Life Sciences
All scientific publications in Area Science Park
Ultrasensitive detection and quantification of bovine Deltapapillomavirus in the semen of healthy horses.
Abstract BPV1, BPV2, BPV13, and BPV14 are all genotypes of bovine delta papillomaviruses (δPV), of which the first three cause infections in horses and are associated with equine sarcoids. However, BPV14 infection has never been reported in equine species. In this study, we examined 58 fresh and thawed commercial semen samples from healthy stallions. In 34 (58.6%), bovine δPV DNA was detected and quantified using droplet digital polymerase chain reaction (ddPCR). Real time quantitative PCR (qPCR) was able to identify bovine δPV DNA in 5 samples (8.6%). Of the BPV-infected semen samples, 15 were positive for BPV2 (~ 44.1%) on ddPCR and 4 (~ 11.7%) on qPCR; 12 (~ 35.3%) for BPV14 on ddPCR and 1 (~ 3%) by qPCR; 4 (~ 11.7%) for BPV1 on ddPCR, whereas qPCR failed to reveal this infection; 3 (~ 8.8%) for BPV13 on ddPCR; and BPV13 infection was not detected by qPCR. Our study showed for the first time that BPV14 is an additional infectious agent potentially responsible for infection in horses, as its transcripts were detected and quantified in some semen samples. Large-scale BPV14 screening is necessary to provide substantial data on the molecular epidemiology for a better understanding of the geographical divergence of BPV14 prevalence in different areas and how widespread BPV14 is among equids. Authors Anna Cutarelli, Francesca De Falco, Francesco Serpe, Simona Izzo, Giovanna Fusco, Cornel Catoi, Sante Roperto Journal Scientific Report Publication date 04/01/2025 Consult the publication
Limiting serine availability during tumor progression promotes muscle wasting in cancer cachexia
Abstract Cancer cachexia is a multifactorial syndrome characterized by a progressive loss of body weight occurring in about 80% of cancer patients, frequently representing the leading cause of death. Dietary intervention is emerging as a promising therapeutic strategy to counteract cancer-induced wasting. Serine is the second most-consumed amino acid (AA) by cancer cells and has emerged to be strictly necessary to preserve skeletal muscle structure and functionality. Here, we demonstrate that decreased serine availability during tumor progression promotes myotubes diameter reduction in vitro and induces muscle wasting in in vivo mice models. By investigating the metabolic crosstalk between colorectal cancer cells and muscle cells, we found that incubating myotubes with conditioned media from tumor cells relying on exogenous serine consumption triggers pronounced myotubes diameter reduction. Accordingly, culturing myotubes in a serine-free medium induces fibers width reduction and suppresses the activation of the AKT-mTORC1 pathway with consequent impairment in protein synthesis, increased protein degradation, and enhanced expression of the muscle atrophy-related genes Atrogin1 and MuRF1. In addition, serine-starved conditions affect myoblast differentiation and mitochondrial oxidative metabolism, finally inducing oxidative stress in myotubes. Consistently, serine dietary deprivation strongly strengthens cancer-associated weight loss and muscle atrophy in mice models. These findings uncover serine consumption by tumor cells as a previously undisclosed driver in cancer cachexia, opening new routes for possible therapeutic approaches. Authors Erica Pranzini, Livio Muccillo, Ilaria Nesi, Alice Santi, Caterina Mancini, Giulia Lori, Massimo Genovese, Tiziano Lottini, Giuseppina Comito, Anna Caselli, Annarosa Arcangeli, Lina Sabatino, Vittorio Colantuoni, Maria Letizia Taddei, Paolo Cirri, Paolo Paoli Journal Cell Death Discovery Publication date 21/12/2024 Consult the publication
Molecular simulations to investigate the impact of N6-methylation in RNA recognition: Improving accuracy and precision of binding free energy prediction
Abstract N6-Methyladenosine (m6A) is a prevalent RNA post-transcriptional modification that plays crucial roles in RNA stability, structural dynamics, and interactions with proteins. The YT521-B (YTH) family of proteins, which are notable m6A readers, functions through its highly conserved YTH domain. Recent structural investigations and molecular dynamics (MD) simulations have shed light on the mechanism of recognition of m6A by the YTHDC1 protein. Despite advancements, using MD to predict the stabilization induced by m6A on the free energy of binding between RNA and YTH proteins remains challenging due to inaccuracy of the employed force field and limited sampling. For instance, simulations often fail to sufficiently capture the hydration dynamics of the binding pocket. This study addresses these challenges through an innovative methodology that integrates metadynamics, alchemical simulations, and force-field refinement. Importantly, our research identifies hydration of the binding pocket as giving only a minor contribution to the binding free energy and emphasizes the critical importance of precisely tuning force-field parameters to experimental data. By employing a fitting strategy built on alchemical calculations, we refine the m6A partial charge parameters, thereby enabling the simultaneous reproduction of N6 methylation on both the protein binding free energy and the thermodynamic stability of nine RNA duplexes. Our findings underscore the sensitivity of binding free energies to partial charges, highlighting the necessity for thorough parametrization and validation against experimental observations across a range of structural contexts. Authors Valerio Piomponi, Miroslav Krepl, Jiri Sponer, Giovanni Bussi Journal The Journal of Physical Chemistry B, Vol 128, Issue 37 Publication Date 06/09/2024 Consult the paper
