Search Results (73)

Cancer immunotherapy has transformed the clinical management of several malignancies; however, its efficacy remains limited in tumors with low mutational burden and restricted availability of classical mutation-derived neoantigens. In this context, increasing evidence indicates that the tumor immunopeptidome extends far beyond canonical protein-coding regions, incorporating peptides derived from non-coding transcripts through non-canonical translation mechanisms. Long non-coding RNAs (lncRNAs), traditionally regarded as transcriptional or post-transcriptional regulators, have recently emerged as an unexpected source of small open reading frame-encoded peptides (lncPEPs). A subset of these peptides is processed and presented by major histocompatibility complex class I molecules, generating tumor-specific neoantigens capable of eliciting CD8⁺ T cell responses. Owing to the high tissue and context specificity of lncRNA expression, lncRNA-derived neoantigens offer unique advantages over mutation-based targets, including increased tumor selectivity and potential recurrence across patient subsets. In this review, we synthesize current knowledge on the biogenesis, detection, and immunogenic potential of lncRNA-derived peptides, highlighting experimental and computational strategies for their identification within the cancer immunopeptidome. We discuss the challenges associated with their validation and clinical translation, as well as their relevance for the development of vaccines and adoptive T cell–based therapies. Finally, we illustrate these concepts using epithelial ovarian cancer as a representative model of low-mutational-burden tumors, where lncRNA-derived neoantigens may help overcome current limitations of immunotherapy and enable patient stratification for personalized treatment approaches. Full article

Extracellular vesicles, which carry bioactive cargos such as proteins, RNAs, and lipids, represent promising drug delivery vehicles owing to their biocompatibility, low immunogenicity, and inherent tissue-targeting capabilities. To address the current limitations in controlled cargo loading, we developed an abscisic acid (ABA)-inducible proximity system that directs proteins into exosomes during biogenesis. We engineered exosomal scaffolds by fusing the ABA receptor PYL1 to EV-enriched proteins—including BASP1, CD9, PTGFRN, and a truncated form PTGFRNΔ687—thereby creating docking sites within the exosomal lumen, while the target cargo (e.g., EGFP, firefly luciferase, or Cas9) was tagged with the ABI1 phosphatase domain. We demonstrate that ABA administration in producer cells induces PYL1–ABI1 complex formation, which recruits ABI1-fused cargo for selective encapsulation into EVs. Among the scaffolds tested, BASP1–PYL1 proved the most effective, enabling robust, ABA-dependent enrichment of cargo proteins. Purified EVs maintained canonical morphology, size, and marker expression (CD63, syntenin-1, CD9), confirming preserved biogenesis. Critically, these loaded exosomes efficiently delivered functional cargo to recipient cells, enabling Cas9/sgRNA-mediated genome editing. Together, our findings establish an ABA-triggered molecular switch for controllable EV protein loading, providing a versatile platform for next-generation therapeutic delivery. Full article

Chemotherapeutic agents targeting ribosome biogenesis induce profound reorganization of nucleolar architecture, yet how the tumor suppressor p53 governs these structural responses remains unclear. Here, we show that loss of p53 leads to NF-κB-dependent disappearance of nucleolar caps induced by doxorubicin (DOXO). Under these conditions, fibrillarin (FBL), which is normally confined to the nucleolus, relocates to the nucleoplasm and forms foci that partially associate with G-quadruplex (G4) structures, non-canonical nucleic acid secondary structures enriched at transcriptionally active genomic regions. To examine whether this redistribution is linked to transcriptional changes, we integrated publicly available transcriptomic datasets and identified genes that were upregulated in p53-deficient cells under DOXO treatment and downregulated upon FBL depletion. Given that casein kinase 2 alpha (CK2α) is a nuclear binding partner of FBL, we further analyzed CK2α-dependent gene programs. This analysis revealed that a fraction of FBL-responsive genes overlapped with CK2α-dependent signatures and were enriched for promoter-proximal G4 structures. Among candidate regulators, the G4-binding transcription factor MAZ emerged as a potential mediator linking nucleoplasmic FBL and CK2α to G4-associated transcriptional regulation. Together, our findings identify a mechanism linking loss of p53 to G4-associated transcriptional reprogramming through nucleolar architectural disruption mediated by an FBL–CK2α–MAZ axis during DOXO treatment. Full article

