Autism spectrum disorder (ASD) and related neurodevelopmental disorders (NDDs) are characterized by a complex genetic architecture involving both rare high-impact variants and cumulative low-effect alterations. The contribution of borderline copy number variants (CNVs) to disease susceptibility and phenotypic variability remains incompletely understood. Reflecting a real-world clinical setting rather than a prospectively recruited research cohort, we performed a retrospective analysis of a tertiary care registry comprising 328 individuals referred for suspected ASD or NDDs who underwent array comparative genomic hybridization (a-CGH), identifying 612 CNVs classified according to ACMG/ClinGen guidelines. CNVs were evaluated by type, size, genomic distribution, and clinical correlation, and patients were stratified based on CNV burden and the presence of borderline variants. Duplications were more frequent than deletions, and medium-sized CNVs (100-500 kb) were the most common. Borderline CNVs represented the largest category. Within this descriptive cohort, increased CNV burden, in both number and cumulative genomic size, co-occurred more frequently in individuals with severe phenotypes, including ASD with intellectual disability, epilepsy, and broader NDDs. Individuals with isolated ASD showed a lower burden and enrichment of small borderline variants, which often co-occurred alongside CNVs. In contrast, severe phenotypes were frequently associated with single large pathogenic variants. Recurrent loci included 15q11.2-q13, 16p11.2, and 22q11.21. Overall, these exploratory observations from a real-world clinical registry illustrate how cumulative genomic burden and borderline CNVs co-occur across different neurodevelopmental presentations, providing descriptive context for future mechanistic studies.

Objectives: Adenoviruses (Ads) are among the most used vectors for gene therapy; human Ad serotype 5-derived (HuAd5) vectors are the most frequently used for gene transfer applications. However, Ad5 infection is endemic in humans, and 20% of the Western population has neutralizing antibodies (nAbs). Pre-existing immunity against HuAd5 represents a major issue for many gene therapy applications. In our study, we evaluated several Ad serotypes derived from chimpanzees (ChAds) in vitro and in vivo to assess their transduction efficiency in various cell types and tissues. We aimed at identifying Ad serotypes able either to transduce "challenging" cell types or to represent a possible alternative to Ad5-derived vectors with comparable infectivity and tropism. Methods: We evaluated the efficacy of transduction of twelve ChAds vectors expressing enhanced green fluorescent protein (EGFP) in human embryonic kidney cells, as well as human leukemic and human mesenchymal stem cells, using flow cytometry to determine the percentage of EGFP-expressing cells and their mean fluorescent intensity (MFI). We observed the highest transduction efficiency in the serotype CV1 ChAd; therefore, we proceeded to evaluate toxicity and biodistribution in vivo. Results: After in vitro evaluation of twelve ChAds serotypes, we observed that the CV1 serotype was the most efficient in transducing both leukemia cell lines (HL-60 and NB-4) and human mesenchymal stem cells. Furthermore, in vivo analysis of the CV1 serotype induced an inflammatory reaction similar to what was observed after HuAd5 administration. Conclusions: ChAds vectors represent an effective alternative for the transduction of cells resistant to HuAd5 infection, such as mesenchymal stem cells and leukemic cells. In addition, we observed that the CV1 ChAd serotype presented a transduction profile similar to HuAd5 in vitro and induced a similar inflammatory response in vivo; therefore, CV1 ChAd-derived vectors represent an interesting alternative for gene therapy applications.

