is a cytosolic innate immune sensor that detects PAMPs and DAMPs and, together with ASC, activates caspase-1 to drive IL-1β/IL-18 release and pyroptotic cell death. A major open question is how such diverse triggers funnel into a single molecular switch within NLRP3. Emerging evidence points to lipids as central integrators: they act as direct NLRP3 ligands, post-translational modifiers, and membrane scaffolds that choreograph inflammasome priming and assembly. Palmitoylation dynamically tunes NLRP3 stability, localization, and activation thresholds, while cardiolipin and PI4P function as organelle-specific lipid cues that recruit and activate NLRP3 at mitochondria and Golgi/endosomal membranes. NLRP3 also senses shifts in cholesterol, fatty acids, and ceramides, mechanistically linking lipid imbalance to cardiometabolic and inflammatory disease. In this review, we spotlight how specific lipid-NLRP3 interactions and lipid-driven post-translational modifications orchestrate inflammasome priming and activation across cellular membranes.
Envenomation by Loxosceles spiders produces a condition termed loxoscelism, which may manifest in cutaneous and systemic forms. Cutaneous loxoscelism is characterized by intense inflammation and dermonecrosis, driven mainly by phospholipase D (PLD) toxins in Loxosceles venom. This study investigated the molecular mechanisms and cellular contributions underlying these effects. Human dermal and epidermal cells, including keratinocytes, fibroblasts, and endothelial cells, were used. Recombinant PLD from L. intermedia venom, LiRecDT1, was employed as the toxin model. Functional assays evaluated leukocyte adhesion to endothelial cells exposed to LiRecDT1 and toxin-treated media from keratinocytes and fibroblasts. Further analysis explored a systems biology approach to model cellular signaling networks activated by PLDs. Finally, the cells were exposed to LiRecDT1, and gene expression was quantified via RT-qPCR to assess cellular responses. LiRecDT1 and conditioned media from treated keratinocytes and fibroblasts promoted leukocyte adhesion to endothelial cells. Based on in silico predictions, key pathway nodes were identified and validated experimentally. The increased expression of inflammatory mediators IL-1β, IL-6 and cellular stress TNF-α genes in all cells peaked at 4 h, consistent with a pattern of acute inflammation. Cell-type-specific differences became apparent by 24 h in the distinct modulation of STAT3, IL1β, TNF-α and AKT1, as well as in the unexplored targets RELA, TP53, STAT3, and c-JUN. Prolonged exposition to the toxin demonstrated modulation of some of these pathways related to chronic/unresolved inflammation, immune modulation and fibrotic remodeling. These findings highlight the coordinated contributions of skin-resident cells to cutaneous loxoscelism and highlight novel potential targets for therapeutic intervention.
Circadian rhythms are physiological, biochemical and behavioural processes with a 24-h period molecularly regulated by clock genes. Cardiovascular physiology is subject to circadian variations in heart rate, blood pressure and contractility to optimize its function according to the rest-activity phases. Adrenergic receptors (ARs), activated by endogenous catecholamine hormones, are crucial in the regulation of cardiac functions; however, controllable in vitro models to study the intrinsic cardiomyocyte circadian clock with minimal systemic timing cues remain limited. Here, we use HL-1 cardiomyocyte cell line (HL-1 cells), in which serum shock induces synchronized oscillations of core clock gene transcripts compared with unsynchronized cultures. Different ARs activators onto HL-1 cells were applied under non-synchronized or synchronized conditions and circadian oscillation of representative clock genes was determined. We show that alpha- and beta-AR activation differentially modulates clock gene mesor, amplitude and phase. These findings support HL-1 cells as a convenient in vitro platform to investigate interactions between adrenergic signaling and cardiomyocyte clock gene oscillations.