Colorectal cancer (CRC) cachexia is a multifactorial, treatment-limiting syndrome characterized by progressive loss of skeletal muscle with or without loss of fat mass, accompanied by systemic inflammation, anorexia, metabolic dysregulation, and impaired treatment tolerance. Despite decades of work, cachexia remains clinically underdiagnosed and therapeutically underserved, in part because canonical models treat tumor-derived factors and host inflammatory mediators as a largely ‘host-only’ network. In parallel, CRC is strongly linked to intestinal dysbiosis, barrier disruption, and microbial translocation. Extracellular vesicles (EVs)—host small EVs, tumor-derived EVs, and bacterial extracellular vesicles (including outer membrane vesicles)—may provide a mechanistically plausible, information-dense route by which these domains could be coupled. Here, we synthesize emerging evidence suggesting that cross-kingdom EV signaling may operate as a vesicular ecosystem spanning gut lumen, mucosa, circulation, and peripheral organs. We propose the “vesicular intersection layer” as a unifying framework for how heterogeneous EV cargos converge on shared host decoding hubs (e.g., pattern-recognition receptors and stress-response pathways) to potentially contribute to muscle catabolism. We critically evaluate what is known—and what remains unproven—about EV biogenesis, trafficking, and causal mechanisms in CRC cachexia, highlight methodological constraints in microbial EV isolation and attribution, and outline minimum evidentiary standards for cross-kingdom claims. Finally, we translate the framework into actionable hypotheses for EV-informed endotyping, biomarker development (including stool EV assays), and therapeutic strategies targeting shared signaling nodes (e.g., TLR4–p38) and endocrine mediators that are predominantly soluble but may be fractionally vesicle-associated (e.g., GDF15). By reframing CRC cachexia as an emergent property of tumor–host–microbiota vesicular communication, this review provides a roadmap for mechanistic studies and clinically tractable interventions. Full article

Brown adipose tissue (BAT) and beige adipocytes play a crucial role in adaptive thermogenesis, primarily via uncoupling protein 1 (UCP1)-driven heat production. Once considered physiologically irrelevant in adults, BAT is now recognized as an active tissue that contributes to energy expenditure and metabolic homeostasis and represents a potential therapeutic target for obesity and metabolic disorders. This review provides an integrated overview of the molecular regulation of thermogenic adipocytes, emphasizing both canonical UCP1-dependent as well as non-canonical UCP1-independent mechanisms of heat generation. Key transcriptional and epigenetic regulators are discussed in the context of mitochondrial biogenesis, substrate utilization, and thermogenic gene programs. Major upstream signaling routes are further summarized, encompassing classical β-adrenergic pathways, as well as alternative regulatory nodes including AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) together with diverse nutrient- and hormone-responsive cues that converge to activate brown and beige adipocytes. Finally, the cross-talk among neuronal, endocrine, immune, and gut microbiota-derived signals is highlighted as a key determinant of thermogenic adipocyte function. Together, these multilayered regulatory inputs provide a comprehensive framework for understanding how thermogenic adipose tissue integrates environmental, metabolic, and microbial cues to regulate systemic energy balance—knowledge that is essential for developing targeted therapies to combat obesity and metabolic diseases. Full article