Familial hypercholesterolemia (FH) is a genetic disorder caused by mutations in the low-density lipoprotein (LDL) receptor, leading to impaired uptake of LDL and its accumulation in arterial walls and other tissues. This accumulation results in cardiovascular disease and early mortality. Treatments, including statins, ezetimibe, PCSK9 inhibitors, and bempedoic acid, are often insufficient in homozygous FH patients, particularly those with null mutations in the LDL receptor (LDLR). To address this unmet need, we have developed two helper-dependent adenoviral (HD-Ad) vectors for the expression of the murine and human versions of a protein composed of the extracellular portion of the LDLR fused to transferrin. In both cassettes, expression is driven by the murine creatine kinase promoter to obtain high levels of expression restricted to muscle cells, to mitigate host response to the fusion protein. Both human and murine proteins restored LDL uptake in Ldlrf-deficient cells, correcting the phenotype in vitro. A single intramuscular administration of the HD-Ad vector induced the expression of the murine fusion protein, leading to a 12-month improvement in the lipid profile, with a reduction in aortic atherosclerosis in Ldlr-deficient mice. Furthermore, we observed no major systemic toxicity, indicating that the present strategy may represent more effective therapy for FH patients.

IntroductionGrowing evidence from microbiome studies has demonstrated associations between dysbiosis and cancers, including bladder cancer (BCa). Our recent works on urobiome revealed a different microbial composition in BCa patients compared to controls. The aim of this work was to create a Weighted Composite Index (WCI) to distinguish BCa-affected patients (mBCa) from healthy controls (mHC) in a cohort of male aged over 50 years.MethodsUrobiome data from 51 subjects (27 mBCa and 24 mHC) were analyzed. Random Forest (RF) classifier was trained to identify genera and species which significantly contributed to discriminating between mBCa and mHC group. A weighted normalization approach was used to compute separate WCIs at genus and species levels and in-silico validation test were performed to assess the models’ robustness.Resultsthe WCI was calculated for each patient at both genera and species levels, showing a significant difference between the two groups (p < 0.0001) in both comparisons. WCIs showed superior discriminative performances compared to any individual taxon used for the model construction. Despite the need for validation in larger independent cohorts, the in-silico validation pipeline showed a stable high sensitivity of the models.ConclusionsOur findings identified a candidate urinary microbial signature in a biomarker discovery setting associated with bladder cancer. This hypothesis-generating approach may contribute to the identification of a non-invasive biomarker, which requires validation in larger, independent cohorts before clinical application.

Background: Neurodevelopmental disorders (NDDs) are a heterogeneous group of conditions characterized by cognitive, behavioral, and developmental impairments, frequently linked to structural genomic alterations. Copy number variants (CNVs) involving chromosome 16, particularly the short arm 16p, are recognized contributors to neurodevelopmental variability. Despite increasing international evidence, data from Italian clinical cohorts are still limited. Methods: We investigated 1200 patients referred for genetic evaluation due to suspected NDDs, including autism spectrum disorder (ASD), intellectual disability (ID), global developmental delay, and language impairment. All individuals underwent array comparative genomic hybridization (a-CGH) analysis, and identified variants were correlated with detailed clinical, cognitive, and behavioral assessments. The analysis focused on recurrent CNVs at 16p11.2, 16p13.3, and 16p13.11, regions containing dosage-sensitive genes relevant to neurodevelopment. Results: CNVs involving the 16p region were identified in 96 patients (8% of the cohort), encompassing both deletions and duplications. Deletions were mainly associated with developmental delay, language deficits, and ASD-related features, whereas duplications were more frequently linked to behavioral dysregulation, attentional deficits, and variable cognitive impairment. Marked phenotypic variability was observed among individuals carrying similar CNVs, suggesting the contribution of modifying genetic or environmental factors. In a subset of patients, additional CNVs were identified, potentially exacerbating clinical severity, consistent with the two-hit model. Conclusions: This study confirms a strong association between recurrent 16p CNVs and a wide spectrum of neurodevelopmental phenotypes in an Italian clinical cohort. The findings emphasize the diagnostic utility of systematic genomic screening and the importance of an integrated genotype-phenotype approach to improve clinical interpretation, management, and genetic counseling in NDDs.

