Workshop - Abstract

Paola Petrone

Computer-assisted human erythrocyte morphometry by scanning electron microscopy for validation of an early autism spectrum disorder biomarker

Abnormal red blood cell (RBC) shapes have been found in patients with autism spectrum disorders (ASD), neurodevelopmental disorders characterized by social and behavioural impairments. This condition is associated with a high social impact and strong suggestions of a dramatically rising prevalence in the general paediatric population over the last decades; currently its diagnosis can be made by expert operators, on a clinical-behavioural basis, in about 36 months old subjects. For the vast majority of cases, neither causes nor therapies are still unknown; however, several rehabilitative interventions of proven efficacy in reducing the symptoms of the disorder are currently available and its clinical course depends fundamentally on how early the intervention is established.

The aim of the project is to test the potential value of RBC morphology as a biomarker candidate for an early ASD diagnosis, carrying out a SEM (Scanning Electron Microscopy) analysis of blood micro-samples from ASD patients. As part of the project, a new protocol, based on the acupuncture method, for the preparation of blood samples aimed at the morphological analysis of RBCs by SEM was developed and optimized. The project concerning the study of RBC morphologies in school-age ASD patients obtained official authorization by a local ethical committee. First ASD patients were recruited and the related blood samples were processed and analysed in the laboratory. A new software has also been developed with the aim of speeding up and making more reproducible RBC identification and classification in SEM micrographs. For the validation of the biomarker "altered erythrocyte morphometry (AEM)" in school-age children, ASD patients and negative control subjects will be recruited and their blood micro-samples will be analysed by SEM technologies. Furthermore, the software efficiency for RBC phenotyping in SEM micrographs will be implemented through ad-hoc artificial intelligence methodologies. If confirmed by ongoing research, identification of an early ASD biomarker could allow ASD patients and their families to be directed early towards a rehabilitation-behavioural path with positive effects both in terms of patients' neurodevelopmental outcome and reduction of the economic-social impact

Luisa Affatigato

Ferritin-coated SPIONs as a smart magnetic nanocarrier for site targeted theranostic applications

The versatility of the Superparamagnetic Iron Oxide Nanoparticles (SPIONs) allows the production of theranostic and multifunctional devices that can be used for simultaneous drug delivery and imaging. We used the Humanized Archaeoglobus fulgidus ferritin (HumAfFt) as a coating material for 10 nm SPIONs, in order to produce a new magnetic nanocarrier able to discriminate cancer cells from normal cells and maintain the potential theranostic properties of SPIONs. HumAfFt is an engineered ferritin characterized by the peculiar salt-triggered assembly-disassembly of the hyperthermophile Archaeoglobus fulgidus ferritin and which is successfully endowed with the human H homopolymer recognition sequence by the transferrin receptor, overexpressed in cancer cells in response to the increased demand of iron. The new complex HumAfFt-SPIONs was exhaustively characterized in terms of size, morphology, composition and cytotoxicity by using different biophysical and biological techniques. The magnetization analysis revealed that the HumAfFt-SPIONs were fully superparamagnetic, which means that the HumAfFt-SPIONs can be used in the magnetic hyperthermia. The imaging properties of the prepared HumAfFt-SPIONs were further evaluated using magnetic resonance imaging (MRI). The new system can be applied both for tumor diagnosis and tumor therapy.

Simone Bonelli

Proteomic characterization of ubiquitin carboxyl-terminal hydrolase 19 deficient cells reveals a multifaceted role for USP19 in protein secretion

USP19 is a unique deubiquitinase, characterized by a C-terminal transmembrane domain that anchors it to the endoplasmic reticulum (ER). Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from ER-associated protein degradation in a catalytic-independent manner, promote autophagy and address proteins to lysosomal degradation via chaperone-mediated autophagy. In addition, USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson’s disease associated protein α-synuclein. However, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by loss of USP19, to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired release of lysosomal proteins, including legumain, collagens and matricellular proteins.

In conclusion, we identified a collection of proteins that are regulated by USP19, which link USP19 to lysosome homeostasis and other biological processes.

Francesco Cancilla

Synthesis, characterization and processing by electrospinning of redox-responsive waterborne polyurethanes to produce nanofibrous scaffolds

Waterborne polyurethanes (WPUs), unlike traditional organic solvent-based polyurethanes, are synthesized by a water environmentally friendly process. These versatile polymers offer a wide range of applications in the biomedical field, e.g., drug delivery systems (DDS), tissue engineering scaffolds, and coatings. In this work, the synthesis of novel redox-sensitive WPUs with increasing extent of hydrophilic character involves the reaction of PCL-PEG-PCL triblock copolymer, 2,2-bis(hydroxymethyl)propionic acid (DMPA) as emulsifier, isophorone diisocyanate and L-glutathione oxidized as a redox-responsive chain extender. The emulsifier enables the dispersion in water of the hydrophobic polyurethane segments, resulting in a stable colloidal system. The synthesized WPUs were characterized by FTIR, 1H-NMR, SEC, DSC, AFM and DLS. Interestingly, starting from lyophilized samples, through autoclaving process, aqueous dispersions of WPUs, which exhibit the ability to undergo a reversible sol-gel transition in response to external oxidative/reductive stimuli, were easily produced. These aqueous dispersions were processed by electrospinning to produce nanofibrous scaffolds. Thanks to their strong affinity for water, WPUs can undergo processing without the need for organic and potentially harmful solvents. Processing with water enhances the capability to incorporate bioactive molecules, such as protein bio-effectors, thereby safeguarding them from denaturation.

