Abstracts of the 3rd International Online Conference on Cells: Charming Micro-Insights into Health and Diseases (Cells 2025), 25–27 March 2025

Abstract

The 3rd International Online Conference on Cells: Charming Micro-Insights into Health and Diseases (Cells 2025) took place online from 25 to 27 March 2025. This conference report is an abstract collection from sessions of Cells 2025.

1. Introduction

The 3rd International Online Conference on Cells (Cells 2025), 25–27 March 2025 provided a highly interactive platform for researchers all over the world to discuss major achievements as well as challenges in cell biology, aimed to cover a wide range of topics and to receive feedback from seasoned researchers and from young investigators. The goal was to facilitate interaction between participants and to further advance the highly collaborative spirit of cell biology science. The conference attracted experts and scholars from all fields of cell science to discuss a wide range of topics: cellular pathology of cancers, neural cell biology, cellular antivirus immune responses, cell therapies, cellular signaling, cell research in animal models, cellular metabolism, protein quality control, proteasomal degradation and autophagy. The conference mainly focused on complex mechanisms underlying cellular physiology and suggested novel treatment strategies for a wide spectrum of life-threatening pathologies.

The main topics of this conference included:

• Cellular Pathology of Cancers;
• Neural Cell Biology;
• Cellular Antivirus Immune Responses;
• Cell Therapies;
• Cellular Signaling;
• Cell Research in Animal Models;
• Cellular Metabolism;
• Protein Quality Control, Proteasomal Degradation and Autophagy.

2. Cellular Pathology of Cancers

2.1. Bergapten Affects Apoptosis Through Notch Family in Colorectal Cancer Cells

Giuseppina Daniela Naimo, Giulia Venneri, Martina Forestiero, Adele Elisabetta Leonetti, Loredana Mauro, Maria Luisa Panno, Francesca Giordano

• Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy

Introduction: Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. Its incidence is influenced by genetics, age, lifestyle, and diet factors. Indeed, a balanced diet rich in fiber and regular physical activity can significantly reduce the risk of developing CRC. Functional Foods have aroused considerable interest due to their antiproliferative and anti-cancer effects. Interestingly, bergapten, the main furanocoumarin in bergamot, exhibiting antioxidant properties, has been particularly studied for its ability to inhibit proliferation and induce apoptosis in different tumour cell lines, including CRC. Paradoxically, several reports have highlighted the pro-oxidant effects of natural antioxidant compounds, which increase free radicals and exacerbate oxidative stress. Based on this evidence, we investigated the molecular mechanism by which bergapten may affect survival and ROS production in CRC cells.

Methods: CaCo-2 colorectal adenocarcinoma cells were used as a model to evaluate bergapten anti-cancer effects. ROS production and mitochondrial membrane potential were evaluated through flow cytometry. mRNA levels of stemness, the epithelial-to-mesenchymal transition (EMT), the cell cycle and apoptosis markers were quantified by qRT-PCR. Western blotting was carried out to detect protein levels.

Results: Low doses of bergapten induced a reduction in intracellular ROS levels in CaCo-2 cells, as is generally reported. Unexpectedly, high doses of the furanocumarin promoted a time-dependent significant increase in ROS levels. In CaCo-2 bergapten-treated cells, a reduction in mitochondrial membrane potential, a down-regulation of anti-apoptotic markers, and an increase in pro-apoptotic markers were observed, leading to cytochome C release. In agreement with this, bergapten induced a lowered expression of Cyclin D1, Notch family members, and Survivin compared to untreated cells. Interestingly, the bergapten-induced inhibition of Notch signalling also led to the down-regulation of the EMT, reducing CRC growth and progression.

Conclusions: These results suggest that bergapten, at high doses, may act as a promoter of oxidative stress, favoring antiproliferative effects through the activation of intrinsic apoptosis.

2.2. Clinicopathological Significance of DNA Damage Signalling and Repair (DDR) Regulating E3 Ubiquitin Ligases and De-Ubiquitinases in Ovarian Cancer

Amera Sheha ^{1, 2, 3}, Emad Rakha ^{1, 2}, Srinivasan Madhusudan ^{1, 4}

¹ 

Naaz-Coker Ovarian Cancer Research Centre, Biodiscovery Institute, School of Medicine, University of Nottingham, Nottingham, NG7 2UH, UK

² 

Department of Pathology, Nottingham University Hospitals, Nottingham, NG5 1PB, UK

³ 

Histopathology Department, South Egypt Cancer Institute, Assiut University, Assiut, 71515, Egypt

⁴ 

Department of Oncology, Nottingham University Hospitals, Nottingham, NG5 1PB, UK

Introduction: E3 ubiquitin ligases (EUL) and de-ubiquitinases (DUB) that regulate DDR proteins may influence ovarian cancer pathogenesis. We evaluated DDR-regulating EULs and DUBs in ovarian cancers.

Methods: EULs (DDB2, CUL4A, HLTF, RAD18 and HUWE1) and DUBs (USP5, USP7, USP11 and PSMD14) were immunohistochemically profiled in 331 tumours, and the clinicopathological outcome were analysed. Transcriptomic analysis was completed on a publicly available data set (n = 424).

Results: DDB2, USP7 and USP11 expression was nuclear. CUL4A, HLTF, RAD18, HUWE1, USP5 and PSMD14 showed both nuclear and cytoplasmic expression. EULs [low nuclear DDB2 and CUL4A cytoplasmic overexpression] and DUBs [USP5 (nuclear and cytoplasmic) overexpression] were associated with high-grade serous carcinoma (p = 0.02, 0.003, 0.001, 0.03, respectively). EULs [low DDB2 nuclear expression (p = 0.01) and CUL4A nuclear overexpression (p = 0.05)] and DUBs [USP5 cytoplasmic overexpression (p = 0.05) and PSMD14 cytoplasmic overexpression (p = 0.02)] were associated with advanced-stage tumours. In terms of survival outcomes, EULs [nuclear and cytoplasmic CUL4A (p = 0.05, 0.001, respectively), nuclear HLTF (p = 0.003) and low-nuclear DDB2 (p = 0.01)] and DUBs [overexpression of USP7 (p = 0.005), nuclear and cytoplasmic USP5 overexpression (p = 0.004, 0.04, respectively), cytoplasmic PSMD14 overexpression (p = 0.01), nuclear USP11 overexpression (p = 0.006)] were associated with poor progression-free survival (PFS). In a multivariate analysis, advanced stage, cytoplasmic CUL4A overexpression and nuclear USP5 overexpression remain independently associated with worse PFS (p = 0.02, 0.03, respectively). At the transcriptomic level, in p53 mutant advanced-stage tumours that received platinum-based chemotherapy (n = 424), high USP5 remains associated with poor PFS.

Conclusion: Our data suggest a complex role of EULs and DUBs in ovarian cancers. USP5 may be an attractive target for patient stratification and therapeutics in ovarian cancer.

2.3. Modulation of Cathepsin D Expression Drives the Growth of Neuroblastoma Cells in 2D or 3D Cultures in Response to EGF: Analysis in a Bona Fide Model Mimicking Tumor Heterogeneity and Metastatic Progression

Chiara Lualdi, Eleonora Secomandi, Andrea Esposito, Giulia Camurani, Chiara Vidoni, Amreen Salwa, Letizia Vallino, Alessandra Ferraresi, Ciro Isidoro

• Laboratory of Molecular Pathology, Department of Health Sciences, Università del Piemonte Orientale “A. Avogadro”, Via Solaroli 17, 28100 Novara, Italy

Neuroblastoma (NB) is a pediatric malignancy originating from the neural crest cells of the sympathetic nervous system, and it often progresses aggressively due to the influence of epidermal growth factor (EGF). Our earlier research demonstrated that cathepsin D (CD) disrupts EGF-driven proliferation in NB cells cultured in 2D by inhibiting the EGFR/MAPK signaling pathway. While this highlights CD’s regulatory function in tumor growth, its potential role in metastasis remains unexplored. In order to reproduce tumor heterogeneity, we engineered neuroblastoma (NB) clones with either silenced or overexpressed CD expression and co-cultured the clones in different proportions. We then analyzed the growth of the mixed populations in 2D (adherent) and 3D (suspension) culture conditions to mimic the stages of the metastatic process in response to EGF stimulation. We unexpectedly observed an opposite behavior in 2D and 3D cells with different levels of CD. In particular, cells overexpressing CD demonstrated a greater adaptability to growth in suspension cultures. In contrast, cells with suppressed CD expression exhibited enhanced growth under adherent conditions in two-dimensional environments. Different CD levels lead to different behaviors even during the transition from 3D to 2D: cells that overexpress CD demonstrated an increase in N-cadherin levels, while cells with silenced CD showed a greater propensity to revert to a mesenchymal-to-epithelial-like phenotype, as evidenced by the elevated expression of E-cadherin. The contrasting roles of CD in driving cancer cell growth under 2D and 3D conditions indicate that clonal evolution may favor the emergence of subpopulations with distinct CD levels, optimizing their adaptability to various metastatic niches. This highlights the potential epigenetic regulation of CD as a mechanism supporting survival and proliferation in both substrate-adherent and neurosphere-forming NB cells. Consequently, disrupting the epigenetic control of CD could represent a promising therapeutic approach to hinder NB progression and metastasis.