Probing conformational dynamics of EGFR mutants via SEIRA spectroscopy: potential implications for tyrosine kinase inhibitor design
Abstract Missense mutations in EGFR’s catalytic domain alter its function, promoting cancer. SEIRA spectroscopy, supported by MD simulations, reveals structural differences in the compactness and hydration of helical motifs between active and inactive EGFR conformations models. These findings provide novel insights into the biophysical mechanisms driving EGFR activation and drug resistance, offering a robust method for studying emerging EGFR mutations and their structural impacts on TKIs efficacy. Authors Emiliano Laudadio, Federica Piccirilli, Henrick Vondracek, Giovanna Mobbili, Marta Stefania Semrau, Paola Storici, Roberta Galeazzi, Elena Romagnoli, Leonardo Sorci, Andrea Toma, Vincenzo Aglieri, Giovanni Birarda, Cristina Minnelli Journal Physical Chemistry Chemical Physics (PCCP) Publication Date 19/08/2024 Consult the publication
Molecular findings and virological assessment of bladder papillomavirus infection in cattle.
Abstract Bovine and ovine papillomaviruses (BPVs – OaPVs) are infectious agents that have an important role in bladder carcinogenesis of cattle. In an attempt to better understand territorial prevalence of papillomavirus genotypes and gain insights into their molecular pathway(s), a virological assessment of papillomavirus infection was performed on 52 bladder tumors in cattle using droplet digital polymerase chain reaction (ddPCR), an improved version of conventional PCR. ddPCR detected and quantified BPV DNA and mRNAs in all tumor samples, showing that these viruses play a determinant role in bovine bladder carcinogenesis. OaPV DNA and mRNA were detected and quantified in 45 bladder tumors. BPV14, BPV13, BPV2, OaPV2, OaPV1, and OaPV3 were the genotypes most closely related to bladder tumors. ddPCR quantified BPV1 and OaPV4 DNA and their transcripts less frequently. Western blot analysis revealed a significant overexpression of the phosphorylated platelet derived growth factor β receptor (PDGFβR) as well as the transcription factor E2F3, which modulate cell cycle progression in urothelial neoplasia. Furthermore, significant overexpression of calpain1, a Cys protease, was observed in bladder tumors related to BPVs alone and in BPV and OaPV coinfection. Calpain1 has been shown to play a role in producing free transcription factors of the E2F family, and molecular findings suggest that calpain family members work cooperatively to mutually regulate their protease activities in cattle bladder tumors. Altogether, these results showed territorial prevalence of BPV and OaPV genotypes and suggested that PDGFβR and the calpain system appeared to be molecular partners of both BPVs and OaPVs. Authors Francesca De Falco, Anna Cutarelli, Francesca Luisa Fedele, Cornel Catoi, Sante Roperto Journal Veterinary Quarterly Publication date 04/08/2024 Consult the publication
Enhancing predictions of protein stability changes induced by single mutations using MSA-based language models
Abstract Protein language models offer a new perspective for addressing challenges in structural biology, while relying solely on sequence information. Recent studies have investigated their effectiveness in forecasting shifts in thermodynamic stability caused by single amino acid mutations, a task known for its complexity due to the sparse availability of data, constrained by experimental limitations. To tackle this problem, we introduce two key novelties: leveraging a protein language model that incorporates Multiple Sequence Alignments to capture evolutionary information, and using a recently released mega-scale dataset with rigorous data preprocessing to mitigate overfitting. Authors Francesca Cuturello, Marco Celoria, Alessio Ansuini, Alberto Cazzaniga Journal Bioinformatics, 2024, 40 (7) Consult the paper
Molecular detection of transcriptionally active ovine papillomaviruses in commercial equine semen