The liver stage of Plasmodium infection represents a critical bottleneck in malaria pathogenesis and a unique interface between parasite development and hepatocyte-intrinsic immunity. Recent evidence suggests that hepatocytes do not eliminate liver-stage parasites through canonical xenophagy, as previously assumed, but instead employ a noncanonical autophagy response known as the conjugation of ATG8 to single membranes (CASM). CASM drives rapid lipidation of LC3 onto the parasitophorous vacuole membrane (PVM) via a V-ATPase-ATG16L1-dependent mechanism, thereby activating the Plasmodium-associated autophagy-related (PAAR) response. This process represents a major hepatocyte-intrinsic mechanism that limits early liver-stage parasite development. Plasmodium liver-stage parasites have evolved specialized strategies to counteract this host defense. The PVM proteins UIS3 and UIS4 enable parasite evasion by sequestering LC3 and remodeling perivacuolar actin, thereby preventing endolysosomal fusion and inhibiting PAAR execution. In parallel, parasites selectively exploit host autophagy components—particularly GABARAP paralogs—to activate TFEB, promoting lysosomal biogenesis and improving access to host-derived nutrients. These interactions highlight autophagy as both a protective and parasite-supportive pathway, depending on the molecular context. Understanding how CASM, PAAR, and parasite evasion mechanisms intersect is crucial for designing pathway-selective interventions that amplify hepatocyte-intrinsic clearance while avoiding the inadvertent enhancement of parasite-supportive autophagy programs. Selective modulation of noncanonical autophagy offers a promising avenue for host-directed therapies that restrict liver-stage development while limiting the emergence of antimalarial resistance. This review synthesizes recent advances in the mechanistic interplay between Plasmodium liver stages and hepatocyte autophagy, identifies major knowledge gaps, and outlines future directions for translating these discoveries into therapeutic innovation. Full article

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, with a substantial proportion of events occurring prematurely. Atherosclerosis (AS), the central driver of cardiovascular pathology, results from the convergence of metabolic disturbances, vascular inflammation, and organelle dysfunction. Among intracellular organelles, mitochondria have emerged as critical regulators of vascular homeostasis. Beyond their canonical role in adenosine triphosphate (ATP) production, mitochondrial dysfunction—including impaired mitochondrial oxidative phosphorylation (OXPHOS), excessive generation of reactive oxygen species (ROS), accumulation of mitochondrial DNA (mtDNA) damage, dysregulated dynamics, and defective mitophagy—contributes to endothelial dysfunction, vascular smooth muscle cell (VSMC) phenotypic switching, macrophage polarization, and ultimately plaque initiation and destabilization. These insights have established the rationale for mitochondrial “reprogramming”—that is, the restoration of mitochondrial homeostasis through interventions enhancing biogenesis, dynamics, and quality control—as a novel therapeutic paradigm. Interventions that enhance mitochondrial biogenesis, restore mitophagy, and rebalance fission–fusion dynamics are showing promise in preclinical models of vascular injury. A growing array of translational strategies—including small-molecule activators such as resveratrol and Mitoquinone (MitoQ), gene-based therapies, and nanoparticle-mediated drug delivery systems—are under active investigation. This review synthesizes current mechanistic knowledge on mitochondrial dysfunction in ASand critically appraises therapeutic approaches aimed at vascular protection through mitochondrial reprogramming. Full article

Background: Mitoxantrone (MX) is regularly used to treat several cancers. Despite its long history in the clinic, recent studies continue to unveil novel protein targets. These targets may contribute to the cytotoxic effects of the drug, as well as potential non-canonical antitumor activity. A better understanding of MX’s cellular targets is required to fully comprehend the molecular consequences of treatment and to interpret MX sensitivity in homologous recombination (HR)-deficient cancer. Methods: Here, we evaluated MX activity in HR-deficient UWB1.289 (BRCA1−) ovarian cancer cells and surveyed the binding profile of MX using TMT-labeled quantitative proteomics and chemoproteomics. Results: Mass spectrometry (MS) analysis of cellular extracts from MX-treated BRCA1−UWB1.289 cells revealed unique downregulation of pathways instrumental in maintaining genomic stability, including single-strand annealing. Moreover, the BRCA1− cells exhibited a significant upregulation of proteins involved in ribosome biogenesis and RNA processing. Additional MS analyses following affinity-purification using a biotinylated-mitoxantrone probe corroborated these findings, which showed considerable targeting of proteins involved in genome maintenance and RNA processing. Conclusions: Our results suggest that an interplay of both canonical and non-canonical MX-antitumor activity overwhelms the BRCA1− UWB1.289 cells. Furthermore, this study characterizes the target landscape of MX, providing insights into off-target effects and MX action in HR-deficient cancer. Full article

