Search

Despite advancements in cancer treatments, therapies frequently exhibit high cytotoxicity, and surgery remains the predominant method for treating most solid tumors, often with limited success in preventing post-surgical recurrence. Implantable biomaterials, designed to release drugs at a localised site in response to specific stimuli, represent a promising approach for enhancing tumour therapy. In this study, a redox-responsive glutathione extended polyurethane urea (PolyCEGS) was used to produce paclitaxel (PTX) and gold nanorods (AuNRs) loaded electrospun membranes for combined redox/near-infrared (NIR) light-responsive release chemotherapy and hyperthermic effect. Electrospinning conditions were optimized to fabricate AuNR-loaded scaffolds, at three different AuNRs concentrations. The obtained membranes were characterized by scanning electron microscopy (SEM) analyses and photothermal profiles were evaluated by a thermocamera, showing a temperature increase, up to 42.5 °C, when exposed to NIR light (810 nm) at 3 W/cm 2 . The AuNRs/PTX loaded scaffolds exhibited sustained PTX release, with 15 % released over 30 days and almost 1.8 times more in a simulated reductive environment. Moreover, their excellent photothermal effects and NIR light-triggered release led to significant synergic cytotoxicity in human colon cancer (HCT-116) and human breast cancer (MCF-7) cell lines. This system potentially enables controllable locoregional PTX release at the tumour site post-surgery, preventing recurrence and enhancing cytotoxicity through combined drug and PTT effects, highlighting its potential for future anticancer treatments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The presence of a guanine quadruplex in the opening reading frame of the messenger RNA coding for the transmembrane serine protease 2 (TMPRSS2) may pave the way to original anticancer and host-oriented antiviral strategy. Indeed, TMPRSS2 in addition to being overexpressed in different cancer types, is also related to the infection of respiratory viruses, including SARS-CoV-2, by promoting the cellular and viral membrane fusion through its proteolytic activity. The design of selective ligands targeting TMPRSS2 messenger RNA requires a detailed knowledge, at atomic level, of its structure. Therefore, we have used an original experimental-computational protocol to predict the first resolved structure of the parallel guanine quadruplex secondary structure in the RNA of TMPRSS2, which shows a rigid core flanked by a flexible loop. This represents the first atomic scale structure of the guanine quadruplex structure present in TMPRSS2 messenger RNA., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

The benzoxazole is one of the most widely exploited heterocycles in drug discovery. Natural occurring and synthetic benzoxazoles show a broad range of biological activities. Many benzoxazoles are available for treating several diseases, and, to date, a few are in clinical trials. Moreover, an ever-increasing number of benzoxazole derivatives are under investigation in the early drug discovery phase and as potential hit or lead compounds. This perspective is an attempt to thoroughly review the rational design, the structure-activity relationship, and the biological activity of the most notable benzoxazoles developed during the past 5 years (period 2019-to date) in cancers, neurological disorders, and inflammation. We also briefly overviewed each target and its role in the disease. The huge amount of work examined suggests the great potential of the scaffold and the high interest of the scientific community in novel biologically active compounds containing the benzoxazole core., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications., (© 2024. The Author(s).)

While platinum(II)-based drugs continue to be employed in cancer treatments, the escalating occurrence of severe side effects has spurred researchers to explore novel sources for potential therapeutic agents. Notably, cobalt(III) has emerged as a subject of considerable interest due to its ubiquitous role in human physiology. Several studies investigating the anticancer effects of Salphen complexes derived from cobalt(III) have unveiled intriguing antiproliferative properties. In a bid to enhance our understanding of this class of compounds, we synthesized and characterized two novel half Salphen cobalt(III) complexes. Both compounds exhibited notable stability, even in the presence of physiologically relevant concentrations of glutathione. The application of spectroscopic and computational methodologies unravelled their interactions with duplex and G4-DNAs, suggesting an external binding affinity for these structures, with preliminary indications of selectivity trends. Importantly, antiproliferative assays conducted on 3D cultured SW-1353 cancer cells unveiled a compelling anticancer activity at low micromolar concentrations, underscoring the potential therapeutic efficacy of this novel class of cobalt(III) complexes.

