The frequent conceptual overlap between aging and cellular senescence has generated ambiguities in experimental approaches, biomarker usage, and therapeutic strategy development within aging research. Cellular senescence and aging are biologically distinct but interconnected processes, frequently misunderstood or used interchangeably within biomedical research and theoretical discussions. Thus, aging refers to the progressive decline in physiological integrity and function at the organismal level, while cellular senescence is a stable cell-cycle arrest triggered by various forms of stress, including DNA damage, telomere shortening, epigenetic dysregulation, and mitochondrial dysfunction. This review dissects the molecular underpinnings of both phenomena, highlighting their key differences, mechanistic overlaps, and roles in health and disease, with particular emphasis on how the accumulation of senescent cells actively contributes to age-related functional decline. In particular, we discuss emerging biomarkers and therapeutic strategies, such as senolytics and partial reprogramming. Furthermore, a deeper understanding of these processes may contribute to the identification of innovative interventions for age-related disorders, such as therapies targeting specific components of the senescence-associated secretory phenotype or selectively modulating senescent cell burden.

Tau is a protein associated with microtubules principally expressed in neuronal cells, where it plays a fundamental role in cytoskeleton stabilization and axonal transport. Several diseases collectively named tauopathies, such as Alzheimer's disease, have been associated with an imbalance in the expression of alternative spliced Tau transcripts and the accumulation of hyperphosphorylated Tau, causing dysfunction and death of neuronal cells. Therefore, understanding the Tau exon splicing mechanisms may contribute to elucidating molecular factors that could underlie the development of neurodegenerative disorders. The aim of this study was to define the role of selected splicing factors in regulating Tau exon expression in cell lines and neuronal organoids. We demonstrated the role of the RNA-binding motif protein 20 (RBM20) splicing factor in regulating Tau exon 6 and exon 10, applying RNA-binding assay and qPCR analyses. Furthermore, we demonstrated that Tau expression was regulated during cerebral organoid differentiation, recapitulating in vivo Tau expression. These results suggest the feasibility of using brain organoid technology to study Tau alternative splicing during neural development, confirming that 3D cellular models could be used to study and characterize pathological processes taking place in Tau-related pathologies.

Genetic mutations, altered RNA regulation, and protein aggregation are the main hallmarks of Parkinson's disease (PD), a neurodegenerative disorder. Investigation into the molecular basis of the disease revealed that post-transcriptional regulation, specifically RNA processing, contributes to neuronal vulnerability in PD. Alterations in alternative splicing affecting genes involved in neuronal function and cellular homeostasis have been reported in PD, including SNCA, LRRK2, MAPT, PRKN, and BIN1. These alterations impact central neuronal pathways, including cytoskeletal maintenance, mitochondrial function, synaptic activity, oxidative stress, and intracellular trafficking. This review aims to provide an overview of alternative splicing in key PD gene transcripts, with a focus on their roles in pathogenesis and disease progression. Emerging data suggest that dysregulation of RNA binding proteins (RBPs) may influence RNA processing in PD. We will examine current evidence on the RNA regulatory networks in PD, highlighting the role of transcript isoforms and RBPs in neuronal dysfunction. Finally, we will discuss emerging experimental models such as 3D-brain organoids that offer new opportunities to investigate splicing regulation.

Background/Objectives: Different risk factors are involved in the initiation and progression of melanoma. In particular, genetic and epigenetic pathways are involved in all stages of melanoma and are exploited in therapeutic approaches. This study investigated the role of circular RNA circ_0001591 in melanoma cell migration. Methods: Three different melanoma cell lines were transfected with siRNA targeting circ_0001591 and with mimic or inhibitor molecules for miR-20a-3p and miR-34a-5p. Gene and protein expression levels were analyzed by RT-qPCR and Western blot, respectively. Dual luciferase reporter assays were performed to confirm the direct interaction of miR-20a-3p and miR-34a-5p with circ_0001591, as well as with the 3’UTRs of AXL (for both miRNAs) and FOSL1 (miR-34a-5p only). Wound healing assays were conducted to assess cell migration velocity. Results: The silencing of circ_0001591 significantly reduces the migration ability of melanoma cell lines. This downregulation was associated with an increased expression of miR-20a-3p and miR-34a-5p. Dual luciferase reporter assays confirmed the direct binding of both miRNAs to circ_0001591, supporting its role as a molecular sponge. The same assays also verified that miR-20a-3p directly targets the 3’UTR of AXL, while miR-34a-5p binds the 3’UTRs of both AXL and FOSL1. Western blot analysis showed that the modulation of this axis affects the expression levels of the AXL and FRA1 oncoproteins. Conclusions: Our findings demonstrate that circ_0001591 promotes melanoma migration by sponging miR-20a-3p and miR-34a-5p, thereby indirectly modulating the expression of AXL and FRA1 oncoprotein. Further investigations of this new regulatory network are needed to better understand its role in melanoma progression and to support the development of targeted therapies.