Olfactory receptors (ORs) are seven transmembrane domains G protein-coupled receptors (GPCRs) located in the olfactory sensory neurons (OSNs) of the nasal olfactory epithelium. Although OR expression was initially hypothesized to be restricted to the OSNs, an ecnomotopic expression has been identified and associated with the modulation of different physiological functions, such as glucose and lipid metabolism, hypoxia sensing, wound healing and sperm chemiotaxis. However, the role of most ORs in non-olfactory tissues is still a matter of debate, as well as their specific ligands and mechanisms of action. High-density lipoproteins (HDL) are heterogenous, and multifunctional nanoparticles constituted primarily of proteins and lipids. Their main structural protein, namely, apolipoprotein A-I (A-I) has been recognized as the major determinant of the biological activities of HDL. Recently, our group, by using unique mouse models and microarray methodology, has demonstrated that human A-II (hA-II) and A-IMilano (A-IM), a molecular variant of A-I, strongly modulate the hepatic expression of different genes involved in lipid metabolism and immune/inflammatory pathways. Therefore, aiming at investigating the impact of these apolipoproteins on the hepatic expression of mouse ORs (Olfrs), we have performed a new bioinformatic analysis of the differentially expressed genes (DEGs) found in the liver of hA-II/A-I k-in versus hA-II/A-IM k-in; A-IM k-in versus hA-II/A-IM k-in; A-I k-in versus A-IM k-in; A-I k-in versus hA-II/A-I k-in mice. Our results suggest that the presence of A-IM, either alone or in combination with hA-II, is critical for the efficient trafficking and functional expression of Olfrs at the cell surface. Moreover, co-expression of hA-II with A-I resulted in down-regulation of previously uncharacterized Olfrs genes an up-regulation of several known Olfrs, which are likely responsive to short-chain fatty acids and signal through the cAMP/CREB pathway.
Afatinib is an irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) used to treat EGFR-mutant non-small cell lung cancer. It frequently causes gastrointestinal toxicity that perturbs intestinal homeostasis, and its impact on Paneth-like lineage differentiation along the crypt-villus axis remains unclear. Using a controlled differentiation-stage framework in Caco-2 cells, we examined how afatinib affects Paneth-like differentiation markers. We incubated undifferentiated Caco-2 cells with afatinib (10-5000 nM) for 24 h during early culture and evaluated downstream DNA methylation and differentiation-associated readouts up to day 14. We quantified proteins (western blotting), mRNAs (RT-qPCR), and promoter methylation (methylation-sensitive restriction enzyme-qPCR). Afatinib increased DNA methylation at the SRY-box transcription factor 9 (SOX9) and defensin alpha 5 (DEFA5) promoters and reduced their protein expression. Immunostaining indicated reduced expression of Paneth-like differentiation markers in Caco-2 cells. In contrast, mRNA levels of the SOX9 regulators odd-skipped related transcription factor 1 (OSR1) and receptor-interacting protein 140 (RIP140) were upregulated without changes in promoter methylation at the analyzed sites, indicating DNA methylation-independent regulation at these promoters. These findings suggest that DNMT1/3B-skewed methylation at the SOX9/DEFA5 promoters may be counteracted by ten-eleven translocation-mediated counter-demethylation. Collectively, our data indicate that afatinib modulates Paneth-like differentiation markers via DNA methylation-dependent repression of SOX9/DEFA5 and DNA methylation-independent induction of OSR1/RIP140 in Caco-2 cells, which may be relevant to crypt-associated epithelial function and gastrointestinal safety.
Pulmonary arterial hypertension (PAH) is a progressive and fatal vascular disorder for which reliable and non-invasive diagnostic biomarkers remain limited. This study aims to screen and verify the hub genes related to PAH by integrating bioinformatics and experimental verification. We analyzed four GEO datasets (GSE117261, GSE24988, GSE53408, GSE113439) to identify differentially expressed genes and co-expressed modules. Functional enrichment analysis revealed the pathways related to vascular smooth muscle contraction and organ development. Protein-protein interaction network analysis screened out 4 hub genes. These genes showed different expression dysfunctions in the training set, an independent validation set and a Single-cell sequencing dataset (GSE33463, GSE228644), and demonstrated good diagnostic value through ROC curve analysis. Crucially, in the nicotine-induced PAH mouse model, RT-qPCR experiments confirmed the significant upregulation of these 4 genes. Our research results have established CDK1, TPX2, IGF1 and VCAM1 as robust polygenic markers related to PAH, providing potential evidence for clarifying their molecular mechanisms and developing non-invasive diagnostic tools.