Fanny Claire Caprì

Actinomycetes: a still-to-tap source of bioactive secondary metabolite

Antibiotic resistance represents the ability of bacteria to survive and replicate in the presence of an antimicrobial drug. According to the World Health Organization, novel antibiotics are urgently needed to tackle the growing worldwide problem of antimicrobial resistance. Secondary metabolites, characterized by low molecular weight, possess complex chemical structures and exhibit a range of biological activities, including antimicrobial, antitumoral, immunomodulatory, etc. Actinomycetes are a diverse group of Gram-positive bacteria known as a prolific source of secondary metabolites of industrial interest. We aim to explore diverse environments (i.e., organic farming soil, hydrothermal vents, saltern ponds, activated sludge, and cultural artefacts) to search for bacteria-producing secondary metabolites and activate cryptic or silent gene clusters devoted to their biosynthesis. We assembled a collection of 500 bacterial strains, encompassing Gram-positive and Gram-negative microorganisms. We selected more than 100 Actinomycetes and conducted the metabolomic profiling of ten antibiotic-producing bacteria, unravelling metabolites active against Gram-positive multi-resistant strains. In addition, novel cultivation strategies and epigenetic approaches have been undertaken to enhance the discovery of new secondary metabolites holding intriguing antimicrobial activities.

Veronica Concetta Ciaramitaro

Polysaccharide-based biodegradable films

Over the past 20 years, the world's population has grown exponentially. Consequently, the demand for agricultural products has increased annually. To meet this need, the prolonged use of mulching films based on low-density polyethene resulted in significant environmental pollution events, leading to serious side effects on human health. Based on this, there is a great interest in developing biodegradable polymeric films that can be tilled directly into the soil after use to guarantee greater environmental sustainability. Here, we present the preparation and characterization of biodegradable sodium carboxymethyl cellulose, chitosan and sodium alginate-based composite films in the presence of glycerol as a plasticizer and calcium chloride as cross-linker and their enrichment with the NH4H2PO4 salt, as N and P are generally the most deficient nutrients in the soil. The effects of the composition and the cross-linking agent on some water interaction properties and the thermal and mechanical properties were evaluated. To rationalise the macroscopic behaviour of the films, infrared spectroscopy and X-ray diffractometry were applied to gain information about interactions and structural changes induced by salt and crosslinker. Moreover, the release kinetics of NH4+ and PO43- ions as a function of film thickness were studied. The latter aspect is of great importance as the release of N and P helps to improve the nutrient supply to the soil, reducing the use of synthetic fertilisers.

Adele Cicio

Chemical Composition and Antioxidant Activity of methanolic extract of Brassica macrocarpa

Brassica genus is an important source of food in the Mediterranean diet from centuries and several studies have documented the nutritional and the medicinal properties of the edible wild plants. Since few studies investigated the phytochemical composition and the biological activity of wild Sicilian taxa, we aimed to study the chemical profile and the antioxidant potential in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells, of a methanolic extract of leaves of Brassica macrocarpa, a specie endemic to Favignana island (Sicily). Chemical profile of the extract was evaluated by HPLC-MS/MS. Free radical scavenging activity was examined by DPPH assay. The toxicity on RAW 264.7 cells was assessed by MTT assay. On LPS (0.1μg/mL) -stimulated RAW 264.7 cells were investigated nitric oxide (NO) concentration (Griess reaction) by spectrometry and reactive oxygen species (ROS) levels by flow cytometry. The chemical profile showed a large amount of glucosinolates, being sinigrin, the major component. Brassica extract did not affect cell viability and exhibited a concentration-dependent antioxidant activity in DPPH assay, as well as in LPS-stimulated RAW 264.7 cells, where it was able to reduce NO and ROS levels, at all concentrations tested. Our study demonstrated that Brassica macrocarpa is good source of bioactive molecules with antioxidant activity. Future studies are required to characterize the action mechanism and the possible application in human health.

Federica Corrao

Exploring the suppression therapy approach in rare genetic diseases in a precision medicine vision

11% of the inherited genetic disorders are caused by nonsense mutations, which are due to a single-base substitution in the mRNA bringing to the onset of a PTC in codifying sequence. This leads to the premature termination of the protein translation and the production of a truncated and non-functional protein, with severe physiological consequences. One of the most promising strategies to overcome this defect relies on the translational readthrough (TR) of PTCs.