Abstract Virological evaluation was performed on equine semen to detect the presence of papillomaviruses (PVs) using droplet digital polymerase chain reaction (ddPCR) as the aim of this study was to investigate whether the sperm from asymptomatic stallions harbors ovine papillomaviruses (OaPVs). Twenty-seven semen samples were analyzed, 18 of which were commercially acquired. The remaining nine samples comprising semen and peripheral blood, were collected from nine stallions with no apparent signs of PV-related diseases during clinical examination at the Didactic Veterinary University Hospital (DVUH) of Naples. OaPV was detected in 26 semen samples. OaPV1 was the most prevalent virus infecting equine semen. OaPV1 infected 21 semen samples (~80.8%) and showed a high number of DNA and RNA copies per microliter. qPCR was used to detect OaPV1 DNA in the 18 semen samples. ddPCR was used to detect and quantify the expression of OaPV2, OaPV3, and OaPV4. qPCR failed to detect DNA for these genotypes. Additionally, ddPCR was used to detect the transcriptionally active OaPV1 in six blood and semen samples from the same stallion. ddPCR failed to detect any nucleic acids in OaPVs in peripheral blood samples from the three stallions. In one semen sample, ddPCR detected OaPV1 DNA but failed to detect any nucleic acid in the remaining two semen samples, and peripheral blood from the same animals of the remaining 18 semen samples was not available, OaPV1 and OaPV4 were responsible for nine and five single infections, respectively. No single infections with either OaPV3 or OaPV4 were seen. Authors Anna Cutarelli, Francesca De Falco, Roberta Brunetti, Michele Napoletano, Giovanna Fusco, Sante Roperto Journal Frontiers in Veterinary Science Publication Date 03/07/2024 Consult the publication
Genome-wide DNA methylation and transcriptomic analysis of liver tissues subjected to early ischemia/reperfusion injury upon human liver transplantation
Abstract Introduction and Objectives Epigenetic changes represent a mechanism connecting external stresses with long-term modifications of gene expression programs. In solid organ transplantation, ischemia-reperfusion injury (IRI) appears to induce epigenomic changes in the graft, although the currently available data are extremely limited. The present study aimed to characterize variations in DNA methylation and their effects on the transcriptome in liver transplantation from brain-dead donors. Patients and Methods 12 liver grafts were evaluated through serial biopsies at different timings in the procurement-transplantation process: T0 (warm procurement, in donor), T1 (bench surgery), and T2 (after reperfusion, in recipient). DNA methylation (DNAm) and transcriptome profiles of biopsies were analyzed using microarrays and RNAseq. Results Significant variations in DNAm were identified, particularly between T2 and T0. Functional enrichment of the best 1000 ranked differentially methylated promoters demonstrated that 387 hypermethylated and 613 hypomethylated promoters were involved in spliceosomal assembly and response to biotic stimuli, and inflammatory immune responses, respectively. At the transcriptome level, T2 vs. T0 showed an upregulation of 337 and downregulation of 61 genes, collectively involved in TNF-α, NFKB, and interleukin signaling. Cell enrichment analysis individuates macrophages, monocytes, and neutrophils as the most significant tissue-cell type in the response. Conclusions In the process of liver graft procurement-transplantation, IRI induces significant epigenetic changes that primarily act on the signaling pathways of inflammatory responses dependent on TNF-α, NFKB, and interleukins. Our DNAm datasets are the early IRI methylome literature and will serve as a launch point for studying the impact of epigenetic modification in IRI. Authors Giraudi PJ, Laraño AA, Monego SD, Pravisani R, Bonazza D, Gondolesi G, Tiribelli C, Baralle F, Baccarani U, Licastro D. Journal Ann Hepatol. Date 01/07/2024 Consult the publication
Age-dependent regulation of ELP1 exon 20 splicing in Familial Dysautonomia by RNA Polymerase II kinetics and chromatin structure
Abstract Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20. Authors Riccardi F, Romano G, Licastro D, Pagani F. Journal PLoS One Date 03/06/2024 Consult the paper
Protein family annotation for the Unified Human Gastrointestinal Proteome by DPCfam clustering
Abstract Technological advances in massively parallel sequencing have led to an exponential growth in the number of known protein sequences. Much of this growth originates from metagenomic projects producing new sequences from environmental and clinical samples. The Unified Human Gastrointestinal Proteome (UHGP) catalogue is one of the most relevant metagenomic datasets with applications ranging from medicine to biology. However, the low levels of sequence annotation may impair its usability. This work aims to produce a family classification of UHGP sequences to facilitate downstream structural and functional annotation. This is achieved through the release of the DPCfam-UHGP50 dataset containing 10,778 putative protein families generated using DPCfam clustering, an unsupervised pipeline grouping sequences into single or multi-domain architectures. DPCfam-UHGP50 considerably improves family coverage at protein and residue levels compared to the manually curated repository Pfam. In the hope that DPCfam-UHGP50 will foster future discoveries in the field of metagenomics of the human gut, we release a FAIR-compliant database of our results that is easily accessible via a searchable web server and Zenodo repository. Authors Federico Barone, Elena Tea Russo, Edith Natalia Villegas Garcia, Marco Punta, Stefano Cozzini, Alessio Ansuini, Alberto Cazzaniga Journal Scientific Data 11, 568 (2024) Date 01/06/2024 Consult the paper
Vertical Intrauterine Bovine and Ovine Papillomavirus Coinfection in Pregnant Cows.