2.4. Photoactivated Aluminium Phthalocyanine Drives Oxidative Stress and Apoptosis in Human Oesophageal Cancer Stem Cells

Onyisi Christiana Didamson, Rahul Chandran, Heidi Abrahamse

• Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Johannesburg, 2028, South Africa

Introduction: Oesophageal cancer is the eleventh most diagnosed malignancy and the seventh most common cause of cancer-associated mortality globally. Studies have shown that cancer stem cells (CSCs) are the main drivers for resistance in oesophageal cancer. Photodynamic therapy (PDT), a light-based treatment strategy, has demonstrated efficiency against several cancers. However, the therapeutic effect of aluminium phthalocyanine chloride tetra sulfonate (AlPcS₄Cl)-mediated PDT in inducing oxidative stress and potentiating apoptotic activity in oesophageal CSCs is limited. This study examined the effects of AlPcS₄Cl in promoting oxidative stress and apoptotic cell death in HKESC-1 human oesophageal CSCs.

Method: The CSCs were isolated from HKESC-1 cells and grouped into control and treatment groups. The CSC treatment group was exposed to AlPcS₄Cl-PDT at 5 J/cm². Twenty-four hours after PDT, the anticarcinogenic actions of AlPcS₄Cl on oesophageal CSCs in inhibiting cell growth and promoting oxidative stress and cell death were evaluated. Cell viability was assessed using a viability assay; a cellular ROS assay was used to determine the induction of oxidative stress, rhodamine-123 flow cytometry analysis was used for mitochondria membrane potential, Annexin V-FITC/PI double-staining flow cytometry analysis for cell death mechanism, and Caspase-Glo 3/7 fluorometric assay for caspase activities. All treatment and control cells were conducted in biological and technical replicates. GraphPad Prism (v5) was used to collate the results and perform statistical analysis. The treatment groups were compared relative to the control cells. One-way ANOVA was used, and a p-value of 0.05 indicated statistical significance.

Results: Findings from our study showed that AlPcS₄Cl-PDT with 5 J/cm² irradiation significantly inhibited cell growth, induced oxidative stress via increased intracellular ROS production, altered the integrity of mitochondria membrane potential, and induced caspase 3/7-mediated apoptosis in oesophageal CSCs.

Conclusion: This study demonstrates the promising use of AlPcS₄Cl-PDT in eradicating oesophageal CSCs and improving prognosis.

2.5. Targeting Glycolysis-Dependent Inhibition of Autophagy Reduces Ovarian Cancer Cell Migration and Impairs CAF Phenoconversion

Alessandra Ferraresi ¹, Letizia Vallino ¹, Chinmay Maheshwari ¹, Carlo Girone ¹, Amreen Salwa ¹, Beatrice Garavaglia ¹, Danny N. Dhanasekaran ², Ciro Isidoro ¹

¹ 

Laboratory of Molecular Pathology, Department of Health Sciences, Università del Piemonte Orientale, via Solaroli 17, 28100 Novara, Italy

² 

Stephenson Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

Introduction: Cancer cells exploit aerobic glycolysis, also known as the Warburg effect, which consists of the fermentation of pyruvate into lactate, even in the presence of oxygen and functioning mitochondria. This metabolic rewiring fuels the uncontrolled growth of the tumor mass and promotes cancer progression. Glycolytic reprogramming occurs not exclusively in cancer cells but also in the stroma, especially in cancer-associated fibroblasts (CAFs), a process that has been named the “reverse Warburg effect”. Autophagy, an evolutionary conserved catabolic process devoted to macromolecular turnover, is often dysregulated in cancer and plays a “double-edged sword” role in regulating the tumor microenvironment.

Methods: To mimic the tumor microenvironment in vitro, we employed IL-6, a pro-inflammatory cytokine abundant in ovarian cancer patients, and we set up an indirect co-culture method by cultivating fibroblasts with the conditioning medium of ovarian cancer cells.

Results: Our work aimed to dissect how the glycolysis/autophagy interplay affects ovarian cancer cell motility and the phenoconversion of CAFs. Our data show that IL-6 induces cancer cell migration only in cases of active glycolysis. On the other hand, nutraceutical resveratrol (RV) inhibits glucose uptake and metabolism while promoting autophagy, collectively hampering cancer cell motility. Moreover, our bioinformatic interrogation of TCGA data shows that patients with a low expression of glycolytic markers and displaying active autophagy exhibit favorable clinical outcomes. Next, we show that the glycolysis-dependent inhibition of autophagy promotes CAF activation. The conditioning medium of the ovarian cancer cells induces the glycolytic reprogramming that is required for CAF activation. Notably, glycolysis inhibition using 2-deoxy-D-glucose (2DG) or RV induces autophagy, which reprograms the CAFs into quiescent fibroblasts.

Conclusions: Taken together, our data support the use of glycolysis inhibitors and/or autophagy inducers as an adjuvant strategy to improve the therapy success rate and limit the risk of metastasis.

3. Neural Cell Biology

3.1. Differential miRNA Expression in the Hippocampus Following Aerobic Exercise: Implications for Adult Neurogenesis

Julia Si ¹, Anais Herrera ¹, Connor Wilusz ², Michal Toborek ¹, Minseon Park ¹

¹ 

Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA

² 

College of Arts and Sciences, University of Miami, Coral Gables, FL 33146, USA

Regular exercise reduces the risk of cardiovascular diseases, stroke, hypertension, and diabetes and enhances bone, muscle, and mental health. Recent evidence strongly suggests that aerobic exercise promotes adult neurogenesis in the hippocampus, improving synaptic plasticity. This study aimed to identify target miRNA candidates regulated by aerobic exercise that may contribute to enhanced hippocampal neurogenesis.

Male and female C57BL/6 mice (13 weeks old) were divided into two groups: a control group housed in locked-wheel cages and an exercise group housed in wheel-equipped cages for two weeks. The mice in the exercise group had free access to the running wheel in each cage, while the control mice were unable to use the wheel, as it was secured with a cable tie. Each group consisted of four mice. Total RNA was extracted from the dissected mouse hippocampi and used for RNA sequencing. Differentially expressed miRNAs were then identified based on gender and/or the exercise conditions.

A total of 698 miRNAs were identified as differentially expressed across the groups. A two-way ANOVA revealed that 67 miRNAs were differentially expressed by sex and 87 miRNAs by exercise (p < 0.05). Additionally, 55 miRNAs were influenced by both factors. The expression levels of miR-7b-3p, miR-212-3p, and miR-132-3p were significantly increased by exercise in both the male and female groups, while miR-12200-5p expression was reduced by exercise (p = 0.0135) but was lower in the male group overall.

This study shows that aerobic exercise alters the expression of specific miRNAs in the hippocampus, with some differences between males and females. miR-7b-3p, miR-212-3p, and miR-132-3p were significantly increased by exercise in both sexes, while miR-122000-5p was reduced. These miRNAs may play important roles in exercise-induced neurogenesis and brain health. Future research will focus on examining the effects of these target miRNAs on hippocampal neurogenesis.