Mechanistic target of rapamycin (mTOR: aka mammalian target of rapamycin), a serine threonine kinase, functions by forming two multiprotein complexes designated mTORC1 and mTORC2. This signaling cascade of PI3K/AKT/mTOR is often upregulated due to frequent loss of the tumor suppressor PTEN, a phosphatase that functions antagonistically to PI3K. mTORC1 is sensitive to nutrients and mTORC2 is regulated via PI3K and growth factor signaling. Aberrant signaling of mTOR is shown to be associated with tumorigenesis of numerous malignancies including glioblastoma (GBM). mTORC1 and mTORC2 activate downstream substrates that execute cellular and metabolic functions. Experimental models have provided evidence of the existence of cancer stem cells (CSCs), also known as tumor-initiating cells within the tumor mass, that may play an active role in development, progression and reformation of GBM. In addition, presence of highly infiltrative CSCs in the peritumoral region of GBM may appear to play an important role in recurrence of disease. Since rapamycin and its analogues are less effective in treatment of GBM, the use of ATP-competitive dual inhibitors of mTORC1 and mTORC2 have been increasingly investigated. These attempt to suppress GBM growth by pharmacodynamically inhibiting phosphorylation of the mTORC1 substrates S6K Ser235/236 and 4E-BP1 Thr37/46. These inhibitors also cause down-regulation of mTORC2 substrate AKT Ser473. These reactions result in reduction of cell growth and migration. Notably, these inhibitors of mTOR also alter self-renewal and growth of CSC of GBM. The aim of this review is to reiterate the use of mTOR inhibitors in the treatment of GBM and its stem cells associated with progression and recurrence of the disease. In addition, understanding the peritumor area of GBM is a crucial means to control the recurrence of the disease.
The presence of inositol lipids in the nucleus has been shown in the late 1980s and since then a considerable amount of interest has been raised about the role of these molecules in an autonomous nuclear signalling system different from that at both the plasma membrane and the cytoplasm. Here we review the main issues of nuclear structure and of nuclear inositol lipids and their related enzymes in cellular signaling, taking into account also the possible role in some human pathologies.
The immune system is central to the prevention and control of cancer, yet tumors evolve multiple strategies to subvert immune surveillance. Checkpoint inhibitors targeting CTLA-4, PD-1, and PD-L1 have revolutionized oncology by demonstrating that therapeutic restoration of T cell activity can yield durable remissions. However, their efficacy remains limited by the profoundly immunosuppressive tumor microenvironment (TME), where regulatory immune cells, suppressive cytokines, and metabolic stressors converge to dampen effector function. As interest in integrative and complementary approaches grows, plant-derived compounds - long used in traditional medicine - have been identified as bioactive agents capable of modulating immune function. This review focuses on three key phytochemicals: piperlongumine, berberine, and epigallocatechin gallate (EGCG). Piperlongumine, a pro-oxidative alkaloid from Piper longum, suppresses T cell activation and promotes regulatory T cell differentiation, suggesting potential for chronic inflammation but raising caution in oncology. Berberine, an isoquinoline alkaloid from Berberis vulgaris, reduces PD-L1 expression via CSN5 inhibition, thereby mimicking checkpoint blockade and enhancing cytotoxic T cell activity in preclinical models. EGCG, the major polyphenol in green tea, downregulates PD-L1 expression and augments anti-tumor immunity in murine melanoma. We critically assess the promise and pitfalls of these compounds in cancer immunotherapy, emphasizing mechanistic insights, pharmacokinetics, translational hurdles, and potential risks of interfering with established therapies. A precision immunology framework - integrating immune monitoring, patient stratification, and controlled clinical trials - will be essential to determine whether phytochemicals can be safely and effectively incorporated into oncology. Far from being benign, plant-derived agents exert potent immune effects that could either complement or compromise modern immunotherapy, underscoring the need for rigorous evaluation.