Previous studies in sports science suggested that regular exercise has a positive impact on human health. However, the effects of endurance sports and their underlying mechanisms are still not completely understood. One of the main debates regards the modulation of immune dynamics in high-intensity exercise. As part of the "Run 4 Science" project in Verona, Italy, we conducted a single-cell RNA sequencing analysis on half-marathon amateur runners to investigate the transcriptional dynamics of peripheral blood mononuclear cells following endurance exercise. Blood samples were collected from four participants before and after running a half-marathon to carry out a comprehensive transcriptomic analysis of immune cells at the single-cell level. Our analysis revealed significant alterations in the transcriptional profiles following endurance physical exercise. Modulations in myeloid cells suggested the activation of stress response (6 related pathways, p < 0.04) and pathways related to viral processes (4 related pathways, p < 0.03), while in lymphoid cells they hinted to a shift towards immune activation (24 related pathways, p < 0.01). Additionally, transcriptional changes in platelets point to an activation of the coagulation process (5 related pathways, p < 0.005). Single-cell data was also analyzed following a pseudo-bulk approach (i.e., simulating a bulk RNAseq experiment) to gain further biological insights. Our findings suggest that a pseudo-bulk analysis could offer complementary findings to classical single-cell analysis methods and demonstrate that endurance physical exercise, such as running a half-marathon, induces substantial changes in the transcriptional dynamics of immune cells. These insights contribute to a better understanding of the immune modulation mediated by endurance exercise and may inform future training routines or nutritional guidelines based on individual gene expression levels.

Background/Objectives: Non-coding microRNA-34a (miR-34a) regulates the expression of key factors involved in several cellular processes, such as differentiation, apoptosis, proliferation, cell cycle, and senescence. Deregulation of the expression of these factors is implicated in the onset and progression of several human diseases, including cancer, neurodegenerative disorders, and pathologies associated with viral infections and inflammation. Despite numerous studies, the molecular mechanisms regulated by miR-34a remain to be fully understood. The present study aimed to generate miR-34a knockout cell lines to identify novel genes potentially regulated by its expression. Methods: We employed the CRISPR-Cas9 gene editing system to knock out the hsa-miR-34a gene in HeLa and 293T cell lines, two widely used models for studying molecular and cellular mechanisms. We compared proliferation rates and gene expression profiles via RNA-seq and qPCR analyses between the wild-type and miR-34a KO cell lines. Results: Knockout of miR-34a resulted in a decreased proliferation rate in both cell lines. Noteworthy, the ablation of miR-34a resulted in increased expression of the long non-coding RNA MALAT1. Additionally, miR-34a-5p silencing in the A375 melanoma cell line led to MALAT1 overexpression. Conclusions: Our findings support the role of the miR-34a/MALAT1 axis in regulating proliferation processes.

Background: Pleural mesothelioma (PM) is a rare and highly aggressive tumor, primarily caused by asbestos exposure. Its long latency period and late-stage diagnosis severely limit therapeutic options. Aim: Recent evidence suggests that non-coding RNAs (ncRNAs)—including microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs)—play key roles in PM biology. This review aims to synthesize current knowledge on ncRNA dysregulation in PM and explore their diagnostic, prognostic, and therapeutic potential. Scope: We summarize studies addressing the expression and function of ncRNAs in tumors and in circulating biofluids of PM patients. Particular attention is given to how ncRNAs regulate proliferation, apoptosis, and migration, and how competing endogenous RNA (ceRNA) networks shape gene regulation in PM. Novelty and Conclusion: Unlike previous reviews, this work integrates findings across different classes of ncRNAs and their interactions, highlighting the emerging concept of ceRNA networks in PM. By bridging molecular mechanisms with potential clinical applications, we provide an updated and comprehensive framework that may inform future strategies for diagnosis, prognosis, and targeted therapy in PM.