3.2. p75NTR Modulation Reverses Oxidative Stress and Metabolic Derangements in a Cell Model of Parkinson’s Disease

Daniele Pensabene ^{1, 2}, Noemi Martella ¹, Giuseppe Scavo ¹, Michela Varone ¹, Emanuele Bisesto ¹, Francesca Cavicchia ¹, Mayra Colardo ¹, Sandra Moreno ², Marco Segatto ¹

¹ 

Department of Biosciences and Territory, University of Molise, Contrada Fonte Lappone, 86090 Pesche (IS), Italy

² 

Department of Science, University Roma Tre, Viale Marconi 446, 00146 Rome, Italy

Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease (AD), mainly affecting the elderly population with an incidence of 1–3%. It is characterized by motor symptoms closely linked to dopaminergic cell death in the Substantia nigra pars compacta. From a molecular standpoint, the deposition of Lewy bodies, namely protein aggregates particularly enriched with misfolded α-synuclein, is a typical hallmark of the disease. In addition, recent findings have highlighted cholesterol dysmetabolism as another common peculiarity of PD profoundly contributing to changes in membrane composition and integrity. Notably, p75 neurotrophin receptor (p75NTR) was found to be upregulated in the brains of post-mortem PD patients and associated with a reduction in the expression of pivotal neuroprotective effectors, strongly suggesting a role for this receptor in PD. Given its ability to favor both pro-survival and pro-apoptotic cascades, the synthesis of small p75NTR-binding molecules (i.e., LM11A-31) promoting cell viability over death was of particular interest in research. Hence, the aim of this study was to evaluate whether the pharmacological modulation of p75NTR by LM11A-31 provides neuroprotection in a rotenone-induced cellular model of PD. Our data showed that LM11A-31 promoted cell viability and reduced rotenone-dependent neuromorphological aberrations, as well as α-synuclein downregulation. We also observed that p75NTR modulation counteracted rotenone-dependent cholesterol alterations: specifically, LM11A-31 normalized free cholesterol content, as well as the expression of proteins involved in cholesterol uptake and trafficking. Furthermore, LM11A-31 reduced oxidative stress damage upon macromolecules by both boosting the expression of transcriptional regulators of the antioxidant response and reducing the expression of NADPH-oxidase (NOX) modulatory subunits. Even though further studies are required to better dissect the molecular mechanisms linking p75NTR to PD physiopathology, our data point to p75NTR modulation as a promising therapeutic avenue in PD treatment.

3.3. Preliminary Investigation of Cerebellar Alterations Induced by Cachexia and Endurance Training in C26 Tumor-Bearing Mouse Model

Francesco Paolo Zummo, Rosario Barone, Filippo Macaluso, Daniela D’Amico, Francesco Carini, Radha Santonocito, Margherita Geraci, Vahid Saqagandomabadi, Valentina Di Felice, Sabrina David

• Biomedicine, Neuroscience and Advanced Diagnostics, University of Palermo, 90127 Palermo, PA, Italy

Cachexia is a syndrome characterized by significant weight loss and is a major complication in cancer development. Despite its importance, there are no effective treatments available. Recently, resistance training has been suggested as a non-pharmacological therapy to prevent muscle atrophy and functional decline in preclinical models. The C26 tumor-bearing mouse model is commonly used, showing progressive muscle and fat loss along with systemic inflammation. However, the impact of these changes on the cerebellum, which is crucial for motor function and balance, remains unclear. In this study, we combined the C26 tumor model with an endurance protocol to investigate cerebellar alterations in sedentary (SED T+) and trained (TR T+) tumor groups compared to sedentary (SED T−) and trained (TR T−) non-tumor groups. The tumor groups were sacrificed at the onset of cachexia to assess the impact of proactive endurance training. After confirming cachexia onset through body weight and strength loss, histological analysis revealed that the cerebellar area was similar across groups. However, a significant reduction in the Purkinje cell layer (PCL) thickness was observed in SED T+ mice. Since PCs are the sole efferent neurons in the cerebellum, we examined their condition using histological analysis, immunohistochemistry, and immunofluorescence by ZIC4 labelling. The PC body size decreased in the tumor groups, while their number was reduced only in TR T+ mice. Interestingly, the SED T+ group exhibited the highest number of PC with abnormal morphology. Additionally, to assess the effects on cerebellar vascularity, we found that tumor groups had an increased number of vessels in the white matter. Altogether, these results suggest that in the C26 tumor mouse model, the cerebellum is affected by PCL hypotrophy with smaller and abnormal PC. Endurance training only partially counteracts these alterations. Further studies are needed to clarify Purkinje cell aberrations due to their crucial role in motor function.

3.4. Synergistic Interaction Between Copper Overload and Aβ Peptide Enhances Microglial Pro-Inflammatory Response

Marlene Zubillaga ^{1, 2}, Xenia Abadin Calaf ¹, Maria José Bellini ², Anna Colell ¹, Nathalie Arnal ²

¹ 

Department of Cell Death and Proliferation, Institute of Biomedical Research of Barcelona (IIBB), Spanish National Research Council (CSIC), August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Biomedical Research Center, Barcelona, 08036, Spain

² 

Neurosciences Laboratory, Institute of Biochemical Research of La Plata “Prof. Dr. Rodolfo R. Brenner” (INIBIOLP), La Plata, Buenos Aires, B1900, Argentina

Background: Copper (Cu) binds to amyloid beta (Aβ), generating reactive oxygen species (ROS) and altering cholesterol metabolism, leading to accumulation that worsens Alzheimer’s disease (AD). Cu near amyloid plaques can mediate inflammation via the NOD-like receptor protein 3 (NLRP3) inflammasome, which activates caspase-1 and interleukin-1β (IL-1β). Whether Cu exacerbates Aβ-induced inflammasome activation and microglial dysfunction remains unclear. Methods: Mouse microglia (SIM-A9) were treated with 1 µM Aβ and 100 µM Cu for 24 h. ROS production was measured using dihydroethidium (DHE). Cholesterol levels were assessed via confocal microscopy and the Amplex Red kit. Inflammasome and phagocytosis proteins were analyzed by Western blot and PCR. Phagocytosis was measured using fluorescent microbeads, while inflammasome assembly was visualized with ASC-GFP speckle formation. Results: Cu increased IL-1β and NLRP3 expression, with a synergistic effect observed during Cu + Aβ treatment. Cu and Cu + Aβ elevated total and mitochondrial cholesterol levels while reducing glutathione (GSH). ROS production increased but was mitigated by glutathione ester (GSHee) and mitochondrial antioxidants (MitoTEMPO and MitoQ). Microglial phagocytosis, impaired by Cu and Cu + Aβ, was partially restored by GSHee. Cu also reduced phagocytosis proteins (ABCA7 and CD36) and caused their cytosolic redistribution, leading to decreased phagocytic activity. Conclusions: Elevated Cu levels exacerbate Aβ-induced microglial dysfunction by increasing mitochondrial oxidative stress, cholesterol accumulation—both at the cellular and mitochondrial level—and inflammasome activation. These effects impair phagocytosis, contributing to Aβ plaque accumulation and worsening AD pathology.

This research was funded by MCIN/AEI/10.13039/501100011033 and co-funded by NextGenera-tionEU/PRTR and “ERDF A way of making Europe”; grants PID2022-143279OB-100 and RED2022-134786-T, AGAUR, and grant 2021-SGR00490, respectively; and by the CIBERNED (Convocatoria 2022 Proyectos Colaborativos 2020/21).

3.5. Unlocking the Potential of Non-Neuronal Cell-Derived Extracellular Vesicles in Pain Relief and Neuroprotection

Parisa Gazerani

• Department of Life Sciences and Health, Oslo Metropolitan University, Pilestredet 50, Oslo, 0167, Norway

Introduction: Extracellular vesicles (EVs), including exosomes, are emerging as key mediators of intercellular communication, transferring bioactive molecules such as microRNAs, proteins, and lipids. Among non-neuronal cells, Schwann cells, oligodendrocytes, and satellite glial cells (SGCs) release EVs with distinct neuroprotective and pain-relieving properties. These EVs play critical roles in modulating inflammation, supporting neuronal repair, and regulating pain pathways, offering innovative therapeutic avenues for chronic pain management and neural repair.

Methods: A scoping review was conducted by systematically searching the PubMed, Scopus, and Web of Science databases. Studies published up to November 2024 were screened to identify evidence on Schwann cell-, oligodendrocyte-, and SGC-derived EVs. A total of 15 key studies were included, focusing on their cargo, functional mechanisms, and therapeutic applications.