Herein, the present study is focused on the use of three new optimized lead drugs classified as TR inducing drugs (TRIDs), namely NV848, NV914 and NV930. Particularly, this TRIDs have been tested on human primary fibroblasts harbouring the nonsense mutation c. 5047 C>T (R1683*) in the LPS Responsive Beige-Like Anchor (LRBA) gene, causing a rare Primary Immunodeficiency disease (PID). The activity of the three compounds and their efficiency have been evaluated in this UGA-stop codon genetic context, by classical molecular approaches. Besides, knowing that the readthrough of UGA PTC often induce Trp, Arg or Cys insertion, in-silico structure assembly simulations have been performed to predict protein structure of the corrected proteins. Finally, mRNA next-generation sequencing (NGS) has been carried on, verifying the fidelity in the transcription under the treatment, to exclude any eventual interference by molecules, showing the correct production of the transcript.

Cesare D'Amico

Elucidate the role and targets of MBP-1 transcription factor in breast cancer cells

ENO1 is the gene that encodes for the glycolytic enzyme α-enolase and, by alternative translation, a shorter form of α-enolase known as MBP-1. Both proteins are involved in tumorigenesis, but with different functions. Alpha-enolase has a major role in glycolytic pathways and is involved in cell growth and hypoxia tolerance, acting as an oncogene under the control of the c-MYC gene. In contrast, MBP-1 acts has a transcription factor controlling genes such as c-MYC, ERBB2, COX2 and FOXP3. In this way, MBP-1 suppresses cell proliferation and cancer cell migration. In many cancers, including breast cancer, when α-enolase levels increase there is a substantial reduction of MBP-1 levels. To elucidate MBP-1 mechanisms of action as an onco-suppressor and the molecular pathways through which it inhibits cell growth, cancer cells were transfected with two different kind of recombinant plasmids. Transient and stable transfections of MBP-1 were performed in breast cancer cells to identify the induced patterns of gene expression. Lipofectamine and Xfect reagents for transient and stable transfections were used, respectively, in SkBr3, MCF-7, MDA-MB231 and HeLa cancer cell lines. Expression of a Flag-tagged MBP-1 protein, constitutively or controlled in a Tet-On 3G system, was obtained. Upon transfection, gene expression profiles by MicroArray technology were obtained and studied. In conclusion, the above molecular strategy will provide new insights into the role of MBP-1 in tumorigenesis.

Giuseppe De Luca

Surface-triggered LLPS phenomena during amyloid self-assembly in microscale compartments

Liquid-liquid phase separation (LLPS) has emerged as a key phenomenon in the formation of membraneless structures within the cell. These structures appear as liquid biomolecular condensates in dynamic equilibrium with the cellular environment. Protein condensates are the most studied and have the tendency to evolve, following a phase transition. Thus, they are often analysed in the framework of protein aggregation processes and amyloid formation.

We present an experimental study on the supramolecular association of Human Insulin in microscale aqueous compartments at room temperature by means of quantitative microscopy techniques. In the presented conditions it is possible to image the formation of spherical amyloid-like superstructure. Their formation and development are a combination of a LLPS-driven process and a liquid-to-solid transition. The process is triggered at the interface between the aqueous compartment and the glass coverslip onto which it is cast. The effects of surface are explored by increasing the surface-volume ratio of the compartments, showing an increase in the aggregation rate and a reduction of the particles' size. FLIM and FRAP data shows that the particles present an amorphous solid-like core, and fluid-like edges, characterised by a higher affinity towards Thioflavin T. Our study sheds light on the intricate interplay between liquid LLPS, surface effects, and the formation of amyloid-like superstructures in the context of protein aggregation.

Ylenia Di Leto

Modulation of sewage sludge microbiome for enhancing the transition from wastewater treatment plants into biorefineries in the circular economy era

The sewage sludge (SS) microbiota operating in wastewater treatment plants (WWTPs) is determinant in pollutant degradation to obtain clean water. According to the WIDER-UPTAKE project , based on the principle of WWTP conversion into biorefineries, SS microorganisms can produce valuable materials, like polyhydroxyalkanoates (PHAs), using pollutants as nutrients. PHAs are promising candidates as alternatives to petroleum plastics. PHAs can be produced by SS microorganisms through a process that involves the supplementation of volatile fatty acids (VFA). Furthermore, VFAs can also be produced in specific WWTP fermenters by SS microorganisms through acidogenic fermentations. Therefore, studies on the relationships between operating conditions and SS microbiome can maximize VFA and consequently, PHA production. In this study different conditions were assayed in batch-cultivation reactors and at pilot scale to improve VFA production, revealing microbiota structure changes by metataxonomics. In particular, in batch-cultivation reactors, initial pH 10 determined methanogenic bacteria decrease and VFA producer increase; the addition of KMnO₄ caused microbial diversity decrease and VFA producers strains survival. In pilot scale, fermenter headspace reduction promoted VFA production, and studies on SS microbiota are in progress. Thus, metataxonomics is a useful approach to highlight relationships between operating conditions, microbiomes, and the achievement of specific outputs.