The unfolded protein response (UPR) is a central regulator of proteostasis, coordinating cellular adaptation to endoplasmic reticulum (ER) stress. It is comprised of three signaling branches: ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1), and PERK (protein kinase RNA-like ER kinase), which mediate transcriptional and translational reprogramming of the proteostasis network. These pathways display both functional redundancy and branch-specific activities. Dysregulated UPR signaling contributes to diverse pathologies: in cancer, UPR activation supports uncontrolled proliferation and treatment resistance, whereas in aging, proteostasis decline and diminished UPR responsiveness are hallmarks. Traditional approaches, including transcriptomics and western blotting, have been widely used to monitor UPR activity, but they offer limited insight into its regulation at the protein level. In contrast, liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics allows comprehensive, branch-specific profiling of UPR signaling. Recent advances, including data-independent acquisition (DIA) MS and automated sample preparation, have further improved sensitivity, reproducibility, and detection of low-abundance UPR target proteins. Proteomics thus provides a systematic and scalable framework to interrogate UPR regulation across cell types and disease models. When integrated with complementary datasets, protein-level measurements can uncover context-dependent molecular signatures of UPR activity, offering insights into disease mechanisms and guiding the rational design of targeted pharmacological interventions. Future work integrating high-resolution LC-MS/MS proteomics with tissue and single-cell analyses will further clarify the role of the UPR in health and disease.
Nuclear receptors are lipid-regulated transcription factors that respond to the changing metabolic and signaling requirements of animal cells and tissues. Steroidogenic Factor 1 (SF-1, NR5A1) is a nuclear receptor and master regulator of steroidogenic gene expression. SF-1 is required for development and adult function of steroidogenic tissues, hyperactivation of SF-1 associates with adrenocortical carcinoma, while hypomorphic loss-of-function polymorphisms associate with disorders of sexual development. Many of these physiological functions of SF-1 are broadly understood, however the identity of the endogenous regulatory lipid ligands for SF-1 have yet to be well established, preventing progress on therapeutic development for human diseases, such as adrenocortical carcinoma. Several signaling lipids have been put forth as potential regulatory ligands of SF-1, including sphingosine, lyso-sphingomyelin, sphingomyelin, ceramide and several phosphoinositide species including PI(4,5)P2 and PI(3,4,5)P3. Here, we review the evidence linking the ability of these potential phospholipid ligands to regulate SF-1 mediated gene expression in metazoan cells, and discuss how lipid ligands regulate SF-1 from a structural perspective.
Diacylglycerol kinases (DGKs) are key enzymes that integrate lipid metabolism with multiple signaling pathways. DGKs regulate the conversion of diacylglycerol (DAG) into phosphatidic acid (PA), two essential bioactive lipids that promote the activation of distinctive proteins controlling cell growth, proliferation and differentiation. The variety of DGK isoforms enables them to perform specialized functions in different tissues, and dysregulation of DGK activity and expression contributes to diverse pathological conditions. DGKs exert potent inhibitory functions in T cells and are aberrantly expressed in a wide range of cancer types, which make DGKs attractive therapeutic targets for cancer immunotherapy. In recent years, the development of novel and highly isoform-specific inhibitors has opened exciting opportunities to further explore the fundamental functions of lipid metabolism in the maintenance of immune cell homeostasis and in the progression of several diseases. Besides T cells, DGKs play important roles in regulating inflammatory processes across distinct immune populations. The therapeutic potential of these drugs has been translated in several ongoing clinical trials. Therefore, it is crucial to delineate DGK-controlled signaling hubs to better understand their impact on immune signatures. In this work, we aimed to recapitulate the effects of modulating DAG/PA balance on immune cells that are relevant in the tumor microenvironment. By dissecting how DGK-mediated lipid signaling shapes immune cell behavior in the tumor microenvironment, we seek to provide mechanistic insights that may guide the rational use of drugs targeting DGKs to improve antitumor immunity.