Schizophrenia (SCZ) is a complex mental disorder, whose pathogenesis involves both environmental and genetic factors. Genetic risk is conferred through a combination of common variants and rare mutations, with point mutations and copy number variants (CNVs). Many of the genetic variants associated with SCZ have pleiotropic effects, influencing brain development and being shared with other neurodevelopmental disorders (NDDs), such as intellectual disability (ID). This overlap supports the concept of a neurodevelopmental continuum, suggesting shared genetic risk, at least between SCZ and ID, and most presumably among SCZ and many other NDDs. Here, we describe the case of a male patient whose clinical features align with this hypothesis. He presented cognitive and behavioral impairments preceding psychotic symptoms, further reinforcing the genetic and clinical interaction between SCZ and other NDDs. The patient’s genetic profile was analyzed using array comparative genomic hybridization (a-CGH) and whole-exome sequencing (WES) to investigate the genetic determinants underlying his clinical condition. The genetic testing identified variants in loci associated with both SCZ and NDDs. Our findings highlight the need to integrate genetic assessments into psychiatrists’ clinical practice. Moreover, this report contributes to the current body of evidence supporting the thesis on the neurodevelopmental continuum of SCZ.

Over the last decade, immunotherapy has revolutionized lung cancer treatments, particularly in non-small cell lung cancer, where immune checkpoint inhibitors have achieved significant clinical success. However, high percentages of patients do not respond initially or eventually develop a resistance to these therapies. This review explores the evolution and challenges of immunotherapy in lung cancer, highlighting its clinical milestones and intrinsic and extrinsic resistance mechanisms. We investigate tumor-intrinsic resistance factors, including alterations in antigen presentation, the loss of Beta-2 microglobulin function, impaired interferon signaling, immune editing, epigenetic modifications, and tumor-extrinsic resistance, such as an immunosuppressive lung tumor microenvironment, dysregulated cytokine profiles, and the upregulation of immune checkpoints. Then, we focus on the emerging role of resistance biomarkers and the development of personalized treatment strategies to overcome these challenges. The complex interplay between tumor biology and immune modulation in lung cancer paves the way for novel approaches for improving the effectiveness of immunotherapeutic treatments.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that first develops in early childhood and is characterized by restricted interests, activities, and behaviors, as well as difficulties with social interactions and communication. ASD arises from a complex interaction between environmental factors and genetic inheritance, influenced by epigenetic mechanisms. With an estimated heritability of 70–90%, ASD is highly familial, indicating that genetic factors play a significant role in its development. This shows how hundreds of genetic variants contribute to ASD, whose risk effects are highly variable and are often related to other conditions; these genetic alterations are at different levels, which include single gene mutations, monogenic disorders, genomic variants, and chromosomal abnormalities. Copy number variants (CNVs) appear to contribute significantly to understanding the pathogenesis of this complex disease. In some cases, single CNVs in genomic DNA are pathogenic and causative, supporting the hypothesis that some sporadic cases of ASD may result from rare mutations with significant clinical impact. However, in many cases, there are common genomic variants that increase the risk of developing ASD but are insufficient by themselves to determine an ASD phenotype, and rare genomic variants, of various sizes, inherited from a parent or de novo, that can be associated with the ASD phenotype. Therefore, the aim of this review is to deepen the concept of ASD inheritance through the two-hit theory of CNVs, in which the concomitant presence of two alterations could determine the clinical phenotypes, the concept of incomplete penetrance for inherited CNVs with pathogenic clinical significance, and the presence of compound heterozygosity. These aspects represent important mechanisms underlying the pathogenesis of autism, contributing to a better elucidation for the understanding of the genetic contribution to the ASD phenotype.