Gender disparity in melanoma is a complex issue where sex hormones could be engaged. Differences in genetic variations are important in understanding the mechanisms of sex disparity in melanoma. Post-transcriptional regulation of prostaglandin-endoperoxide synthase (PTGS2) mRNA occurs through a complex interplay of specific trans-acting RNA-binding proteins and microRNAs. MiR-146a is a key player in melanoma, modulating immune responses and tumor microenvironment (TME). Polymorphisms in PTGS2 gene rs20415GC have been associated with an increased risk of melanoma. Epistasis between polymorphisms rs20415GC was investigated by genotyping 453 melanoma patients and 382 control individuals. The effects of testosterone and 17β-estradiol were analyzed in keratinocytes and two melanoma cell lines. The rs2910164GG showed a higher risk in the presence of the genotype rs20417CC in the male population. Testosterone and 17β-estradiol act differently on PTGS2 and miR-146a expression, depending on the cell type. Testosterone augments PTGS2 gene expression in keratinocytes and miR-146a in melanoma cells. While 17β-estradiol only increases miR-146a expression in HaCaT cells. The present study indicates a sex-specific relation between miR-146a and PTGS2 polymorphisms with melanoma cancer risk. Testosterone and 17β-estradiol act differently on the expression of PTGS2 and miR-146a depending on the skin cell type.

Background/Objectives: microRNAs (miRNAs) and circular RNA (circRNAs) show a close interconnection in the control of fundamental functions, such as cell proliferation and tumor development. A full understanding of this complex and interconnected network is essential for better understanding the mechanisms underlying cancer progression. Hsa_circ_0079593 is a circRNA highly expressed in melanoma and is associated with increased metastasis and progression of malignancy, whereas miR516b-5p is a microRNA whose expression is lower in several tumor types, including melanoma; its overexpression inhibits cell proliferation, migration, and invasion. In this study, we tested whether circ_0079593 is involved in the progression of melanoma aggressiveness by regulating CHAF1B and MCAM via the inhibition of miR-516b-5p. Methods: We first verified the expression of the key components in both healthy melanocyte lines and melanoma metastases, subsequently using in vitro assays such as scratch tests, Western blot, qRT-PCR, and dual luciferase report assay; we verified their interconnected regulatory effect. Results: Our results showed that circ_0079593-miR516b-5p interactions are involved in the increase in the migration of metastasis melanoma cells by exploiting their binding to MCAM and CHAF1B mRNAs. Conclusions: This study provides two other regulatory networks in which circ_0079593 may exert its oncogenic function by increasing the speed of movement of metastatic cells through the sponge of miR-516b-5p, which cannot regulate MCAM and CHAF1B expression.

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed.

Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of protein-truncating variants (PTVs), missense variants and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 genes associated with ASD at false discovery rate (FDR) ≤ 0.001 (185 at FDR ≤ 0.05). De novo PTVs, damaging missense variants and CNVs represented 57.5%, 21.1% and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD) (n = 91,605) yielded 373 genes associated with ASD/DD at FDR ≤ 0.001 (664 at FDR ≤ 0.05), some of which differed in relative frequency of mutation between ASD and DD cohorts. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells, whereas genes showing stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophrenia-associated genes, emphasizing that these neuropsychiatric disorders may share common pathways to risk.