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

An amine derivative of hyaluronic acid (HA) was crosslinked to obtain a 3D dried sponge. The sponge was subsequently rehydrated using secretome from human mesenchymal stromal cells (MSCs), resulting in the formation of a hydrogel. The release kinetics analysis demonstrated that the hydrogel effectively sustained secretome release, with 70% of the initially loaded wound-healing-associated cytokines being released over a 12-day period. Tuning the hydrogel properties through heparin crosslinking resulted in a biomaterial with a distinct mechanism of action. Specifically, the presence of heparin enhanced water uptake capacity of the hydrogel and increased its sensitivity to enzymatic degradation. Notably, the heparin crosslinking also led to a significant retention of cytokines within the hydrogel matrix. Overall, the secretome-rehydrated HA hydrogel holds promise as a versatile device for regenerative medicine applications: the non-heparinized hydrogel may function as a biomaterial with low reabsorption rates, sustaining the release of bioactive molecules contained in MSC secretome. In contrast, the heparinized hydrogel may serve as a depot of bioactive molecules with faster reabsorption rates. Given its patch-like characteristic, the HA-based hydrogel appears suitable as topical treatment for external organs, such as the skin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

There is still much unknown about the fluid mechanical response to cardiac valve scaffolds, even as their implementation in the clinic is on the horizon. Specifically, while degradable polymer valve scaffolds are currently being tested in the pulmonary valve position, their material and mechanical properties have not been fully elucidated. Optimizing these properties are important determinants not only of acute function, but long-term remodeling prospects. This study aimed to characterize fluid profiles downstream of electrospun valve scaffolds under dynamic pulmonary conditions. Valve scaffold design was changed by either blending poly(carbonate urethane) urea (PCUU) with poly(ε-caprolactone) (PCL) to modulate material stiffness or by changing the geometric design of the valve scaffolds. Specifically, two designs were utilized: one modeled after a clinically used bioprosthetic valve design (termed Mk1 design), and another using a geometrically "optimized" design (termed Mk2) based on anatomical data. Particle image velocimetry results showed that material stiffness only had a mild impact on fluid mechanics, measured by velocity magnitude, vorticity, viscous shear stress, Reynolds shear stress, and turbulent kinetic energy. However, comparing the two geometric designs yielded a much greater impact, with the Mk2 valve groups containing the highest PCUU/PCL ratio demonstrating the overall best performance. This report highlights the easily manipulable design features of polymeric valve scaffolds and demonstrates their relative significance for valve function., (© 2023. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Adolescent Idiopathic Scoliosis (AIS) is the most common form of three-dimensional spinal disorder in adolescents between the ages of 10 and 18 years of age, most commonly diagnosed in young women when severe disease occurs. Patients with AIS are characterized by abnormal skeletal growth and reduced bone mineral density. The etiology of AIS is thought to be multifactorial, involving both environmental and genetic factors, but to date, it is still unknown. Therefore, it is crucial to further investigate the molecular pathogenesis of AIS and to identify biomarkers useful for predicting curve progression. In this perspective, the relative abundance of a panel of microRNAs (miRNAs) was analyzed in the plasma of 20 AIS patients and 10 healthy controls (HC). The data revealed a significant group of circulating miRNAs dysregulated in AIS patients compared to HC. Further bioinformatic analyses evidenced a more restricted expression of some miRNAs exclusively in severe AIS females. These include some members of the miR-30 family, which are considered promising regulators for treating bone diseases. We demonstrated circulating extracellular vesicles (EVs) from severe AIS females contained miR-30 family members and decreased the osteogenic differentiation of mesenchymal stem cells. Proteomic analysis of EVs highlighted the expression of proteins associated with orthopedic disease. This study provides preliminary evidence of a miRNAs signature potentially associated with severe female AIS and suggests the corresponding vesicular component may affect cellular mechanisms crucial in AIS, opening the scenario for in-depth studies on prognostic differences related to gender and grade.