Matilda Iemmolo

Inflammation response and CX3CL1 in Alzheimer’s disease

Alzheimer's disease (AD) is the most common form of dementia characterized by the accumulation of β-amyloid plaques, tau tangles, neuroinflammation, and synaptic/neuronal loss, the latter being the strongest correlating factor with memory and cognitive impairment. The cerebrospinal fluid (CFS) obtained from AD and not AD patients were analyzed for the presence of the soluble form of CX3CL1, together with the canonical markers by ELISA test. The results obtained highlighted the increase of expression of CX3CL1 in patients with AD compared to non-AD patients. Through an in vitro study on a neuron-astrocyte-microglia co-culture system, we have analyzed the effects of cerebrospinal fluid (CSF) samples from non AD and AD patients. The treatment of the NAM co-colture with the CSF of AD patients through microscopy analyses, immunofluorescence analyses, Zymography analyses and Q-PCR analyses showed the activation of elements involved in inflammation pathways including CX3CL1. In addition, “in vivo” studies were conducted. Alzheimer’s was induced in rats through intracerebroventricular stereotaxis microinjection of Streptozotocin (STZ). Dot Blot and Western Blot analyses confirm the induction of disease noting the presence of the amyloid fragment 1-42 and hyperphosphorylated tau. Other elements, including CX3CL1, were evaluated by western blot analysis to identify elements that may be up and downregulated In the metabolic pathways after the induction of Alzheimer’s disease.

Francesco La Monica

Regenerative medicine approach in the treatment of chronic skin wounds

Chronic wounds, named ulcers, are a serious social and health problem, affecting millions of people every year and costing the world’s healthcare system huge sums of money. They originate from the interference of internal and external factors with the physiological healing processes and heal slowly or not at all. Among these, diabetic ulcers have attracted the scientific community for their prevalence. The goal of the project is to find a therapeutic approach using regenerative medicine to treat skin ulcers, especially diabetic ones. Fibroblasts isolated from Wistar rat skin have been seeded into PLLA (Poly-L-Lactic Acid) scaffolds treated with collagen fibrils to improve cell adhesion. Furthermore, microvascular fragments have been extracted from adipose tissue (ad-MVFs) of the same rats to be cultured in this system. In this context, fibroblasts cultured in the PLLA scaffold will promote ECM regeneration; while, the ad-MVFs interconnecting with the circulatory system of the host will avoid the hypoxic condition that generally occurs in the ulcer and will allow the right transport of nutrients and gases to the regenerating tissue cells. This construct will be tested in in vivo systems for the treatment of skin ulcers and Streptozotocin diabetes-induced ulcers and analyzed by biochemical, molecular biology and imaging assays. This multifactor approach could be a huge step forward in the treatment of ulcers of different nature.

Silvia La Scala

Plant-growth promoting actinobacteria: their role in plant growth and stimulation of bioactive molecule production

Biofertilizers are gaining a leading position in our present, and plant growth promoting (PGP) bacteria are, in this regard, emerging as excellent candidates, since they exert beneficial effects on plant growth and development by improving, as examples, nutrition uptake, abiotic and biotic stress tolerance in plants. They are naturally occurring in plant crop soil and rhizosphere and, among them, the actinobacteria are considered very promising due to their metabolic versatility and bioactive metabolites production. In this context, two PGP actinobacteria have been selected to investigate on their possible effects on Origanum heracleoticum L., one of the most widespread drought-resistant plants in the Mediterranean area. In particular, origanum cultures were grown in pots and, subsequently, inoculated with actinobacterial cultures or mixtures thereof, using water, bacterial growth medium and a commercial microbial mixture as conditions, respectively. Preliminary results showed that PGP bacterial treatments exerted significant effects on selected morpho-physiological parameters, like plant height. In addition, leaves and flowers were analyzed to reveal possible changes on plant volatile organic compounds that could be ascribed to plant-actinobacteria interaction. Should the results of this research be confirmed by further experiments, the positive effects of PGP bacteria would be extended to a novel field of interest that includes direct positive effects on consumers health.

Gabriele Lo Buglio

Combined effect of collagenase and nanoparticles for synergistic cancer therapy

Solid tumors are characterized by a collagen-rich extracellular matrix (TEM), that limits its accessibility, nullifying the action of classical chemotherapeutic agents. For this purpose, we want to test in vitro and in vivo a two-step therapy composed of ultrapure (>99%) recombinant collagenases (Class I and Class II) (ABIEL s.r.l) and nanoparticles (NPs) in Polyvinylpyrrolidone (PVP). The tertiary structure of collagenases was predicted in silico, to evaluate the presence of free carboxylic groups of glutamic and aspartic acids. The goal is to exploit the free carboxylic groups to decorate recombinant collagenases with Polyethylene glycol (PEG), to enhance their immunocompatibility. They will be administrated in situ to dissociate TEM and increase the accessibility of NPs conjugated to doxorubicin by interposing a glutathione-sensitive linker, to exploit the high concentration of glutathione inside tumor cells and obtain a drug controller release. In parallel, breast cancer was induced in an immunocompetent female Wistar rat via chemical induction. After 5 months, a tumor mass developed and was surgically explanted and treated with a mixture of ultrapure recombinant collagenases by Abiel s.r.l, to isolate the tumor cells. The cell population obtained was used to generate organoids and it will be also used to induce a breast tumor in immunocompetent female Wistar rats. In this way, the combined effect of recombinant collagenases and NPs will be evaluated in vitro and in vivo.

