Biology subjects

Mancini, E.

Publications and source records attributed to Mancini, E..

10 recordsLinked to original sources

bioRxiv2026

Structure-function studies of HRIKD-{triangleup}KI, a Minimal Kinase Domain of Human Heme-Regulated Inhibitor Kinase

EIF2 kinase heme-regulated inhibitor (HRI) is a novel target for haematological malignancies with modulators reported to trigger cell death via the HRI-eIF2-ATF4 pathway. We report a protocol for producing the minimal kinase domain of full-length human HRI, termed HRIKD-{Delta}KI, where the unstructured 140 amino acid (aa) kinase insert (KI) within HRI kinase domain (HRIKD) is replaced with a 2aa glycine/serine (GS) linker. X-ray crystal structures were determined of apo-HRIKD-{Delta}KI and of its complex with ATP at 2.1 & 2.5 [A] resolution respectively. Both structures display a canonical bi-lobal kinase fold. However, they remain in a non-productive state with a displaced C-helix, disassembled R-spine, and a disordered activation segment hindering the substrate site. Biophysical assays (fluorescence based thermal shift & Synchrotron Radiation Circular Dichroism) demonstrate HRIKD-{Delta}KI retains its functional ligand-binding conformation. All together, these findings define structural and ligand-binding features of HRI to support ongoing drug discovery efforts in blood cancer.

biochemistry↗

bioRxiv2024

AGE READINGS AND ASSESSMENT IN COASTAL BATOID ELASMOBRANCHS FROM SMALL-SCALE SIZE-SELECTIVE FISHERY: THE IMPORTANCE OF DATA COMPARABILITY IN MULTISPECIFIC ASSEMBLAGES.

Age readings and assessment of vertebral growth increments were obtained in four batoid elasmobranch species, namely Dasyatis pastinaca, Raja asterias, Torpedo marmorata, and Torpedo torpedo. Samples were obtained opportunistically from the bycatch of a size-selective fishery, such as local small-scale trammel net fishing, in the coastal waters of the Central Tyrrhenian Sea during 2019-21. We analysed the vertebrae by a simple and rapid method preventing staining phase and histological preparation to elucidate band pairs in all species studied. Consistency of age estimates was checked by controlling for agreement on band pair counts between and within observers, and by estimating the relationships between vertebral diameter and height, and body size of the animal. Based on these data, we developed a statistical routine to obtain multiple estimates of age and growth parameters for incomplete samples due to size-selective fishing. The acceptable agreement between and within readers and the increase in vertebral size with body size demonstrated the consistency of the method. Based on the results of Von Bertalanffy and Gompertz growth models, body size was a better predictor of age than vertebral size, and Gompertz models were a better estimator of age and growth parameters than Von Bertalanffy models. The estimated parameters (k and t0, kg and cg) matched the data available for the species studied in the Mediterranean Sea. In fact, D. pastinaca, T. torpedo and R. asterias showed the lowest (k = 0.05-0.12), intermediate (k = 0.112-0.19) and highest (k = 0.18-0.23) growth rate, respectively. Overall, the method proved effective both in delineating band pairs in vertebrae of different species by making use of only minimal optical equipment and a single reagent, and in reliably estimating the age and growth parameters of problematic samples due to size-selective fishing. The replicability of the method will help to collect comparable demographic data in similar samples from other areas of the Mediterranean, as well as in assemblages of different species from other places.

zoology↗

bioRxiv2023

Quantifying mutational synergy using computational models predicts survival in haematological cancers

Genetic heterogeneity and co-occurring driver mutations impact clinical outcomes in blood cancers. Grouping tumours into clusters based on genetic alterations is prognostically informative. However, predicting the emergent effect of co-occurring mutations that impact multiple complex and interacting signalling networks remains challenging. Here, we used mathematical models to predict the impact of co-occurring mutations on cellular signalling and cell fates in diffuse large B cell lymphoma (DLBCL) and multiple myeloma (MM). Simulations predicted adverse impact on clinical prognosis when combinations of mutations induced both pro-proliferative and anti-apoptotic signalling. So, we established a pipeline to integrate patient-specific mutational profiles into personalised lymphoma models. Using this approach, we identified a subgroup (19%) of patients characterised by simultaneous upregulation of anti-apoptotic and pro-proliferative (AAPP) signalling. AAPP patients have dismal prognosis and can be identified within all current genomic and cell-of-origin classifications. Combining personalised molecular simulations with mutational clustering enabled stratification of patients into clinically informative prognostic categories: good (80% progression-free survival at 120 months), intermediate (median progression-free survival of 93 months), and poor (AAPP, median progression-free survival of 26 months). This study shows that personalised computational models enable identification of novel high-risk patient subgroups, providing a valuable tool for future risk-stratified clinical trials.