Results: Schwann cell-derived EVs are enriched with neuroprotective microRNAs such as miR-21 and miR-146a, which reduce pain by regulating inflammation and promoting neuronal survival. They also carry proteins like brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), enhancing neuronal repair. Oligodendrocyte-derived EVs deliver proteins such as myelin basic protein (MBP) and proteolipid protein (PLP), which are essential for neuronal stability and repair, as well as superoxide dismutase (SOD), which mitigates oxidative stress. SGC-derived EVs, while also carrying miR-21 and miR-146a, modulate neuronal hyperexcitability and inflammation uniquely through cytokines such as interleukin-10 (IL-10), amplifying their pain-relieving effects.

Conclusion: This review highlights the distinctive roles of Schwann cell-, oligodendrocyte-, and SGC-derived EVs in alleviating pain and supporting neural health. It identifies key research gaps, including the need for standardized methodologies and deeper characterization of EV cargo. These findings emphasize the therapeutic potential of EVs as innovative tools for chronic pain treatment and neural regeneration, underscoring their translational value for future research and clinical applications.

4. Cell Therapies

Design of a Nasal Prophylactic for Targeting SARS-CoV-2 Variants with B-Escin and Nanoparticles

Ilyes Zatla, Lamia Boublenza

• Laboratory of Microbiology applied to the Food industry, Biomedical and the Environment, Faculty of Natural and Life Sciences, Earth and Universe Sciences. Department of Biology. University of Tlemcen, Tlemcen, 13000, Algeria

The emergence of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) variants continues to challenge global healthcare systems, necessitating the exploration of innovative therapeutic modalities. This study proposes a novel nasal treatment approach leveraging the antiviral properties of Beta-escin (B-escin), a natural compound, in conjunction with gold and silver nanoparticles. B-escin, extracted from horse chestnut seeds, exhibits notable efficacy against coronaviruses, while nanoparticles offer advantageous features for targeted delivery and stability enhancement. Through integrated computational analyses encompassing molecular docking, molecular dynamics simulations, and quantum mechanical calculations, we assess the potential of this combined strategy against prevalent COVID-19 variants, including Alpha, Beta, Gamma, Delta, and Omicron. Specifically tailored for nasal administration, our investigation emphasizes the synergistic interactions between B-escin and nanoparticles, elucidating their collective impact on variant-specific viral targets within the nasal mucosa. The computational results indicate augmented antiviral activity and target specificity when combining B-escin with gold (Au) and silver (Ag) nanoparticles, surpassing individual treatment efficacy. Moreover, we investigate the influence of nanoparticle characteristics, such as size, morphology, and surface functionalization, on the observed synergistic effects. These findings underscore the promise of developing a nasal treatment utilizing natural compounds and nanotechnology, offering a potential frontline defense against the diverse spectrum of SARS-CoV-2 variants.

5. Cellular Signaling

5.1. (Phospho-)proteomic Signaling Responses of Human Male Germ Cell Lines to Simulated Microgravity and Hypogravity

Alessia Di Pauli ¹, Giulia Ricci ², Marco Crescenzi ³, Maria Addolorata Mariggiò ⁴, Caterina Morabito ⁴, Luisa Gesualdi ¹, Serena Camerini ³, Casella Marialuisa ³, Rosanna Chianese ², Marika Berardini ¹, Francesca Ferranti ⁵, Teresa Chioccarelli ², Gilda Cobellis ², Angiolina Catizone ¹, Michele Signore ³

¹ 

Department of Anatomy, Histology, Forensic-Medicine and Orthopedics, Section of Histology and Embryology, “Sapienza” University of Rome, 00161 Rome, Italy

² 

Department of Experimental Medicine, Università degli Studi della Campania “Luigi Vanvitelli”, 80138 Naples, Italy

³ 

Core Facilities, ISS Istituto Superiore di Sanità, 00161 Rome, Italy

⁴ 

Department of Neuroscience, Imaging and Clinical Sciences-CAST, “G. d’Annunzio” University of Chieti-Pescara, 66013 Chieti, Italy

⁵ 

Human Spaceflight and Scientific Research Unit, Italian Space Agency, 00133 Rome, Italy

Introduction: Access to space has significantly increased over the last decade, and with this comes the need for comprehensive knowledge about the effects exerted by altered gravitational conditions on human physiology and reproductive health. However, it should be highlighted that our knowledge about the effects of altered gravitational force on germ cells is still very poor. In this study, we exploited Reverse-Phase Protein microArrays (RPPAs), a biased (phospho-)proteomic approach, to investigate the impact of simulated microgravity (SμG) and hypogravity (ShG) on two human male germ cell lines, TCam-2 and NT2D1. Methods: TCam-2 and NT2D1 cell lines were exposed to SμG and ShG conditions using a Random Positioning Machine for 3, 24, and 72 h. RPPA analysis was conducted using a panel of 130 antibodies selected to investigate a broad number of pathways potentially affected by altered gravity conditions. Results: The data analysis revealed that the exposure of both TCam-2 and NT2D1 cells to altered gravity induced significant (phospho-)proteomic changes. In particular, SμG induced early-phase alterations (3–24 h), mostly characterized by the upregulation of some key regulators of signaling pathways, whereas longer SμG exposure (72 h) resulted in the downregulation of other signaling proteins. ShG elicited minor changes, mostly characterized by reduced protein expression. The key pathways affected included cytoskeletal dynamics, proliferation, apoptosis, and autophagy. Notably, cell viability was not significantly impacted, suggesting compensating adaptation mechanisms to altered gravitational conditions. Conclusions: These findings indicate that (phospho-)proteomic responses to simulated gravity conditions were transient and non-persistent, demonstrating that human male germ cells exhibit resilience and adaptative capacity to cope with altered gravitational environments. This study provides valuable preliminary insights into the cellular and molecular mechanisms involved in gravity sensing and adaptation, which is crucial for developing countermeasures to ensure reproductive health and functionality during long-duration space missions for astronauts but also for the health of their future offspring.

5.2. Aberrant Methylation Events in Arteriovenous Malformation of the Brain

Concetta Scimone ¹, Luigi Donato ¹, Simona Alibrandi ¹, Alfredo Conti ^{2, 3}, Carlo Bortolotti ², Antonino Germanò ¹, Concetta Alafaci ¹, Sergio Lucio Vinci ¹, Rosalia D’Angelo ¹, Antonina Sidoti ¹

¹ 

Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Messina, 98125, Italy

² 

IRCCS Istituto Delle Scienze Neurologiche di Bologna, Via Altura 3, 40123, Bologna, Italy

³ 

Department of Biomedical and NeuroMotor Sciences (DiBiNeM), Alma Mater Studiorum, University of Bologna, Bologna, 40123, Italy

Introduction: Arteriovenous malformation of the brain (bAVM) is a vascular condition affecting brain arterioles. It most likely arises due to an impaired expression of vascular differentiation markers that leads failed capillary bed development and, then, to the arteriolar-to-venule direct shunt. In this context, EFNB2 and ephrin-B4 seem to drive arteriovenous differentiation. Although several pathways have been linked to disease development, knowledge on the molecular basis of this phenotype is still very limited.

Methods: Methylome analysis was performed in endothelial cells purified from bAVM in order to identify aberrant methylation patterns in genes controlling vascular development and endothelial cell/vascular smooth muscle cell (VSMC) cross-talk. Human cerebral microvascular endothelial cells (HCMECs) were used as the control. The results were validated by quantitative methylation-specific PCR and quantitative real-time PCR.

Results: Both CpG- and CHG-aberrant methylation events were identified in bAVM endothelial cells. Most differentially methylated loci included noncoding RNA genes. Genes already known to be linked to bAVM development were identified. Interestingly, we focused our attention on the EPHB1 gene, not yet linked to bAVM onset. Likewise, other differentially methylated genes included transcription factors expressed in VSMCs that regulate the expression of genes involved in endothelial cell differentiation.

Conclusion: Our data allow to identify aberrant methylation events occurring in bAVM endothelial cells when compared to HCMECs. Notably, these genes are clustered in pathways related to vascular homeostasis, as well as to VSMC-- endothelial cell crosstalk, suggesting that an impairment of this interaction plays a prominent role in the loss of vascular differentiation in bAVM phenotypes.