Lisa Longo

INVESTIGATION ON MMACHC-R161Q PATHOLOGICAL MUTANT FROM cblC DISEASE

The cblC disease is a rare inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by combined methylmalonic aciduria and homocystinuria. The clinical consequences are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The molecular genetic cause of the disease was found in the mutations of the gene coding for MMACHC, a 282 amino acid protein that transports and processes the various forms of Cbl. Here we present the biophysical characterization of wild type MMACHC and a variant, p.R161Q, resulting from the most common missense pathological mutation found in CblC patients. By using a biophysical approach we investigated the stability of the two proteins and their ability to bind and transform the vitamin B12, and to assemble in a dimeric structure. Moreover, interesting indications about the behaviour of the proteins resulted from the Molecular Dynamics (MD) simulations. Overall, our results reveal how a biophysical approach based on the complementarity of computational and experimental methods can offer new insights in the study of the specific effects of the pathological cblC mutation and help prospecting new routes for the cblC treatment.

Sefora Marino

Antisense oligonucleotide strategy to promote MBP1 alternative translation from ENO1 mRNA in breast cancer cells

MBP-1 is an important tumor suppressor, which negatively regulates proto-oncogenes such as cMYC and ERBB2 in normal cells. MBP-1 is the alternative translation product of ENO1 mRNA, which encode the 48 kDa protein α-enolase starting from codon in position 1, and the 37 kDa MBP-1 protein starting from the downstream codon in position 97. However, while α-enolase is a cytoplasmatic glycolytic enzyme, MBP-1 is mainly localized in the nucleus and act as a transcriptional repressor of c-MYC gene, whose high expression is responsible for enhancement of glycolysis (Warburg effect) in cancer cells. The project aims to develop a therapeutic strategy to restore higher levels of MBP-1 in breast cancer cells exploiting the well known RNA interference mechanism. Oligonucleotides capable to pair with ENO1 mRNA in the region containing the AUG in position 1 have been transfected in SkBr3, an ERBB2 positive breast cancer cell line, to block the canonical translation of the α-enolase and verify whether MBP-1 alternative translation is activated. Since α-enolase works as a heterodimer, MBP-1 is able to dimerize but lacks a.a. required for activity and is partially localized in the cytoplasm, another aim is to verify a putative dominant negative effect of MBP-1 on glycolysis. SkBr3 cells have been transfected with a MBP-1/GFP encoding plasmid, the inhibition of glycolysis as been tested by Enolase and ATP assays, and MBP-1/ α-enolase interaction will be analized by BlueGel electrophoresis.

Salvatore Martino

The importance of DNA methylation in repetitive elements

DNA methylation is an epigenetic modification involved in DNA heterochromatinization and gene repression. If DNA methylation plays a role in genes-poor repetitive sequences at centromere is still unclear. Lately, studies revealed that tumour cells reduce DNA methylation at repetitive sequences which has been correlated to a loss of chromatid cohesion and aneuploidy. However, DNA methylation direct involvement for the function of the centromeric sequences is elusive. To disclose this, I used a tumour cell line (DLD-1) engineered to allow an inducible DNA hypomethylation. My results showed that, once hypomethylated, DLD-1 cells activate p21. Moreover, CenpA, which gives identity to the centromere, increases in both mitosis and interphase nuclei. DNA hypomethylation also correlated in metaphase with an enhancement of the recruitment of proteins involved in the signalling of microtubule-kinetochore association errors, p150Glued and Aurora B kinase. The studies I conducted so far revealed that methylation of repetitive DNA is important for cell survival, probably by maintaining centromere stability and function and by impacting microtubule-kinetochore attachments. This confirms the usefulness of hypomethylating drugs to impair tumour cell proliferation. However, these results also suggest cautiousness since the aneuploidy induced by DNA hypomethylation can be a double-edged sword, inducing death but adaptation of the tumour cells and transformation of the normal cells.

Giovanni Carlo Miceli

Conductive bioadhesive coating with electrophoretic properties to enhance drug delivery devices integration and functionality

This work focuses on the development of a conductive bioadhesive (CB) electrophoretic platform that enables the direct transfer of molecules from a solid-state device. We have synthesized, characterized, and processed a novel biodegradable segmented polyurethane urea as a biomaterial. It has a remarkable hydrolytic resistance to degradation, a low melting point (55°C) and high adhesion. These properties suit well the use of this polyurethane urea as a coating interface, between metallic prosthesis or implantable devices and biological tissues, to improve biological tolerance and biointegration. Moreover, they allow us to easily produce precise and defined nano or microfibers and to build up structures with a tuneable microarchitecture and external shape. We aim to develop multifunctional coatings that can actively control the release of therapeutics. Therefore, we chose to incorporate poly(3,4-ethylenedioxythiophene) (PEDOT) doped with polystyrene sulfonate (PSS) inside the bioadhesive polyurethane urea matrix (PU-LDA). The final polymer was characterized by electrochemical impedance spectroscopy (EIS), in both wet and dry conditions, and cyclic voltammetry (CV). Paclitaxel was incorporated in the PEDOT doped polymer dispersion in the DMAc/water blend. Porous titanium rods were dip-coated into the dispersion to produce the devices used to evaluate the voltage-driven drug release of paclitaxel in physiological-like conditions.