Cancer immunotherapy requires a specific antitumor CD8+ T cell-driven immune response; however, upon genetic and epigenetic alterations of the antigen processing and presenting components, cancer cells escape the CD8+ T cell recognition. As a result, poorly immunogenic tumors are refractory to conventional immunotherapy. In this context, the use of viral cancer vaccines in combination with hypomethylating agents represents a promising strategy to prevent cancer from escaping immune system recognition. In this study, we evaluated the sensitivity of melanoma (B16-expressing ovalbumin) and metastatic triple-negative breast cancer (4T1) cell lines to FDA-approved low-dose decitabine in combination with PeptiCRAd, an adenoviral anticancer vaccine. The two models showed different sensitivity to decitabine in vitro and in vivo when combined with PeptiCRAd. In particular, mice bearing syngereceiving the combinatorial treatment compared to single controls in association with a higher expression of MHC class I on cancer cells and reduction in Tregs within the tumor microenvironment. Furthermore, remodeling of the CD8+ T cell infilthe effect of decitabine in enhancing anticancer vaccines in immunotherapy regimens.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a threat to public health and the global economy, necessitating the development of various vaccination strategies. Mutations in the SPIKE protein gene, a crucial component of mRNA and adenovirus-based vaccines, raised concerns about vaccine efficacy, prompting the need for rapid vaccine updates. To address this, we leveraged PeptiCRAd, an oncolytic vaccine based on tumor antigen decorated oncolytic adenoviruses, creating a vaccine platform called PeptiVAX. First, we identified multiple CD8 T-cell epitopes from highly conserved regions across coronaviruses, expanding the range of T-cell responses to non-SPIKE proteins. We designed short segments containing the predicted epitopes presented by common HLA-Is in the global population. Testing the immunogenicity, we characterized T-cell responses to candidate peptides in peripheral blood mononuclear cells (PBMCs) from pre-pandemic healthy donors and ICU patients. As a proof of concept in mice, we selected a peptide with epitopes predicted to bind to murine MHC-I haplotypes. Our technology successfully elicited peptide-specific T-cell responses, unaffected by the use of unarmed adenoviral vectors or adeno-based vaccines encoding SPIKE. In conclusion, PeptiVAX represents a fast and adaptable SARS-CoV-2 vaccine delivery system that broadens T-cell responses beyond the SPIKE protein, offering potential benefits for vaccine effectiveness.

IntroductionSchizophrenia (SCZ) and autism spectrum disorder (ASD) are neurodevelopmental diseases characterized by different psychopathological manifestations and divergent clinical trajectories. Various alterations at glutamatergic synapses have been reported in both disorders, including abnormal NMDA and metabotropic receptor signaling.MethodsWe conducted a bicentric study to assess the blood serum levels of NMDA receptors-related glutamatergic amino acids and their precursors, including L-glutamate, L-glutamine, D-aspartate, L-aspartate, L-asparagine, D-serine, L-serine and glycine, in ASD, SCZ patients and their respective control subjects. Specifically, the SCZ patients were subdivided into treatment-resistant and non-treatment-resistant SCZ patients, based on their responsivity to conventional antipsychotics.ResultsD-serine and D-aspartate serum reductions were found in SCZ patients compared to controls. Conversely, no significant differences between cases and controls were found in amino acid concentrations in the two ASD cohorts analyzed.DiscussionThis result further encourages future research to evaluate the predictive role of selected D-amino acids as peripheral markers for SCZ pathophysiology and diagnosis.

Peptide Nucleic Acids (PNAs) represent a promising tool for gene modulation in anticancer treatment. The uncharged peptidyl backbone and the resistance to chemical and enzymatic degradation make PNAs highly advantageous to form stable hybrid complexes with complementary DNA and RNA strands, providing higher stability than the corresponding natural analogues. Our and other groups’ research has successfully shown that tailored PNA sequences can effectively downregulate the expression of human oncogenes using antigene, antisense, or anti-miRNA approaches. Specifically, we identified a seven bases-long PNA sequence, complementary to the longer loop of the main G-quadruplex structure formed by the bcl2midG4 promoter sequence, capable of downregulating the expression of the antiapoptotic Bcl-2 protein and enhancing the anticancer activity of an oncolytic adenovirus. Here, we extended the length of the PNA probe with the aim of including the double-stranded Bcl-2 promoter among the targets of the PNA probe. Our investigation primarily focused on the structural aspects of the resulting DNA2-PNA heterotriplex that were determined by employing conventional and accelerated microsecond-scale molecular dynamics simulations and chemical-physical analysis. Additionally, we conducted preliminary biological experiments using cytotoxicity assays on human A549 and MDA-MB-436 adenocarcinoma cell lines, employing the oncolytic adenovirus delivery strategy.