5.3. Assessment of Gene Signature Deriving from Prostate Cancer-Associated Fibroblasts (CAFs)

Azzurra Zicarelli ¹, Marianna Talia ¹, Eugenio Cesario ², Francesca Cirillo ¹, Domenica Scordamaglia ¹, Adelina Assunta Mondino ¹, Marika Di Dio ¹, Salvatore De Rosis ¹, Michele Di Dio ³, Anna Maria Miglietta ⁴, Marcello Maggiolini ¹, Rosamaria Lappano ¹

¹ 

Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy

² 

Department of Cultures, Education and Society Department, University of Calabria, 87036 Rende, Italy

³ 

Division of Urology, Department of Surgery, Annunziata Hospital, Cosenza, 87100, Italy

⁴ 

Breast and General Surgery Unit, Annunziata Hospital Cosenza, Cosenza, 87100, Italy

Background: The tumor microenvironment plays a pivotal role in shaping tumor aggressiveness and driving disease progression. In this context, the identification of gene signatures characterizing cancer-associated fibroblasts (CAFs) obtained from the two most frequently diagnosed cancers worldwide, such as breast and prostate tumors, may improve outcome prediction and therapeutic strategies for patients.

Methods: The transcriptomes of CAFs isolated from breast and prostate cancer biopsies were analyzed using RNA sequencing. Data from The Cancer Genome Atlas (TCGA) were used to compare the gene expression profiles of CAFs with those of breast and prostate cancer patients. The clusterProfiler package was employed to perform pathway enrichment analysis, while the gene signature associated withprostate CAFs was identified by applying K-means clustering. Kaplan--Meier curves and log-rank tests were used to assess the prognostic significance of the signature in prostate cancer patients. A decision-tree classification approach validated the clustering results and the prognostic relevance of the gene signature.

Results: The comparisons of either breast and prostate CAFs transcriptomes or the gene expression landscapes of breast and prostate cancer patients allowed us to construct a gene signature counting 11 genes (IL13RA2, GDF7, IL33, CXCL1, TNFRSF19, CXCL6, LIFR, CXCL5, IL7, TSLP, and TNFSF15) with clinical implications in prostate cancer. Notably, the aforementioned genes are implicated in immune-related transduction pathways. Thereafter, clustering and classification analyses revealed that prostate cancer patients exhibiting low expression levels of these 11 genes are characterized by a worse prognosis with a high prediction accuracy.

Conclusions: The prostate CAFs-related gene signature identified here might serve as a prognostic indicator and might offer a valuable set of biomarkers for improving the management of prostate cancer patients.

5.4. Cell Biology of Knee Joint Injuries: Early Mechanical Loading Perspective

Mikołaj Stańczak ¹, Bartłomiej Kacprzak ²

¹ 

Rehabilitation, AECC Univeristy College, Bournemouth 13-15 Parkwood Rd, United Kingdom

² 

Orthopaedics, Orto Med Sport, Łódź 90-640, Poland

Knee joint injuries, including those affecting the anterior cruciate ligament (ACL), meniscus, and cartilage, present significant challenges in sports medicine and orthopedics. Understanding the cellular and molecular mechanisms underlying these injuries is essential for developing effective therapeutic strategies. This systematic review explores the cell biology of knee joint injuries, focusing on the effects of early mechanical loading. We examine diffierent types of knee injuries, cellular responses to mechanical loading, the signaling pathways involved, and implications for treatment and rehabilitation. This comprehensive synthesis aims to provide insights into optimizing rehabilitation protocols and developing novel therapeutic approaches.

Knee joint injuries are prevalent among athletes and the general population, often resulting from trauma, overuse, or degenerative processes. The knee joint, a complex and critical structure for mobility, is susceptible to various injuries, including ligament tears, meniscal damage, and cartilage degradation. Among these, anterior cruciate ligament (ACL) injuries are particularly common and frequently require surgical intervention. Recent research has underscored the importance of early mechanical loading in the rehabilitation process, which can significantly influence cellular responses, tissue repair, and remodeling. This review systematically examines current knowledge on the cellular mechanisms affected by early mechanical loading in knee joint injuries, providing valuable insights into potential therapeutic strategies.

5.5. Exploiting Mechanotransductive Processes to Promote Pancreatic β-Cell Differentiation and Function

Alessandra Galli ¹, Elisa Maffioli ², Nevia Dule ¹, Monica Galbusera ¹, Ilaria Fagnani ¹, Sveva Del Maffeo ¹, Marika Visioli ¹, Michela Castagna ¹, Carla Perego ¹

¹ 

Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, 20122, Italy

² 

Department of Veterinary Medicine, University of Milan, Lodi, 26900, Italy

Introduction: Pancreatic β-cells, by releasing insulin, play a critical role in the control of glucose homeostasis. In vivo, they reside in the islet niche, which provides a myriad of stimuli derived from the extracellular matrix (ECM) and the neighbouring cells. This multifaceted environment plays a pivotal role in the regulation of pancreas development and in the control of β-cell function. Even though the contribution of chemical stimuli has been widely investigated, the mechanical signals are poorly known. Therefore, the aim of the proposed research was to explore the role of nanotopography in the regulation of β-cell functionality and to investigate the underlying molecular mechanisms.

Methods: Human islets of Langerhans were grown on cluster-assembled zirconia substrates with a tailored roughness mimicking the ECM nanotopography, and flat zirconia substrates were used as controls. The β-cell functionality was evaluated by means of super-resolution fluorescence microscopy, Western blot, and ELISAs and confirmed by shot-gun proteomics.

Results: Quantitative immunofluorescence revealed that β-cells are mechanosensitive and respond to nanotopography through mechanotransduction, which impacts on focal adhesions and cytoskeletal and nuclear organization. These modifications are paralleled by a profound gene reprogramming which promotes the expression of pro-survival and pro-differentiation factors and proteins involved in the regulation of granule trafficking in the islets grown on the nanostructure. In line with these observations, we found that the nanotopography preserves β-cell differentiation and function in long-term cultured islets, as suggested by increased β-cell number, reduced β-cell death, and potentiated glucose-stimulated insulin secretion.

Conclusions: This study provides a better understanding of how mechanical forces contribute to β-cell fate, offering the possibility to harness these mechanisms for promoting β-cell function in physiological and pathological conditions.

5.6. GPER Is Involved in the Resistance to Palbociclib in Breast Cancer Cells

Salvatore De Rosis, Marianna Talia, Francesca Cirillo, Domenica Scordamaglia, Marika DI Dio, Azzurra Zicarelli, Marcello Maggiolini, Rosamaria Lappano

• Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende 87036, Italy

Background: The cyclin dependent kinase (CDK) 4/6 inhibitor palbociclib plus endocrine therapy is an effective treatment for patients with estrogen receptor (ER)-positive and HER2-negative breast cancer (BC). Notwithstanding promising clinical outcomes, intrinsic or acquired resistance frequently arises. Given the established role of cancer-associated fibroblasts (CAFs) in the resistance to chemotherapeutics and the implication of the G-protein estrogen coupled receptor (GPER) in mediating stromal functions, we sought to investigate whether CAFs may contribute to palbociclib resistance in BC cells via GPER.

Methods: MCF7 and T47D BC cells, as well as CAFs isolated from breast tumor tissues, were used as model systems. Docking experiments allowed us to evaluate the potential interaction of palbociclib with GPER. Immunoblotting, gene expression arrays, and gene silencing experiments along with 2D and 3D co-culture assays were performed to evaluate the activation of GPER signaling by palbociclib in CAFs. Investigations on large cohorts of BC patients served to assess new potential stromal targets involved in palbociclib resistance.

Results: First, docking studies showed that palbociclib can interact with the binding site of GPER, like its well-known ligands. Next, we found that in CAFs, palbociclib stimulates the main sensors of GPER activation like ERK1/2 activation and c-Fos up-regulation. To assess the transcriptional changes mediated by palbociclib through GPER in CAFs, we performed a human chemokine gene expression array. Notably, we found that the palbociclib-induced increase of pro-inflammatory genes is no longer evident upon silencing GPER expression. Finally, GPER silencing in CAFs interfered with the capacity of palbociclib to reduce the viability of BC cells in co-culture assays.

Conclusions: Our findings suggest that the activation of GPER signaling in CAFs may contribute to palbociclib resistance in breast cancer cells. On this basis, therapeutic strategies targeting CAFs might enhance the efficacy of palbociclib in BC patients.