Class IA phosophoinositide kinases (PI3Ks) are master regulators of growth, metabolism, and immunity. The class IA PI3Ks are a heterodimer composed of a p110 catalytic subunit and one of five possible regulatory subunits (p85α, p85β, p55γ, p55α, p50α). The regulatory subunit plays critical roles in stability, inhibition, and activation of the p110 catalytic subunit. The p110α catalytic subunit frequently contains activating mutations in human cancer, with many of these mutations altering the interaction between catalytic and regulatory subunits. It has been found that different regulatory subunits play unique roles in human disease, but it is unknown how these different subunits regulate p110α. Here, using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS) we examined how the five different regulatory subunits inhibit, activate, and interact with the p110α catalytic subunit. We find that there are no significant differences in lipid kinase activity or in membrane recruitment between the different heterodimer complexes. HDX-MS in the presence and absence of an activating phosphopeptide also showed only minor conformational differences between different regulatory subunit complexes. Overall, our work reveals that the different regulatory subunits interact with and inhibit p110α in a similar fashion at a molecular level.
Tepsin is an accessory protein in Adaptor Protein 4 (AP-4) coated vesicles responsible for trafficking cargo from the trans-Golgi network (TGN). AP-4 vesicles recognize and sort multiple cargoes including ATG9A, a lipid scramblase essential for autophagosome maturation. In cultured cells, tepsin loss alters ATG9A distribution and autophagosome morphology, and tepsin has been shown to contain a canonical LC3-interacting region (LIR) motif required for proper ATG9A distribution. Computational modeling in AlphaFold Multimer combined with biochemical and biophysical experiments identified three additional LC3B binding motifs within tepsin disordered regions. Structural models paired with bio-layer interferometry (BLI) uncovered and confirmed specific residues involved in each interaction and indicated all four motifs independently engage the LC3B LIR docking site (LDS). Thermodynamic and kinetic properties associated with each motif found in full-length tepsin were quantified. BLI and biochemical data reveal all four motifs in tepsin must be mutated to abrogate binding to LC3B in vitro, while stoichiometry data estimate one tepsin likely binds two LC3B at one time on a surface or membrane. Together, data suggest tepsin could respond dynamically to LC3B concentrations on membranes by leveraging multivalency to modulate binding strength.
Esophageal squamous cell carcinoma (eSCC) is an aggressive malignancy with poor prognosis and limited therapeutic options. The phosphoinositide 3-kinase (PI3K)/AKT pathway is frequently activated in eSCC, but clinical use of PI3K or AKT inhibitors is restricted by toxicity and compensatory signaling. SHIP2, an inositol 5-phosphatase encoded by INPPL1, modulates this pathway by converting PI(3,4,5)P3 to PI(3,4)P2, thereby regulating AKT activation. We previously identified INPPL1 amplification as recurrent in eSCC and demonstrated that SHIP2 inhibition suppresses tumor growth and synergizes with PLK1 inhibition. Here, we extend these findings and show that SHIP2-PLK1 synergy is not confined to eSCC but is also observed in multiple colorectal cancer cell lines, revealing a conserved vulnerability across tumor types. Mechanistic analyses demonstrate that this synergy depends on PI3K/AKT signaling, with SHIP2 inhibition producing stronger effects than direct PI3K blockade, suggesting additional regulatory functions beyond canonical PI3K control. Furthermore, SHIP2 inhibition enhances the cytotoxic activity of standard chemotherapies, including 5-fluorouracil and paclitaxel, in eSCC cells. Importantly, these effects occur at sub-cytotoxic drug concentrations, indicating potential therapeutic benefit with reduced toxicity. Collectively, our results identify SHIP2 as a central regulator of the PI3K/AKT axis in eSCC and colorectal cancer and highlight its value as a combinatorial target. SHIP2 inhibition represents a promising strategy to potentiate existing chemotherapies and targeted agents, opening new avenues for the treatment of refractory gastrointestinal cancers.