Antimicrobial peptides (AMPs), also referred to as host defense peptides, are promising molecules in the development of the next generation of antibiotics against drug-resistant bacterial pathogens. Thanatin comprises a family of naturally occurring cationic AMPs derived from several species of insects. The first thanatin, 21 residues long, was identified from the spined soldier bug, and more thanatin peptides have been discovered in recent studies. The 16-residue thanatin from Anasa tristis, or Ana-thanatin, represents the shortest sequence in the family. However, the antimicrobial activity and mechanistic process underpinning bacterial cell killing have yet to be reported for Ana-thanatin peptide. In this work, we examined the antibacterial activity, structures, and target interactions of Ana-thanatin. Our results demonstrated that Ana-thanatin exerts potent antibiotic activity against strains of Gram-negative and Gram-positive bacteria. Biophysical studies demonstrated that Ana-thanatin interacts with LPS outer membrane and can permeabilize the OM barrier in the process. Atomic-resolution structures of the peptide in free solution and in complex with lipopolysaccharide (LPS) micelle were solved by NMR, determining canonical β-sheet structures. Notably, in complex with LPS, the β-sheet structure of the peptide was better defined in terms of the packing of amino acid residues. Further, MD simulations demonstrated rapid binding of the Ana-thanatin peptide with the LPS molecules within the lipid bilayers. These studies have revealed structural features which could be responsible for LPS-OM disruption of the Gram-negative bacteria. In addition, NMR heteronuclear single quantum coherence (HSQC) studies have demonstrated that Ana-thanatin can strongly interact with the LPS transport periplasmic protein LptA_m, potentially inhibiting OM biogenesis. Taken together, we surmise that the Ana-thanatin peptide could serve as a template for the further development of novel antibiotics. Full article

DROSHA protein is widely known for its essential role in the microRNA (miRNA/miR) biogenesis pathway where, together with its co-factor DGCR8, it forms the “Microprocessor” complex and catalyzes the primary miRNA (pri-miRNA) processing in the nucleus. Nevertheless, DROSHA also seems to participate in several miRNA-independent cellular mechanisms, such as transcriptional regulation, RNA processing and genome integrity maintenance. Hence, the present study aims to further investigate novel miRNA-independent activities of DROSHA protein, with potentially regulatory roles in the oncogenesis of human cancer cells. Our results reveal a new, strong profile of microprocessor-independent DROSHA localization at the Golgi apparatus in several human cancer cell lines of different tissue origin, with hepatic carcinoma, thyroid cancer, urothelial bladder cancer, colon carcinoma and melanoma being the cellular model systems herein examined. Notably, oncogenic activity, malignancy grade and metastatic capacity are shown to be strongly associated with DROSHA’s compartmentalization at Golgi, a phenotype that does not seem to rely on p53 protein’s functionality. Taken together, through employment of advanced confocal laser scanning microscopy (CLSM) and molecular modeling, we herein unveil the ability of DROSHA, but not AGO2 and DICER, to reside at Golgi, where DROSHA can physically interact with the GM130 Golgi-specific component, thus indicating DROSHA’s engagement in non-canonical and miRNA-independent—but also Golgi apparatus-dependent—novel mechanisms that can be tightly coupled with malignancy dynamics and beneficially utilized as potential biomarkers and therapeutic targets for human cancer. Full article