Neurodevelopmental disorders are a group of complex multifactorial disorders characterized by cognitive impairment, communication deficits, abnormal behaviour, and/or motor skills resulting from abnormal neural development. Copy number variants (CNVs) are genetic alterations often associated with neurodevelopmental disorders. We evaluated the diagnostic efficacy of the array-comparative genomic hybridization (a-CGH) method and its relevance as a routine diagnostic test in patients with neurodevelopmental disorders for the identification of the molecular alterations underlying or contributing to the clinical manifestations. In the present study, we analysed 1800 subjects with neurodevelopmental disorders using a CGH microarray. We identified 208 (7%) pathogenetic CNVs, 2202 (78%) variants of uncertain significance (VOUS), and 504 (18%) benign CNVs in the 1800 patients analysed. Some alterations contain genes potentially related to neurodevelopmental disorders including CHRNA7, ANKS1B, ANKRD11, RBFOX1, ASTN2, GABRG3, SHANK2, KIF1A SETBP1, SNTG2, CTNNA2, TOP3B, CNTN4, CNTN5, and CNTN6. The identification of interesting significant genes related to neurological disorders with a-CGH is therefore an essential step in the diagnostic procedure, allowing a better understanding of both the pathophysiology of these disorders and the mechanisms underlying their clinical manifestations.

To date, the increased awareness of the impact of microbes on human health has promoted scientific interest in microbiome studies for diagnostic and therapeutic purposes, revealing correlations between specific taxa and cancer. In particular, numerous species of Porphyromonas have been associated with several types of tumors. Previously, we studied the urobiome using Next-Generation Sequencing (NGS), and found an increase in Porphyromonas somerae in first morning urine of subjects affected by bladder cancer (BCa). Here, we aimed to confirm the presence of P. somerae in BCa patients by using droplet digital Polymerase Chain Reaction (ddPCR), testing a cohort of 102 male subjects over 50 years. Our findings showed a significant increase in P. somerae in the urine of the BCa group within both ddPCR and NGS, and a correlation between the two methods was observed at a statistical level. Moreover, P. somerae’s identification with ddPCR confirmed a significant association between this bacterium and the presence of BCa, highlighting its potential role as a biomarker. This allows us to propose the ddPCR as a suitable method for first-stage BCa screening and follow-up.

Supplementary Results, Methods, Table 1, Figures 1-4 from A Placental Growth Factor Variant Unable to Recognize Vascular Endothelial Growth Factor (VEGF) Receptor-1 Inhibits VEGF-Dependent Tumor Angiogenesis via Heterodimerization

Infertility incidence is rising worldwide, with male infertility accounting for about 50% of cases. To date, several factors have been associated with male infertility; in particular, it has been suggested that semen microbiota may play a role. Here, we report the NGS-based analyses of 20 semen samples collected from men with (Case) and without (Control) semen alterations. Genomic DNA was extracted from each collected sample, and a specific PCR was carried out to amplify the V4-V6 regions of the 16S rRNA. Sequence reactions were carried out on the MiSeq and analyzed by specific bioinformatic tools. We found a reduced richness and evenness in the Case versus the Control group. Moreover, specific genera, the Mannheimia, the Escherichia_Shigella, and the Varibaculum, were significantly increased in the Case compared to the Control group. Finally, we highlighted a correlation between the microbial profile and semen hyperviscosity. Even if further studies are required on larger groups of subjects to confirm these findings and explore mechanistic hypotheses, our results confirm the correlation between semen features and seminal microbiota. These data, in turn, may open the way to the possible use of semen microbiota as an attractive target for developing novel strategies for infertility management.