systems biology↗

bioRxiv2021

Microlitter pollution in the marine environment and preliminary evidences of in vitro cytotoxic effects on two Mediterranean commercial fish species

Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter. HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets C_LIO_LIThe ingested microlitter contained plastic items C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity C_LI

zoology↗

bioRxiv2020

Aurora-A phosphorylates splicing factors and regulates alternative splicing

Aurora-A kinase is well known to regulate progression through mitosis. However, the kinase also performs additional functions that could explain the failure of its inhibitors to be effective in cancer treatments. To identify these functions, we applied a proteomics approach to search for interactors of Aurora-A. We found a large number of proteins involved in pre-mRNA splicing, strongly suggesting an important role for Aurora-A in this biological process. Consistently, we first report the subcellular localization of Aurora-A in nuclear speckles, the storehouse of splicing proteins. We also demonstrate direct interaction of Aurora-A with RRM domain-containing splicing factors such as hnRNP and SR proteins and their phosphorylation in vitro. Further, RNA-sequencing analysis following pharmacological inhibition of Aurora-A resulted in alternative splicing changes corresponding to 505 genes, including genes with functions regulated by Aurora-A kinase. Finally, we report enrichment of RNA motifs within the alternatively spliced regions affected by Aurora-A kinase inhibition which are bound by Aurora-A interacting splicing factors, suggesting that Aurora-A regulates alternative splicing by modulating the activity of these interacting splicing factors. Overall our work identified Aurora-A as a novel splicing kinase and for the first time, describes a broad role of Aurora-A in regulating alternative splicing.

molecular biology↗

bioRxiv2020

Cdc42 reactivation at growth sites is regulated by cell-cycle-dependent removal of its GAP Rga4 in fission yeast

In fission yeast, polarized cell growth stops during division and resumes after cell separation. We uncoupled these sequential events by delaying cytokinesis with a temporary Latrunculin A treatment. Mitotic cells recovering from treatment initiate end growth without cell separation, displaying a polar elongation sans separation (PrESS) phenotype. PrESS cell ends reactivate Cdc42, a major regulator of polarized growth, before cell separation, but at a fixed time after anaphase B. A candidate screen implicates Rga4, a negative regulator of Cdc42, in this process. We show that Rga4 appears punctate at the cell sides during G2, but is diffuse during mitosis, extending to the ends. While the Morphogenesis Orb6 (MOR) pathway is known to promote cell separation and growth by activating protein synthesis, we find that for polarized growth, removal of Rga4 from the ends is also necessary. Therefore, we propose that growth resumes after division once the MOR pathway is activated and the ends lose Rga4 in a cell-cycle-dependent manner.

cell biology↗

bioRxiv2020

ASpli2: Integrative analysis of splicing landscapesthrough RNA-Seq assays

Genome-wide analysis of alternative splicing has been a very active field of research since the early days of NGS (Next generation sequencing) technologies. Since then, ever-growing data availability and the development of increasingly sophisticated analysis methods have uncovered the complexity of the general splicing repertoire. However, independently of the considered quantification methodology, very often changes in variant concentration profiles can be hard to disentangle. In order to tackle this problem we present ASpli2, a computational suite implemented in R, that allows the identification of changes in both, annotated and novel alternative splicing events, and can deal with complex experimental designs. Our analysis workflow relies on the analysis of differential usage of subgenic features in combination with a junction-based description of local splicing changes. Analyzing simulated and real data we found that the consolidation of these signals resulted in a robust proxy of the occurrence of splicing alterations. While junction-based signals allowed us to uncover annotated as well and non-annotated events, bin-associated signals notably increased recall capabilities at a very competitive performance in terms of precision.

bioinformatics↗

bioRxiv2019

Global transcriptome analysis reveals circadian control of splicing events in Arabidopsis thaliana

O_LIThe circadian clock of Arabidopsis thaliana controls many physiological and molecular processes, allowing plants to anticipate daily changes in their environment. However, developing a detailed understanding of how oscillations in mRNA levels are connected to oscillations in post-transcriptional processes, such as splicing, has remained a challenge. C_LIO_LIHere we applied a combined approach using deep transcriptome sequencing and bioinformatics tools to identify novel circadian regulated genes and splicing events. C_LIO_LIUsing a stringent approach, we identified 300 intron retention, 8 exon skipping, 79 alternative 3 splice site usage, 48 alternative 5 splice site usage, and 350 multiple (more than one event type) annotated events under circadian regulation. We also found 7 and 721 novel alternative exonic and intronic events. Depletion of the circadian regulated splicing factor AtSPF30 homolog, resulted in the disruption of a subset of clock controlled splicing events. C_LIO_LIAltogether, our global circadian RNA-seq coupled with an in silico, event centred, splicing analysis tool offers a new approach for studying the interplay between the circadian clock and the splicing machinery at a global scale. The identification of many circadian regulated splicing events broadens our current understanding of the level of control that the circadian clock has over this posttranscriptional regulatory layer. C_LI

plant biology↗

bioRxiv2018

Increased association between Epstein-Barr virus EBNA2 from type 2 strains and the transcriptional repressor BS69 restricts B cell growth