Translation is the final stage of protein synthesis and involves a broad range of proteins—from those directly participating in the process, such as initiation factors and ribosomal components, to those involved in post-translational regulation. Beyond their canonical functions, many of these proteins also influence key signaling pathways, including those regulating cellular stress responses and tumor suppression. This review explores the current knowledge of translation-associated proteins that modulate the tumor-suppressor protein p53. It highlights the roles of ribosomal proteins, stress arising from impaired ribosome biogenesis (nucleolar stress), and various translation-related factors in influencing p53 stability and activity. By integrating findings from diverse studies, this work provides insight into the intricate interplay between translation and p53 signaling, emphasizing its relevance for cellular homeostasis and stress adaptation. Full article

Magnesium (Mg) alloys, particularly Mg AZ31, have emerged as promising biomaterials for orthopedic applications due to their biodegradability and favorable mechanical characteristics. Among these, the Mg AZ31+SPF alloy, subjected to hydrothermal (HT) treatment, has demonstrated enhanced bioactivity. Our previous research established that this surface modification supports the osteogenic differentiation of human mesenchymal stem cells (hMSCs) by modulating both canonical and non-canonical signaling pathways, including those implicated in osteogenesis, hypoxic response, exosome biogenesis, and lipid metabolism. In the present study, we extended our investigation to assess the effects of Mg AZ31+SPF+HT and Mg AZ31+SPF extracts on murine pre-osteoclasts (RAW 264.7 cells) over 3- and 6-day treatment periods. The primary objectives were to evaluate biocompatibility and to investigate potential impacts on osteoclastogenesis induction and miRNA expression profiles. Methods: To assess cytocompatibility, metabolic activity, DNA integrity, and morphological alterations in RAW 264.7 cells were evaluated. Osteoclast differentiation was quantified using TRAP staining, alongside the assessment of osteoclastogenic marker expression by qRT-PCR and ELISA. The immunomodulatory properties of the extracts were examined using multiplex BioPlex assays to quantify soluble factors involved in bone healing. Additionally, global miRNA expression profiling was performed using a specialized panel targeting 82 microRNAs implicated in bone remodeling and inflammatory signaling. Results: Mg AZ31+SPF+HT extract exhibited high biocompatibility, with no observable adverse effects on cell viability. Notably, a significant reduction in the number of TRAP-positive and multinucleated cells was observed relative to the Mg AZ31+SPF group. This effect was corroborated by the downregulation of osteoclast-specific gene expression and decreased MMP9 protein levels. Cytokine profiling indicated that Mg AZ31+SPF+HT extract promoted an earlier release of key cytokines involved in maintaining the balance between bone formation and resorption, suggesting a beneficial role in bone healing. Furthermore, miRNA profiling revealed a distinct regulatory signature in Mg AZ31+SPF+HT-treated cells, with differentially expressed miRNAs associated with inflammation, osteoclast differentiation, apoptosis, bone resorption, hypoxic response, and metabolic processes compared to Mg AZ31+SPF-treated cells. Conclusions: Collectively, these findings indicate that hydrothermal treatment of Mg AZ31+SPF (resulting in Mg AZ31+SPF+HT) attenuates pre-osteoclast activation by influencing cellular morphology, gene and protein expression, as well as post-transcriptional regulation via modulation of miRNAs. The preliminary identification of miRNAs and the activation of their regulatory networks in pre-osteoclasts exposed to hydrothermally treated Mg alloy are described herein. In the context of orthopedic surgery—where balanced bone remodeling is imperative—our results emphasize the dual significance of promoting bone formation while modulating bone resorption to achieve optimal implant integration and ensure long-term bone health. Full article