We tested the hypothesis that common genetic variability of beta-cell genes responsible for monogenic diabetes may affect beta cell function in type 2 diabetes mellitus (T2DM). We studied 794 drug- naïve GAD-negative patients with newly diagnosed T2DM (age: median=59 years; I.Q. range: 52-66; body mass index: 29.3 kg/m2; 26.6-32.9). Beta-cell function was assessed by state-of-art mathematical modeling of glucose/C-peptide curves during a 240'-300' frequently sampled oral glucose tolerance test, to provide the beta-cell responses to the rate of increase in glucose concentration (derivative control: DC) and to glucose concentration (proportional control: PC). Forty-two single nucleotide polymorphism (SNPs), selected to cover over 90% of common genetic variability, were genotyped in nine monogenic diabetes genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, KCNJ11 and ABCC8. Allelic variants of four SNPs (rs1303722 and rs882019 of GCK, rs7310409 of HNF1A and rs5219 of KCNJ11) were significantly associated with DC of beta-cell secretion (all P < 0.036). Allelic variants of four other SNPs (rs2868094 and rs6031544 of HNF4A, and rs1801262 and rs12053195 of NEUROD1) were associated with PC of beta-cell secretion (P < 0.02). In multivariate models, GCK, HNF1A and KCNJ11 SNPs explained 2.5% of the DC variability of beta-cell secretion, whereas HNF4A and NEUROD1 SNPs explained 3.6% of the PC variability of beta-cell secretion. We conclude that common variability of monogenic diabetes genes is significantly associated with an impaired beta-cell function in patients with newly diagnosed T2DM; thereby, these genes might be targeted by specific treatments in T2DM.

Genome-wide association studies have identified numerous genetic variants for type 2 diabetes (T2D).Most genetic loci discovered to date were studied in Caucasians or Asian ancestry, however, there are no data regarding a quite large Italian sample.Therefore, we investigated T2D genetic susceptibility of 143 single nucleotide polymorphisms (SNPs) within 30 genes involved in glucose metabolism in a large Italian case-control study.For the study, 1875 Caucasian patients were selected from three Italian cohorts.Age, gender, BMI and fasting plasma glucose (FPG) values were collected.Population was split in cases and controls based on FPG values or T2D diagnosis.T2D subjects and whom with FPG higher that 126 mg/dL were recruited as cases whereas subjects with normal values of FPG were considered controls.In each subject 143 SNPs were genotyped.To evaluate the association between genetic variations and diabetes status, a logistic regression analysis, adjusted for age, sex and BMI, was performed.Overall, 948 (50.6%) had T2D.Twenty out of 143 variants within 11 different genes resulted significantly associated to T2D.Four of them were located into TCF7L2 gene and presented the highest odd ratio (from 1.42 to 1.57).At least two SNPs were located within KCNJ11, WFS1, ABCC8, JAZF1 and HNF1B genes and one SNP each was identified in ADAMTS9,

A Correction to this paper has been published: https://doi.org/10.1038/s41588-021-00832-z.

ABSTRACTIndividuals with autism spectrum disorder (ASD) or related neurodevelopmental disorders (NDDs) often carry disruptive mutations in genes that are depleted of functional variation in the broader population. We build upon this observation and exome sequencing from 154,842 individuals to explore the allelic diversity of rare protein-coding variation contributing risk for ASD and related NDDs. Using an integrative statistical model, we jointly analyzed rare protein-truncating variants (PTVs), damaging missense variants, and copy number variants (CNVs) derived from exome sequencing of 63,237 individuals from ASD cohorts. We discovered 71 genes associated with ASD at a false discovery rate (FDR) ≤ 0.001, a threshold approximately equivalent to exome-wide significance, and 183 genes at FDR ≤ 0.05. Associations were predominantly driven by de novo PTVs, damaging missense variants, and CNVs: 57.4%, 21.2%, and 8.32% of evidence, respectively. Though fewer in number, CNVs conferred greater relative risk than PTVs, and repeat-mediated de novo CNVs exhibited strong maternal bias in parent-of-origin (e.g., 92.3% of 16p11.2 CNVs), whereas all other CNVs showed a paternal bias. To explore how genes associated with ASD and NDD overlap or differ, we analyzed our ASD cohort alongside a developmental delay (DD) cohort from the deciphering developmental disorders study (DDD; n=91,605 samples). We first reanalyzed the DDD dataset using the same models as the ASD cohorts, then performed joint analyses of both cohorts and identified 373 genes contributing to NDD risk at FDR ≤ 0.001 and 662 NDD risk genes at FDR ≤ 0.05. Of these NDD risk genes, 54 genes (125 genes at FDR ≤ 0.05) were unique to the joint analyses and not significant in either cohort alone. Our results confirm overlap of most ASD and DD risk genes, although many differ significantly in frequency of mutation. Analyses of single-cell transcriptome datasets showed that genes associated predominantly with DD were strongly enriched for earlier neurodevelopmental cell types, whereas genes displaying stronger evidence for association in ASD cohorts were more enriched for maturing neurons. The ASD risk genes were also enriched for genes associated with schizophrenia from a separate rare coding variant analysis of 121,570 individuals, emphasizing that these neuropsychiatric disorders share common pathways to risk.