Natural variation separates Epstein-Barr virus (EBV) into type 1 and type 2 strains. Type 2 EBV is less transforming in vitro due to sequence differences in the EBV transcription factor EBNA2. This correlates with reduced activation of the EBV oncogene LMP1 and some cell genes. Transcriptional activation by type 1 EBNA2 can be suppressed through the binding of two PXLXP motifs in its transactivation domain (TAD) to the dimeric coiled-coil MYND domain (CC-MYND) of the BS69 repressor protein (ZMYND11). We identified a third conserved PXLXP motif in type 2 EBNA2. We found that type 2 EBNA2 peptides containing this motif bound BS69CC-MYND efficiently and that the type 2 EBNA2TAD bound an additional BS69CC-MYND molecule. Full-length type 2 EBNA2 also bound BS69 more efficiently in pull-down assays. Molecular weight analysis and low-resolution structures obtained using small-angle X-ray scattering showed that three BS69CC-MYND dimers bound two molecules of type 2 EBNA2TAD, in line with the dimeric state of full-length EBNA2 in vivo. Importantly, mutation of the third BS69 binding motif in type 2 EBNA2 improved B-cell growth maintenance. Our data indicate that increased association with BS69 restricts growth promotion by EBNA2 and may contribute to reduced B-cell transformation by type 2 EBV.\n\nAuthor summaryEpstein-Barr virus (EBV) drives the development of many human cancers worldwide including specific types of lymphoma and carcinoma. EBV infects B lymphocytes and immortalises them, thus contributing to lymphoma development. The virus promotes B lymphocyte growth and survival by altering the level at which hundreds of genes are expressed. The EBV protein EBNA2 is known to activate many growth-promoting genes. Natural variation in the sequence of EBNA2 defines the two main EBV strains: type 1 and type 2. Type 2 strains immortalise B lymphocytes less efficiency and activate some growth genes poorly, although the mechanism of this difference is unclear. We now show that sequence variation in type 2 EBNA2 creates a third site of interaction for the repressor protein (BS69, ZMYND11). We have characterised the complex formed between type 2 EBNA2 and BS69 and show that three dimers of BS69 form a bridged complex with two molecules of type 2 EBNA2. We demonstrate that mutation of the additional BS69 interaction site in type 2 EBNA2 improves its growth-promoting function. Our results therefore provide a molecular explanation for the different B lymphocyte growth promoting activities of type 1 and type 2 EBV. This aids our understanding of immortalisation by EBV.

microbiology↗

bioRxiv2018

Old fibroblasts secrete inflammatory cytokines that drive variability in reprogramming efficiency and may affect wound healing between old individuals

Age-associated chronic inflammation (inflammaging) has emerged as a central hallmark of aging1-3, but its impact on specific cells is still largely unknown. Fibroblasts are present in all tissues and contribute to wound healing4-6. They are also the cell type that is mostly used for induced pluripotent stem cell (iPSC) reprogramming7 - a process that has implications for regenerative medicine and rejuvenation strategies8-17. Here we show that primary fibroblasts from old mice secrete inflammatory cytokines and that there is an increased variability in reprogramming efficiency between fibroblast cultures from old individuals. Individual-to-individual variability is emerging as a key feature of old age18-21, which could reflect distinct aging trajectories, but the underlying causes remain unknown. To identify drivers of this variability, we perform a multi-omic assessment of young and old fibroblast cultures with different reprogramming efficiency. This approach, coupled with single cell transcriptomics, reveals that old fibroblast cultures are heterogeneous and show a greater proportion of activated fibroblasts that secrete inflammatory cytokines, which correlates with reprogramming efficiency. We experimentally validate that activated fibroblasts express inflammatory cytokines in vivo and that their presence is linked to enhanced reprogramming efficiency in culture. Conditioned-media swapping experiments show that extrinsic factors secreted by activated fibroblasts are more critical than intrinsic factors for the individual-to-individual variability in reprogramming efficiency, and we identify TNF as a key inflammatory cytokine underlying this variability. Interestingly, old mice also exhibit variability in wound healing efficiency in vivo and old wounds show an increased subpopulation of activated fibroblasts with a unique TNF signature. Our study shows that a switch in fibroblast composition, and the ratio of inflammatory cytokines they secrete, drives variability in reprogramming in vitro and may influence wound healing in vivo. These findings could help identify personalized strategies to improve iPSC generation and wound healing in older individuals.

cell biology↗