This study aims to investigate the effect of the Pd(II) complex on HeLa cells using computational biology and proteomic analysis. [Pd(dach)Cl₂]-treated HeLa cells were subjected to comparative proteomics analysis using label-free data-independent liquid chromatography-tandem mass spectrometry (LC-MS/MS). In parallel, the informational spectrum method (ISM) was used to predict potential protein interactors of the [Pd(dach)Cl₂] complex in HeLa cells. Proteomics analysis revealed 121 differentially abundant proteins (DAPs). Enrichment analysis of Gene Ontology (GO) annotations revealed ATP hydrolysis and RNA/protein binding as the top molecular functions and RNA splicing and protein–RNA complex organization as the top biological processes. Enrichment analysis of altered canonical pathways pointed out spliceosome and ribosome pathways. The top hub proteins with potential regulatory importance encompassed ribosomal proteins, translational and transcriptional factors, and components of the ribosome assembly machinery. ISM and cross-spectral analysis identified the nucleoplasm and sensor of the single-stranded DNA (SOSS DNA) complex. Proteome analysis showed that [Pd(dach)Cl₂] targets proteins involved in ribosomal biogenesis and RNA splicing, whereas theoretical prediction implies also potential effect on p53 signaling pathway, and thus, alterations of the expression of regulatory proteins involved in cell survival and proliferation. These findings underscore the potential of Pd(II) complexes as anti-cancer agents, warranting further exploration and detailed functional validation. Full article

Aberrant DNA methylation is a hallmark of colorectal cancer (CRC), contributing to tumor progression through the silencing of tumor suppressor genes and activation of oncogenes. Indicaxanthin (IND), a dietary betalain pigment from Opuntia ficus indica, has shown antiproliferative effects in CRC models, yet its epigenetic impact remains unexplored. In this study, we investigated the effects of IND on the methylome of Caco-2 cells using Reduced Representation Bisulfite Sequencing (RRBS). IND induced a global hypermethylation profile, particularly at gene promoters and CpG islands. Among the differentially methylated genes, 60% were protein-coding, and 10% encoded transcription factors, including PAX5 and TFAP4, both hypermethylated at active enhancers. Functional enrichment analysis revealed pathways beyond canonical intestinal functions, suggesting altered cell identity and plasticity. Transcription factor targets (SOX10, NFKB1, AHR, ARNT) were significantly enriched among the affected genes, several of which are involved in transdifferentiation processes. Methylation changes also indicated potential reprogramming toward epithelial cell types from pulmonary or neuroectodermal origin. Moreover, IND induced selective hypomethylation of Alu elements on chromosome 21 and hypermethylation of rDNA loci, hinting at suppressed ribosomal biogenesis. Overall, these findings highlight the epigenetic remodeling potential of IND and its possible role in modulating cell fate and metabolism in CRC cells. Full article

MicroRNAs are key regulators of lymphoid differentiation, exhibiting a pivotal role in acute lymphoblastic leukemia (ALL) biology and prognosis. The initial steps of canonical miRNA biogenesis involve the microprocessor complex processing the primary miRNA transcripts into precursor miRNAs via Drosha. DROSHA polymorphisms have been implicated in pediatric ALL and linked with cancer risk. This study investigated the role of rs642321, rs3805500, and rs10035440 DROSHA polymorphisms in ALL susceptibility, relapse, and outcomes in children and adolescents of Greek descent. The study included 252 children and adolescents (115 ALL cases and 137 controls). Genotyping was performed using RT-qPCR and the TaqMan Genotyping Assay. Homozygotes for the minor allele in DROSHA rs642321 were nominally associated with ALL susceptibility (TT vs. CC+CT; OR 4.5; 95% CI: 1.2–21.2; p_adj = 0.034). Likewise, homozygotes for the minor allele in rs3805500 were linked with ALL risk (GG vs. AA+AG; OR 2.7; 95% CI: 1.3–6.1; p_adj = 0.012). A suggestive association was observed between the rs3805500 AG genotype and both relapsed (OR 5.8; 95% CI: 1.6–24.3; p_adj = 0.011) and deceased cases (OR 5; 95% CI: 1.1–26.3; p_adj = 0.038). Patients with the rs3805500 AG and GG genotypes showed a trend toward poorer overall survival rates. In summary, certain haplotypes of DROSHA polymorphisms may be modestly associated with the occurrence of childhood ALL and its outcomes, although these findings require validation in larger, independent cohorts. Full article