5.7. Parental Biomedical Manifestations and Newborn Telomeres: The Ends with a New Beginning

Sadia Farrukh ¹, Saeeda Baig ²

¹ 

Biological and Biomedical Sciences, The Aga Khan University, Karachi 74800, Pakistan

² 

Department of Biochemistry, Ziauddin University, Karachi 74800, Pakistan

Background: Telomere biology, even during fetal development, is influenced by parental manifestations that can imprint parental epigenetic marks onto the newborn. The interplay between genes, immune biology, and oxidative stress contributes to age-related non-communicable diseases (NCDs), and the telomere length (TL) dynamics in utero are not fully known. This study investigated the impact of parental biomedical factors, including NCDs, on newborn TL, as well as telomerase genes and immune biology.

Method: Blood samples (n = 612) were collected from 204 parent–newborn pairs. Their demographics, socioeconomic status (SES), education, and NCD exposure were assessed. TL was quantified using the T/S ratio via qPCR; telomerase gene (TERC, TERT) polymorphisms were identified through Sanger sequencing; and immune senescence was analyzed using flow cytometry. Multivariate regression was used to analyze the paternal–newborn LTL associations, with p0.05 as the significance threshold.

Results: The mean ages of the mothers and fathers were 27 ± 5.12 and 34 ± 6.36 years, respectively. The newborns of parents aged >30 years old had longer TLs (2.31 ± 1.45; p = 0.034). A low SES and blue-collar occupations were correlated with a shorter TL in the parent–newborn pairs (1.5 ± 1.14; p0.05), with higher frequencies of the telomerase CC genotype (TERC: 28%, 41%; TERT: 22%, 37%) compared to those in high-SES and white-collar groups. NCDs and viral pathologies significantly influenced telomere biology (p0.05). The analysis of immune senescence markers showed decreased CD57+ KLRG1+ expression in the newborn T-cells (2.45 ± 4.34; p0.05) compared to those of their parents (3.5 ± 5.49; p = 0.04).

Conclusion: Parental biomedical factors and diseases influence newborn telomere biology and immune development in utero, highlighting the importance of mitigating prenatal risk factors.

5.8. The Silencing of the G Protein-Coupled Estrogen Receptor (GPER) Drives Apoptotic Death in Triple-Negative Breast Cancer Cells

Marianna Talia, Francesca Cirillo, Domenica Scordamaglia, Salvatore De Rosis, Azzurra Zicarelli, Marika Di Dio, Rosamaria Lappano, Marcello Maggiolini

• Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036, Rende, Italy

Introduction: The G protein-coupled estrogen receptor (GPER) is known for its ability to mediate rapid estrogen signaling in diverse normal and malignant cell contexts, including breast cancer (BC). Of note, the role of the GPER in promoting pro-tumorigenic traits in triple-negative breast cancer (TNBC) has been suggested. In this study, we sought to provide novel insights into the transcriptionally guided biological behavior of TNBC cells lacking GPER expression.

Methods: GPER knock-out (KO) MDA-MB-231 TNBC cells were obtained using the CRISPR/Cas9 genome editing technology. RNA sequencing (RNA-seq) and Gene Ontology (GO) enrichment analyses served to assess the differentially expressed genes (DEGs) between the GPER KO and wild-type (WT) cells and their biological role. Chromatin immunoprecipitation assays, real-time PCR, immunoblots, immunofluorescence, ELISA, and flow cytometric experiments, as well as the use of RNA interference techniques, allowed us to uncover the molecular routes implicated in the biological features triggered by GPER silencing in the TNBC cells. Survival analyses were performed on TNBC patients ffrom the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset.

Results: The RNA-seq data revealed that GPER silencing in MDA-MB-231 cells induces a pro-apoptotic gene signature. Accordingly, the biological assays demonstrated that GPER KO cells exhibit enhanced mitochondria-dependent apoptotic death with respect to these levels in WT cells. Mechanistically, we found that reduced cAMP levels in GPER KO cells may trigger the activation of the pro-apoptotic JNK/c-Jun/p53/Noxa pathway. In accordance with these results, the bioinformatics analyses on the TNBC patients revealed that high NOXA gene expression levels are associated with better outcomes.

Conclusions: Collectively, our findings unveil the role of the GPER in sustaining anti-apoptotic signals in TNBC cells, thus suggesting this receptor as a potential valuable therapeutic target for preventing the progression of this malignancy.

6. Cell Research in Animal Models

6.1. Advancing Neuroblastoma Research: A 3D Tumorsphere Model to Study GD2 Immunotherapy and Metastasis

Piotr Krzysztof Jung

• Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia 19104, USA

Metastatic neuroblastoma (NB) is one of the most challenging childhood cancers to treat, with a high rate of relapse despite aggressive therapies. Most NB cells express disialoganglioside GD2, a tumor-specific antigen with limited expression in normal tissues. While GD2-targeting monoclonal antibodies have shown promising clinical success, a significant number of patients still experience relapse. This is thought to be due, in part, to heterogeneous or low GD2 expression, which complicates the effectiveness of immunotherapies. However, studying GD2-targeted treatments and metastasis in NB is hindered by a lack of suitable in vitro models. Current 2D culture systems are limited in their ability to mimic the complex environment of metastatic tumors, and murine NB cells typically lose GD2 expression when cultured. To overcome these challenges, I have developed a novel 3D cell culture system known as “tumorspheres”. This model allows NB cells to grow in a more physiologically relevant environment and better replicates the conditions of tumor metastasis. Preliminary results demonstrate that tumorspheres retain high GD2 expression and show significant growth and motility within a matrix scaffold, mimicking in vivo tumor behavior more accurately than traditional 2D models. Importantly, these tumorspheres can be formed from a mix of GD2-positive and GD2-negative cells, enabling the study of antigenic heterogeneity and its impact on therapeutic resistance. Additionally, tumorspheres can be implanted subcutaneously into mice to promote in vivo tumor growth, further enhancing their utility as a model for studying neuroblastoma metastasis and immunotherapy. This new 3D model offers a more representative platform for investigating GD2-targeted therapies and tumor metastasis, with the potential to advance our understanding of treatment resistance and lead to more effective strategies for combating metastatic neuroblastoma.

6.2. Annexin A1 Deficiency Increases Liver Damage and Metabolic Alterations in Mice with Type I Diabetes

Diego Dias dos Santos ¹, Rafael André da Silva ², Álex Rosini Silva ³, Antônio Thiago Pereira Campos ^{4, 5}, Luiz Philipe Souza-Ferreira ⁶, Carlos Lenz-César ^{4, 5}, Andréia de Melo Porcari ³, Cristiane Damas Gil ^{1, 2}

¹ 

Structural and Functional Biology Graduate Program, Paulista School of Medicine, Federal University of São Paulo (EPM/UNIFESP), São Paulo, SP, Brazil

² 

Biosciences Graduate Program, Institute of Biosciences, Letters and Exact Sciences, Universidade Estadual Paulista (UNESP), São José do Rio Preto, SP 15054-000, Brazil

³ 

MS4Life Laboratory of Mass Spectrometry, Health Sciences Postgraduate Program, Sao Francisco University, Bragança Paulista, SP 12916-900, Brazil

⁴ 

National Institute of Photonic Applied to Cell Biology, Universidade Estadual de Campinas (UNICAMP), Campinas, SP 13083-865, Brazil

⁵ 

Physics Graduate Program, Universidade Federal do Ceará (UFC), Fortaleza, Ceará 60440-900, Brazil

⁶ 

Structural and Functional Biology Graduate Program, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP 04023-900, Brazil