Elevated fasting plasma glucose has been associated with increased risk for development of type 2 diabetes (T2D). The balance between glucokinase (GCK) and glucose-6-phosphate catalytic subunit 2 (G6PC2) activity are involved in glucose homeostasis through glycolytic flux, and subsequent insulin secretion. In this study, we evaluated the association between the genetic variability of G6PC2 and GCK genes and T2D-related quantitative traits. In 794 drug-naïve, GADA-negative, newly diagnosed T2D patients (VNDS; NTC01526720) we performed: genotyping of 6 independent tag-SNPs within GCK gene and 5 tag-SNPs within G6PC2 gene; euglycaemic insulin clamp to assess insulin sensitivity; OGTT to estimate beta-cell function (derivative and proportional control; DC, PC) by mathematical modeling. Genetic association analysis has been conducted using Plink software. Two SNPs within GCK gene (rs882019 and rs1303722) were associated to DC in opposite way (both p < 0.004). Two G6PC2 variants (rs13387347 and rs560887) were associated to both parameters of insulin secretion (DC and PC) and to fasting C-peptide levels (all p < 0.038). Moreover, subjects carrying the A allele of rs560887 showed higher values of 2h-plasma glucose (2hPG) (p = 0.033). Haplotype analysis revealed that GCK (AACAAA) haplotype was associated to decreased fasting C-peptide levels, whereas, the most frequent haplotype of G6PC2 (GGAAG) was associated with higher fasting C-peptide levels (p = 0.001), higher PC (β = 6.87, p = 0.022) and the lower 2hPG (p = 0.012). Our findings confirmed the role of GCK and G6PC2 in regulating the pulsatility in insulin secretion thereby influencing insulin-signaling and leading to a gradual modulation in glucose levels in Italian patients with newly diagnosed T2D.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies.

Notwithstanding several research efforts in the past years, robust and replicable molecular signatures for autism spectrum disorders from peripheral blood remain elusive. The available literature on blood transcriptome in ASD suggests that through accurate experimental design it is possible to extract important information on the disease pathophysiology at the peripheral level. Here we exploit the availability of a resource for molecular biomarkers in ASD, the Italian Autism Network (ITAN) collection, for the investigation of transcriptomic signatures in ASD based on a discordant sibling pair design. Whole blood samples from 75 discordant sibling pairs selected from the ITAN network where submitted to RNASeq analysis and data analyzed by complementary approaches. Overall, differences in gene expression between affected and unaffected siblings were small. In order to assess the contribution of differences in the relative proportion of blood cells between discordant siblings, we have applied two different cell deconvolution algorithms, showing that the observed molecular signatures mainly reflect changes in peripheral blood immune cell composition, in particular NK cells. The results obtained by the cell deconvolution approach are supported by the analysis performed by WGCNA. Our report describes the largest differential gene expression profiling in peripheral blood of ASD subjects and controls conducted by RNASeq. The observed signatures are consistent with the hypothesis of immune alterations in autism and an increased risk of developing autism in subjects exposed to prenatal infections or stress. Our study also points to a potential role of NMUR1, HMGB3, and PTPRN2 in ASD.

The risk of developing adult T-cell leukemia/lymphoma (ATLL) in individuals infected with human T-cell lymphotropic virus 1 (HTLV-1) is about 3–5%. The mechanisms by which the virus triggers this aggressive cancer are still an area of intensive investigation. The viral protein Tax-1, together with additional regulatory proteins, in particular HTLV-1 basic leucine zipper factor (HBZ), are recognized as relevant viral factors required for both viral replication and transformation of infected cells. Tax-1 deregulates several cellular pathways affecting the cell cycle, survival, and proliferation. The effects of Tax-1 on the NF-κB pathway have been thoroughly studied. Recent studies also revealed the impact of Tax-1 and HBZ on microRNA expression. In this review, we summarize the recent progress in understanding the contribution of HTLV-1 Tax- and HBZ-mediated deregulation of NF-κB and the microRNA regulatory network to HTLV-1 pathogenesis.