Abstract There is very little information available about transplacental infections by the papillomavirus in ruminants. However, recent evidence has emerged of the first report of vertical infections of bovine papillomavirus (BPV) in fetuses from naturally infected, pregnant cows. This study reports the coinfection of BPV and ovine papillomavirus (OaPV) in bovine fetuses from infected pregnant cows suffering from bladder tumors caused by simultaneous, persistent viral infections. Some molecular mechanisms involving the binary complex composed of Eras and platelet-derived growth factor β receptor (PDGFβR), by which BPVs and OaPVs contribute to reproductive disorders, have been investigated. A droplet digital polymerase chain reaction (ddPCR) was used to detect and quantify the nucleic acids of the BPVs of the Deltapapillomavirus genus (BPV1, BPV2, BPV13, and BPV14) and OaPVs belonging to the Deltapapillomavirus (OaPV1, OaPV2, and OaPV4) and Dyokappapapillomavirus (OaPV3) genera in the placenta and fetal organs (heart, lung, liver, and kidneys) of four bovine fetuses from four pregnant cows with neoplasia of the urinary bladder. A papillomaviral evaluation was also performed on the bladder tumors and peripheral blood of these pregnant cows. In all fetal and maternal samples, the genotype distribution of BPVs and OaPVs were evaluated using both their DNA and RNA. A BPV and OaPV coinfection was seen in bladder tumors, whereas only BPV infection was found in peripheral blood. The genotype distribution of both the BPVs and OaPVs detected in placentas and fetal organs indicated a stronger concordance with the viral genotypes detected in bladder tumors rather than in peripheral blood. This suggests that the viruses found in placentas and fetuses may have originated from infected bladders. Our study highlights the likelihood of vertical infections with BPVs and OaPVs and emphasizes the importance of gaining further insights into the mechanisms and consequences of this exposure. This study warrants further research as adverse pregnancy outcomes are a major source of economic losses in cattle breeding. Authors Francesca De Falco, Anna Cutarelli, Leonardo Leonardi, Ioan Marcus, Sante Roperto Journal Pathogens Publication date 26/05/2024 Consult the publication
The Deep Proteomics Approach Identified Extracellular Vesicular Proteins Correlated to Extracellular Matrix in Type One and Two Endometrial Cancer
Abstract Among gynecological cancers, endometrial cancer is the most common in developed countries. Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that contain proteins involved in immune response and apoptosis. A deep proteomic approach can help to identify dysregulated extracellular matrix (ECM) proteins in EVs correlated to key pathways for tumor development. In this study, we used a proteomics approach correlating the two acquisitions—data-dependent acquisition (DDA) and data-independent acquisition (DIA)—on EVs from the conditioned medium of four cell lines identifying 428 ECM proteins. After protein quantification and statistical analysis, we found significant changes in the abundance (p < 0.05) of 67 proteins. Our bioinformatic analysis identified 26 pathways associated with the ECM. Western blotting analysis on 13 patients with type 1 and type 2 EC and 13 endometrial samples confirmed an altered abundance of MMP2. Our proteomics analysis identified the dysregulated ECM proteins involved in cancer growth. Our data can open the path to other studies for understanding the interaction among cancer cells and the rearrangement of the ECM. Authors Capaci V, Kharrat F, Conti A, Salviati E, Basilicata MG, Campiglia P, Balasan N, Licastro D, Caponnetto F, Beltrami AP, Monasta L, Romano F, Di Lorenzo G, Ricci G, Ura B. Journal Int J Mol Sci. Date 24/04/2024 Consult the paper