Emanuela Peri

Proteomic and in silico analysis of the epithelial-to-mesenchymal transition (EMT) mediators in breast cancer

The epithelial-to-mesenchymal transition (EMT) represents a cancer hallmark and is characterized by the loss of epithelial traits and the acquisition of mesenchymal characteristics. In breast cancer (BC), EMT is involved in cell survival and cancer progression. To better understand the biological significance of the EMT, proteomic investigation of vimentin and E-cadherin expression, markers of the mesenchymal and the epithelial phenotype respectively, was conducted on a large sample set of BC tissues. High heterogeneity of expression of both proteins, also in terms of the presence of different isoforms, was detected among patients, whose functional significance is under investigation. A positive correlation between vimentin and E-cadherin was found; moreover, considering the vimentin/E-cadherin ratio, most samples show intermediate values, suggesting an incomplete EMT pattern. To unveil a possible EMT-gene signature in BC, a bioinformatic approach was employed by using different databases. A cluster of 49 genes differentially expressed between normal and BC tissues and significantly associated with prognosis was selected. Collectively, the functional pathway analysis revealed their involvement in both EMT and proteoglycans regulation (Syndecan-2 activity), suggesting a possible crosstalk with tumor microenvironment. The obtained results suggest that the EMT in BC could not be characterized by a complete phenotypic switch and is more complex than previously assumed.

Giorgia Puleo

Lignin-based Waterborne Nanofibers for Sustainable Biomedical Applications

Lignin, Earth's second most abundant polymer, is a waste-product of the pulp and paper industry composed of aromatic building blocks. It possesses remarkable biomedical characteristics, including biocompatibility, biodegradability, high stiffness, as well as antibacterial, antioxidant and UV-blocking properties. However, its challenging processability, characterized by poor water-solubility and high heterogeneity, represents a significant obstacle to its widespread use in the biomedical field. To address this problem, blending with polymers and/or surfactants as well as various solubilization techniques are used to produce lignin-based nanoparticles and nanofibers suitable for applications such as wound dressing, antimicrobial coatings and tissue engineering. In this scenario waterborne electrospinning stands out as a highly attractive technique using water as the sole solvent, coupled with precise control of temperature and humidity. This approach reduces the environmental impact of organic solvents and enhances the safety of the final product. Nanofibers of lignin and polyethylene oxide were successfully produced via waterborne electrospinning to produce a biocompatible scaffold, capable of both retaining lignin's properties and serving as a platform for loading drugs. Work is in progress to evaluate its antioxidant capability, mechanical properties and drug loading/release efficiency, with the final aim of testing it as potential treatment for non-healing infected wounds.

Mattia Romeo

Deep learning approach for diffusion correction in Fricke gel dosimeters

Fricke gels are known for their ability of mapping 3D dose distribution, thanks to the presence of ferrous ions which are oxidized to Fe3+ by ionizing radiation and can be readout by magnetic resonance imaging or optical techniques. However, this dosimetry technique is limited by the diffusion processes which involve the ions. As result, the measurements appear to be dependent on the time between irradiation and signal acquisition. In our study we propose the use of the so-called Physics Informed Neural Network (PINN). These models can be optimized using physical equations as loss functions and thus we can predict real-life phenomena such as the diffusion of ions. The idea is to solve the backward diffusion equation using a PINN model and predict the initial condition, providing as input the spatial distribution of ferric ions at measurement time, the boundary conditions and the diffusion coefficient D of Fe3+ ions.

Tests were performed on 1D simulated data, with a total mean squared error about of 0.002 in a step-wise distribution (gap=3.5 signal a.u., D=0.005 a.u.), and on experimental data (optical density of radiated gel as function of the position for step-wise irradiation of a 1D gel 25mm in length), with a mean squared error about of 2x10-4. Tests on 2D and 3D systems will be performed in the next future. The technique here proposed is very promising for overcoming the limits due to the diffusion of ferric ions in Fricke gel dosimeters.