Diabetes mellitus (DM) is a global public health issue causing systemic dysregulations, including severe liver complications. Type 2 diabetes (DM2) patients show elevated annexin A1 (AnxA1) levels, and in murine DM2 models, AnxA1 mitigates insulin resistance effects like hepatosteatosis. However, its role in DM1 is underexplored. This study investigates AnxA1’s role in hepatocyte biology in a streptozotocin (STZ)-induced DM mouse model. Male C57BL/6 mice (WT and AnxA1-/-) were divided into control (CTR) and DM groups. DM was induced via STZ injection (65 mg/kg for 5 days). After 12 weeks, livers were collected for analysis. DM WT and AnxA1-/- mice showed weight loss and increased blood glucose, reflecting typical diabetic metabolic disruptions. Morphological evaluations revealed normal hepatocytes in WT CTR mice, while 70% of AnxA1-/- CTR mice showed cytoplasmic vacuolation. In DM groups, 50% of WT mice had vacuolated, damaged hepatocytes, increasing to 75% in AnxA1-/- DM mice, highlighting AnxA1’s protective role. Hepatocyte glycogen levels were lower in DM mice, especially AnxA1-/-. Collagen deposition in the centrolobular veins and portal triads was higher in AnxA1-/- DM mice, indicating worsened fibrosis without AnxA1. Both DM WT and AnxA1-/- livers showed reduced fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGF-A), suggesting impaired regeneration. Inflammation varied between genotypes: WT DM mice had higher IL-10 and TNF-α, while AnxA1-/- DM mice showed increased monocyte chemoattractant protein-1 (MCP-1). Oxidative stress markers indicated increased reactive oxygen species (ROS) in AnxA1-/- DM hepatocytes, with differing trends in superoxide dismutase (SOD) and catalase (CAT) activities. Metabolites linked to the tyrosine, malate-aspartate, taurine, and hypotaurine pathways stood out in AnxA1 knockout mice, suggesting that these pathways are key in AnxA1-deficient mice. This highlights AnxA1’s role in liver protection under diabetic conditions.

6.3. The Effect of the Lipophilic Fraction of Pequi on Extracellular Matrix Remodeling in Skin Lesions in Mice

Daniely Lisboa Matsnaka ¹, Maria Eduarda Urzêda da Silva ¹, Cristiane Damas Gil ², Kallyne Kioko Oliveira Mimura ², Sergio Marcelino de Oliveira ¹

¹ 

Laboratory of Histophysiology and Animal Reproduction, Federal University of Mato Grosso, CUA/UFMT, Pontal do Araguaia 78698-000, MT, Brazil

² 

Department of Morphology and Genetics, Paulista School of Medicine, Federal University of São Paulo (EPM-UNIFESP), São Paulo 04023-900, SP, Brazil

Introduction: The tissue repair process is regulated by various molecular and cellular elements that must be in balance to result in healthy tissue. Pequi oil (Caryocar brasiliense) possesses therapeutic properties that may contribute to this context. Thus, this study evaluated the action of this oil on skin lesions. Methods: Balb/c mice with induced skin lesions were divided into groups (n = 5) receiving daily topical applications of saline solution (the Control group) or the lipophilic fraction of pequi (the Treated group). After 3, 7, and 14 days, the animals from each group were sacrificed, and fragments of their injured skin were analyzed using polarization microscopy to evaluate collagen; using Western blot to evaluate matrix metalloproteinase 2 (MMP2); and using a multiplex assay to evaluate tumor growth factor-beta 1 (TGF-β1). CEUA–UFMT 23108.080623/2023-50. Results: In the Treated group, a lower amount of type I collagen was observed on days 3 (p ≤ 0.05) and 14 (p ≤ 0.001) and of type III collagen on day 14 (p ≤ 0.001) compared to these levels in the Control group. Additionally, the Treated group exhibited slightly higher MMP2 expression (not significant) and lower TGF-β1 levels (p ≤ 0.05) on day 3 compared to the Control, indicating reduced fibrogenic stimulation. Conclusion: These results demonstrate that treatment with pequi oil promotes lower deposition of type I and III collagen in the late stages of skin regeneration, primarily by inhibiting TGF-β1 production in the early stage, suggesting the reduced activation of pathways that could lead to excessive fibrosis. Meanwhile, the stable levels of MMP2 reflect the necessary balance for tissue remodeling. These findings reveal that pequi oil regulates key factors in tissue remodeling, promoting less fibrosis and preserving extracellular matrix balance, highlighting its therapeutic potential for wound management (Financial Support: FAPEMAT 000547-2023).

6.4. Therapeutic Properties of Pequi Lipophilic Fraction (Caryocar Brasiliense) in Skin Wound Repair: Modulation of Inflammatory and Proliferative Phases

Maria Eduarda Urzêda da Silva ¹, Daniely Lisboa Matsnaka ¹, Cristiane Damas Gil ², Fernanda Regina Casagrande Giachini Vitorino ³, Sergio Marcelino de Oliveira ^{1, 2}, Kallyne Kioko Oliveira Mimura ²

¹ 

Laboratory of Histophysiology and Animal Reproduction, Federal University of Mato Grosso—CUA/UFMT, Pontal do Araguaia 78698-000, MT, Brazil

² 

Department of Morphology and Genetics, Paulista School of Medicine, Federal University of São Paulo (EPM-UNIFESP), São Paulo 04023-900, SP, Brazil

³ 

Laboratory of Vascular Biology and Histopathology, Federal University of Mato Grosso—CUA/UFMT, Barra do Garças 78605-091, MT, Brazil

Introduction: Pequi (Caryocar brasiliense) is a fruit from Cerrado, Brazil, that has important therapeutic properties for the tissue repair of skin wounds. Methods: Balb/c mice were subjected to skin lesions and received daily topical treatment with the lipophilic fraction of pequi (Treated group) or saline solution (Control group). Animals from each group were sacrificed at 3, 7, and 14 days post injury (n = 5). Macroscopic analyses were performed to assess wound contraction, and peripheral blood was collected for leukocyte quantification. Additionally, skin fragments were used for histopathological analysis (Hematoxylin–Eosin) and for quantification of the expression of the proteins Annexin A1 (AnxA1) and phosphorylated ERK (pERK) using the Western blot technique. CEUA-UFMT 23108.080623/2023-50. Results: At 3 days, the Treated animals exhibited a more robust scab on the lesion, providing greater physical and microbiological protection. At 7 days, wound contraction was significantly higher in the Treated group compared to the Control group (p ≤ 0.01), accelerating wound closure and preventing dehydration and infection risks. In peripheral blood, a higher number of neutrophils, eosinophils, and monocytes were identified in the Treated group compared to the Control group (p ≤ 0.0001; p ≤ 0.05; p ≤ 0.05, respectively), particularly during the early 3-day period. No difference was observed in AnxA1 protein expression between the experimental groups; however, the Treated group showed higher pERK expression at 7 days compared to the Control group (p ≤ 0.05). Conclusions: These data suggest that pequi oil acts as an effective modulator of the inflammatory phase, by inhibiting leukocyte transmigration, and of the proliferative phase, through the activation of the ERK pathway, preparing the tissue for regeneration without altering AnxA1 levels. Thus, it is evident that the lipophilic fraction of pequi accelerates tissue repair by modulating the inflammatory and proliferative phases, promoting more efficient healing. (Financial Support: Fapemat 000547-2023; CAPES 88887.976959/2024-00).

7. Cellular Metabolism

7.1. Alpha-Ketoglutarate Dehydrogenase (KGDH): An Update on Its Redox Regulation and Importance in Generating Mitochondrial Reactive Oxygen Species (mtROS) in Hepatocytes

Ryan Mailloux

• School of Human Nutrition, Faculty of Agricultural and Environmental Science, McGill University, Montréal, QC, Canada

The regulated production of mitochondrial reactive oxygen species (mtROS) is vital for maintaining optimal liver health. The mtROS-mediated maintenance of cell function (s) is through the induction of oxidative eustress signals, which are transmitted through the site-specific cysteine modifications. By contrast, the sustained overgeneration of mtROS triggers oxidative distress, resulting in the manifestation of metabolic diseases like non-alcoholic fatty liver disease (NAFLD) and the progression of cancer. Therefore, it is crucial to understand precisely how mitochondria generate mtROS, how its production is regulated, and how dysfunction in these regulatory mechanisms may cause NAFLD and its related disorders. Complexes I and III of the electron transport chain (ETC) are viewed as the primary mtROS sources in mammalian cells. However, we recently generated compelling evidence showing that TCA cycle enzyme α-ketoglutarate dehydrogenase (KGDH), and not the ETC, is the main mtROS supplier in hepatocytes. We found that KGDH is a main mtROS source when hepatic mitochondria are fueled with oxidizable substrates and is a main source of oxidative stress in the livers of mice subjected to dietary fat overload. Additionally, we discovered that mtROS generation by KGDH is controlled by reversible protein S-glutathionylation, which is catalyzed by glutaredoxin-2 (Glrx2). In this context, the oxidation of the glutathione (GSH) pool S-glutathionylates KGDH to shut down mtROS generation. This creates a self-contained negative feedback loop for the redox regulation of mtROS production. Recently, we showed that the manipulation of the Glrx2 pathway protects from diet-induced NAFLD development through the dynamic inhibition of KGDH-mediated mtROS hyper-generation by S-glutathionylation. In this presentation, I will elaborate on the importance of these findings in understanding the manifestation of NAFLD and how the targeted and dynamic redox modification of KGDH with mitochondria-targeted drugs can mitigate the onset of this disease through the dynamic inhibition of mtROS hyper-production by redox modifications.