Alessia Maria Sampino

Molecular approaches to improve antibiotics biosynthesis in Actinomycetes

The widespread use of antibiotics has led to an increase in infections caused by multi-resistant bacterial strains, generating the emergence of antimicrobial resistance. The challenge of the pharmaceutical industry is to develop novel antibiotics. Secondary metabolites are low molecular weight molecules with complex chemical structures and biological activities, such as antibiotics, antitumor agents, etc. Actinomycetes are the most economically and biologically precious bacteria for producing antibiotics. Genes involved in the biosynthesis of the antibiotic are within a biosynthetic gene cluster (BGCs) encoding proteins involved in antibiotic biosynthesis, regulation, immunity, and export. BGCs undergo complex regulation involving cluster-situated, pleiotropic, and global regulators. However, only a few of BGCs are expressed under standard laboratory conditions. Thus, to activate the “silent” BGCs, improve their biosynthetic potential, and discover novel natural products strategies for genetic manipulation are required such as the heterologous expression of regulators of biosynthetic pathways to active silent clusters, and the generation of mutant strains. Specifically, we are investigating the role of DNA cytosine methylation in the model strain Streptomyces coelicolor, demonstrating that methyltransferases can impair the morpho-physiological differentiation and investigating the role of a pleiotropic regulator belonging to the IclR-family in Nonomuraea gerenzanensis.

Martina Sollazzo

Polyglutammine neurodegenerative diseases: the case of Ataxin-3

Neurodegenerative diseases are debilitating and intractable disorders that share a common molecular mechanism consisting in the progressive accumulation inside neurons of insoluble protein aggregates. As model to study pathological protein aggregation, we chose Ataxin-3 linked to Spino-Cerebellar Ataxia type 3. Its propensity to form pathological aggregates results from expansion of CAG triplets in the ATXN3 gene, leading to a protein with expanded C-terminal polyglutamine tract, whose length is correlated with the severity of symptoms and the disease’s onset. Evidence shows a competition between proteins normal function and their tendency to aggregate, thus suggesting that exploring interactions between neurodegeneration-related proteins and their natural binders might offer promising therapeutic perspectives. In this context, we aim at characterize Ataxin-3 in complex with polyubiquitin chains (physiological partner of Ataxin-3), and investigate the effect of these chains in Ataxin-3 aggregation. To solve the complex structure, we employed gold-standard Structural Biology techniques, such as SAXS and X-Ray crystallography. Additionally, in-vitro studies as fluorescent spectroscopy and advanced microscopy techniques were used to characterize the ability of several molecules to interfere with the aggregation process, representing a consistent experimental setup for the screening of potential anti-aggregating compounds.

Donatella Pia Spanò

Characterization of iRhom-mediated substrate selectivity of stimulated ADAM17 by high-resolution proteomics

ADAM17 belongs to the family of disintegrin and metalloproteases (ADAMs) and is implicated in the cleavage of extracellular domain of different transmembrane proteins through a mechanism called shedding. Since its substrates range from cytokines to growth factors and receptors, ADAM17 plays a crucial role in various biological processes but if dysregulated, leads to several pathologies, including inflammatory disorders and cancer. The maturation, trafficking and function of ADAM17 are controlled by the seven-membrane-spanning proteins iRhom1 and iRhom2. But the functional properties of the ADAM17/iRhom1 and ADAM17/iRhom2 complexes differ. To date, the shedding of only 6 ADAM17 known substrates can be supported by iRhom2, whereas iRhom1 can only support stimulated shedding of 1 ADAM17 substrate. In this work, applying an unbiased mass-spectrometry based analysis on a murine and a human cell line to broadly investigate the substrate-selective shedding supported by ADAM17/iRhom1 or ADAM17/iRhom2, we found that the shedding of most ADAM17 substrates was supported by either iRhoms. Moreover, we identified several potential novel substrates, and validated two novel ones: MXRA8, the entry receptor for arthritogenic viruses, and SIRPα, the “do-not eat me” signal protein used by cancer cells to immunoescape. These studies not only provide new insights to the iRhom selectivity, but also confirm proteomics as useful tool in uncovering novel ADAM17 implications in different pathologies.

Fazan Subhan

From Virtual Screening to Lab Validation: Uncovering Translational Read-through- Promoting Drugs

Nonsense mutations that insert a premature stop codon (PTC) into mRNAs and the translation of truncated proteins cause 11% of human genetic diseases. One possible therapeutic strategy is to use PTC readthrough drugs. Herein, an in silico approach was carried out on compounds that showed readthrough activity, to look for novel translational read-through-inducing Compounds (TRIDs). A ligand-based pharmacophore models was generated using LigandScout 4.3. The best model has a score of 0.9 out of 1. The model was validated using the DUD-E tool's decoys and active compounds. The confirmation test identified 29 hits. The early enrichment factor (1%) value of 19.8 and AUC of 1 were observed in the ROC plot. The model was used to filter commercial and in-house libraries with more than 87000 compounds. The screening revealed several new hits. From the hit list, NV2870 was synthesized by reacting methyl 3-amino benzoate with isocyanate moiety. While the NV2877 was obtained from the hydrolysis of NV2870 by NaOH. The products were purified by column chromatography and characterized by IR and 1HNMR spectra, and HPLC/MS analysis. The novel compounds showed a significant result in the FLuc cell-based assay. In conclusion, a Ligand-based pharmacophore approach together with a virtual screening protocol allowed the identification of new promising chemical scaffolds as PTC suppressors.