7.2. Brain Pericytes: From Origins to Implications in Cell–Cell Communication Within the Neurovascular Unit

Julien Saint-Pol

• Laboratoire de la Barrière hémato-Encéphalique (LBHE, UR2465), Université d’Artois, Lens 62307, France

The BBB is a natural barrier located in the endothelial cells of the cerebral microvessels (BMECs) that restricts exchanges between the bloodstream and the cerebral parenchyma. The integrity of the BBB is vital for preserving cerebral homeostasis, and neuroinflammatory processes alter the physical and/or metabolic properties of this barrier. The brain pericyte, which shares a common basement membrane with BMECs, has been shown to be a major cell type involved in the induction and maintenance of the BBB’s main features. This presentation will review the role and intercellular interactions of human brain pericytes (hBPs) with the cells of the neurovascular unit, and particularly BMECs, based on data generated within the lab using proteomics, cell physiology, and studies of extracellular vesicles (EVs). The protein pattern of hBPs is modified once they are cocultured with BMECs, highlighting a metabolic switch induced by BMECs on hBPs. A stimulation of EV biogenesis is observed, indicating a potential need for a hBP-derived cell–cell communication through EVs. The mechanisms of interaction between BMECs and hBP-derived EVs are in favor a clathrin-coated pit-mediated endocytosis. Under inflammation, hBP-derived EVs exhibit a modified protein pattern compared to untreated EVs and induce a dysregulation of trans-endothelial electric resistance (TEER) on BMECs.

Despite their former reputation as a contaminant for BBB modeling in vitro, hBPs remain of importance for maintaining the BBB phenotype. BMECs also modulate the functions and developmental fates of hBPs. This work opens perspectives on the role of hBP-derived EVs on BBB feature regulation.

7.3. Dysfunctional Cellular Energy Metabolism Is a Premorbid Characteristic of Late-Onset Alzheimer’s Disease

Kai C. Sonntag, Bruce M. Cohen

• Department of Psychiatry, Program for Neuropsychiatric Research, McLean Hospital, Harvard Medical School, Belmont, MA 02478, USA

Age is the main risk factor for late-onset Alzheimer’s disease (LOAD) and all processes in normal aging also occur in LOAD pathology. While some factors appear to be more specific to LOAD and may be genetically determined, others are acquired through the aging process. Because bioenergetic metabolism is among the most fundamental features of cell functions, changes thereof have profound effects on every aspect of aging. In both fibroblasts and induced pluripotent stem cell (iPSC)-derived immature and mature brain cells of subjects with LOAD, we observed bioenergetic substrate deficiencies, including reductions in the redox agent nicotinamide adenine dinucleotide (NAD) or glucose uptake and metabolism, and alterations of associated bioenergetic-dependent cell functions. These data suggest that dysfunctional bioenergetics is an inherent cell-specific and cell-autonomous risk factor in LOAD. Together with findings from the brains of normally aging individuals or patients with LOAD, our data support the view that in LOAD, inherent cell-metabolic dysfunctions may occur in development and early life, altering the trajectory of the normal aging process in youth and middle-age, and predisposing neuropathological events that lead to symptomatic features of LOAD later in life. Our studies seek to further identify and characterize the cellular mechanisms underlying LOAD-associated bioenergetic abnormalities. The results of these studies may reveal targets and methods for treating individuals at risk for LOAD before illness develops or delaying disease onset and progression later in life.

7.4. p75NTR Modulation Restores Redox Metabolism and Blunts Inflammation in a Cell Model of Rett Syndrome

Michela Varone ¹, Giuseppe Scavo ¹, Mayra Colardo ¹, Noemi Martella ¹, Daniele Pensabene ^{1, 2}, Emanuele Bisesto ¹, Francesca Cavicchia ¹, Marco Segatto ¹

¹ 

Department of Biosciences and Territory, University of Molise, Fonte Lappone, 86090 Pesche, Italy

² 

Department of Science, University Roma Tre, Viale Guglielmo Marconi 446, 00146 Rome, Italy

Rett syndrome (RTT) is a neurological disorder with an early onset, primarily affecting females, and is characterized by severe cognitive and physical impairments. RTT is no longer regarded as an exclusively neurological disease; rather, it is now considered a multisystem syndrome that affects the brain and several other tissues/organs. Recent research suggests that disruptions in redox homeostasis and heightened inflammatory responses are of pivotal importance with regard to the disease’s clinical manifestations. Notably, emerging evidence points to the p75 neurotrophin receptor (p75NTR) as a regulator of oxidative stress (OS) and inflammation.

The objective of this study is to explore the impact of modulating p75NTR through the use of the small molecule LM11A-31 on fibroblasts derived from RTT patients. Fibroblasts were treated with 0.1 μM of LM11A-31 for 24 h, and analyses were conducted through qPCR, immunofluorescence, ELISA, and Western blotting.

The results reveal that LM11A-31 significantly mitigates OS markers in RTT fibroblasts. Specifically, p75NTR modulation restored protein glutathionylation and reduced the expression of the pro-oxidant enzyme NADPH-oxidase 4 (NOX4). Moreover, LM11A-31 blunted the expression of pro-inflammatory mediators while simultaneously normalizing transcription factors involved in antioxidant response and inflammatory response.

These results suggest that targeting p75NTR with LM11A-31 may offer a potential therapeutic avenue for restoring redox balance and alleviating inflammation in RTT patients. The use of a p75NTR modulator, such as LM11A-31, could be more effective for treating RTT by targeting key processes, addressing multiple aspects of the disease simultaneously. Further research should be directed towards a more detailed investigation of the molecular mechanisms and the corroboration of these findings in in vivo models.

8. Protein Quality Control, Proteasomal Degradation and Autophagy

Intrinsic Disorder in Autophagy-Related Proteins: Insights for Therapeutic Development

Deepak Chaurasiya

• India Institute of Information Technology, Allahabad, 211010, India

Autophagy is a crucial cellular process that maintains homeostasis by degrading and recycling intracellular components. A network of autophagy-related proteins orchestrates this process, many of which exhibit intrinsic disorder. Intrinsically disordered proteins (IDPs), lacking stable three-dimensional structures, possess structural flexibility, which enables dynamic interactions and diverse biological functions.

In this study, we analyzed 95 autophagy-related proteins from the Human Autophagy Database (HADb) and UniProt using sets of bioinformatics tools like ESpritz and leveraging datasets trained on X-ray, NMR, and DisProt. Our findings revealed that these proteins are significantly enriched with intrinsically disordered protein regions (IDPRs), particularly in key functional roles such as cargo recognition (e.g., SQSTM1, NBR1), autophagosome formation (e.g., ATG8, ATG12, WIPI1), and lysosomal degradation. Remarkably, proteins such as Beclin-1, LC3, and ATG9 exhibited high levels of intrinsic disorder, underscoring their critical regulatory roles.

The statistical analysis demonstrated that 80.21% of autophagy-related proteins contain at least one disordered region longer than 30 amino acids, and 65.97% have regions exceeding 50 amino acids. A total of 159 long disordered regions (greater than 30 amino acids) and 100 very long disordered regions (greater than 50 amino acids) were identified, emphasizing their functional relevance. Proteins like SPO95817 and SPP35638 showed extreme levels of disorder, with a mean percentage disorder above 90%, while others, such as SPP51809, exhibited minimal disorder, highlighting the variability within the autophagy-related proteome.

These results reinforce the critical role of IDPs in mediating transient and dynamic interactions that are essential for autophagy. This study advances our understanding of the molecular dynamics of autophagy-related proteins and provides a foundation for developing disorder-targeted therapeutic strategies. Such strategies hold potential for addressing neurodegenerative diseases, cancers, and other disorders linked to autophagic dysregulation.