Simona Titoli

Sequence-specific RNA Editing tools for the correction of CFTR nonsense mutations

Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. In particular, CFTR nonsense mutations generate a premature termination codon (PTC) in the mRNA, leading to the production of a shortened and non-functional CFTR protein. Unfortunately, approved therapies are not efficient against CFTR nonsense mutations. In this regard, we are investigating the possibility to correct the PTC within CFTR mRNA by using RNA-editing tools in Human Bronchial Epithelial cells. These systems exploit the ADAR enzymes to convert the adenosine (A) within the PTC into inosine (I) that will be read as guanosine (G) by the ribosome, allowing the full-length protein synthesis. We used the minixABE base editor (mxABE) based on the truncated (mini) dCAS13x.1 fused with the deaminase domain of ADAR2 (ADAR2DD), which is recruited to the adenosine within the PTC by means of a specifically designed guide RNA. We also tested the RESTORE system which relies on specific antisense RNA oligonucleotides (ASOs) complementary to the CFTR mRNA region with the PTC, except for a cytidine-adenosine mismatch that promotes ADAR recruitment. With both approaches, our results show the restoration of CFTR protein on the plasma membrane by immunofluorescence, and the increase of CFTR full transcript by RT-qPCR in the mutated CFF-16HBEge cell lines. Altogether, these RNA editing-based tools mediated by ADARs are potentially able to correct the nonsense mutations in cystic fibrosis.

Fabiola Vaglica

Matrix metalloproteinases (MMP-2 and MMP-9) in biological fluids of multiple sclerosis patients

In multiple sclerosis (MS), immune cells infiltrate the blood-brain barrier (BBB), causing inflammation and neural damage. This process involves matrix metalloproteinases (MMPs), including gelatinases MMP-2 and MMP-9, which participate in extracellular matrix turnover. Since MS patients are commonly misdiagnosed with other neurological conditions, there's a need for novel biomarkers to improve diagnostic and prognostic accuracy. MMPs hold promise for this purpose, given their potential role in MS pathology. In this study, gelatinase activity levels were screened using gelatin zymography in CSF samples, CSF-derived Extracellular Vesicles (EVs), as well as sera and plasma from MS patients and neurological disease controls (NC). Our findings highlight high variability in expression and activation of MMPs across biological compartments among patients. Specifically, active MMP-2 and MMP-9 were exclusive of CSF samples, showing an enrichment in CSF-derived EVs. Instead, proMMP-9 dimers were exclusive to serum samples. ProMMP-9, proMMP-2, and proMMP-9/TIMP-1 were found in all biological fluids. The clinical significance of MMP isoforms is under investigation. Our findings pointed to MMPs as potential biomarkers in MS diagnosis and prognosis, offering a less invasive and more accurate means of addressing this complex neurological condition.

Emanuele Vitale

Rab Escort Protein-1 rescue by Translational Readthrough Inducing Drugs in CHMR293X-GFP cell model system

Choroideremia is an inherited genetic disorder caused by several mutations in the CHM gene, which codifies for Rep1 protein strictly linked in intravesicular trafficking. The Rep1 lack causes choroid and photoreceptors degeneration, leading firstly to night blindness and at last to complete blindness. About 39% of mutations on the CHM gene are represented by nonsense mutations, which insert a premature termination codon in the reading frame of respective mRNA, with the production of truncated non-functional protein. Nowadays there is no cure for diseases caused by nonsense mutations, but a promising approach is the suppression therapy using TRIDs molecules (translational readthrough-inducing drugs). In our study, a CHMR293X/R293X cell model system was produced to evaluate the activity of three new optimized molecules (NV848, NV914, and NV930) in the rescue of the Rab-Escort-Protein-1 (Rep1) expression.

Sara Volpes

FABRY DISEASE: STUDY OF GENOMIC AND GENIC METHYLATION STATUS

Fabry disease (FD) is a rare and progressive X-linked genetic lysosomal storage disorder caused by mutations in the GLA gene, a 14kb housekeeping gene located on the long arm of X chromosome in position Xq22, that encode for α-galactosidase A (α-GAL). It’s known that inactivation of the X chromosome (XCI), in FD women, significantly influences their symptoms and it also could define as the "onset" of the pathology. Females heterozygous for X-linked diseases have cells that express the normal gene (wild type) and cells that express the mutant gene. Some studies found XCI to be an important factor in determining clinical severity in heterozygous females, while later studies reported that chromosome inactivation, random in heterozygotes, rule out importance in explaining the variable phenotype. The aim is to better characterize the possible contribution of DNA methylation in the FD pathogenesis, since in addition to genetic mutations, even epigenetic variations, linked or not to the XCI, can represent prognostic agents or in the possibility to modulate the gravity of symptoms. Our preliminary results, obtained after the comparison of genomic DNA methylation between FD patients and healthy controls, show a remarkable demethylation of genomic DNA of FD patients. From the methylation status analysis of the GLA gene promoter, we instead obtained a total or almost total methylation of the gene promoter, while healthy subjects showed values around 50% methylation, as we expected.

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