Human mesenchymal stromal cells (MSCs) have gained significant interest as cell-based therapeutics for organ restoration in the field of regenerative medicine. More recently, substantial attention has been directed toward cell-free therapy, achieved through the utilization of soluble factors possessing trophic and immunomodulatory properties present in the MSC secretome. This collection of soluble factors can be found either freely in the secretome or packed within its vesicular fraction, known as extracellular vesicles (EVs). MSCs can be derived from various tissue sources, each involving different extraction methods and yielding varying cell amounts. In this study, we describe a nonenzymatic procedure for a straightforward isolation of MSCs from the fetal dermis and the adult dermis. The results demonstrate the isolation of a cell population with a uniform MSC immunophenotype from the earliest passages (approximately 90% positive for the classical MSC markers CD90, CD105, and CD73, while negative for the hematopoietic markers CD34 and CD45, as well as HLA-DR). Additionally, we describe the procedures for cell expansion, banking, and secretome collection., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In the last years, the field of nanomedicine and drug delivery has grown exponentially, providing new platforms to carry therapeutic agents into the target sites. Extracellular vesicles (EVs) are ready-to-use, biocompatible, and non-toxic nanoparticles that are revolutionizing the field of drug delivery. EVs are involved in cell-cell communication and mediate many physiological and pathological processes by transferring their bioactive cargo to target cells. Recently, nanovesicles from plants (PDNVs) are raising the interest of the scientific community due to their high yield and biocompatibility. This study aims to evaluate whether PDNVs may be used as drug delivery systems. We isolated and characterized nanovesicles from tangerine juice (TNVs) that were comparable to mammalian EVs in size and morphology. TNVs carry the traditional EV marker HSP70 and, as demonstrated by metabolomic analysis, contain flavonoids, organic acids, and limonoids. TNVs were loaded with DDHD1-siRNA through electroporation, obtaining a loading efficiency of 13%. We found that the DDHD1-siRNA complex TNVs were able to deliver DDHD1-siRNA to human colorectal cancer cells, inhibiting the target expression by about 60%. This study represents a proof of concept for the use of PDNVs as vehicles of RNA interference (RNAi) toward mammalian cells.

Background: Lysine demethylase enzymes (KDMs) are an emerging class of therapeutic targets, that catalyse the removal of methyl marks from histone lysine residues regulating chromatin structure and gene expression. KDM4A isoform plays an important role in the epigenetic dysregulation in various cancers and is linked to aggressive disease and poor clinical outcomes. Despite several efforts, the KDM4 family lacks successful specific molecular inhibitors., Results: Herein, starting from a structure-based fragments virtual screening campaign we developed a synergic framework as a guide to rationally design efficient KDM4A inhibitors. Commercial libraries were used to create a fragments collection and perform a virtual screening campaign combining docking and pharmacophore approaches. The most promising compounds were tested in-vitro by a Homogeneous Time-Resolved Fluorescence-based assay developed for identifying selective substrate-competitive inhibitors by means of inhibition of H3K9me3 peptide demethylation. 2-(methylcarbamoyl)isonicotinic acid was identified as a preliminary active fragment, displaying inhibition of KDM4A enzymatic activity. Its chemical exploration was deeply investigated by computational and experimental approaches which allowed a rational fragment growing process. The in-silico studies guided the development of derivatives designed as expansion of the primary fragment hit and provided further knowledge on the structure-activity relationship., Conclusions: Our study describes useful insights into key ligand-KDM4A protein interaction and provides structural features for the development of successful selective KDM4A inhibitors., (© 2023. The Author(s).)

Pancreatic cancer (PCa) is the fifth leading cause of cancer mortality. Recently, our group and others have demonstrated the oncolytic activity of the Zika virus (ZIKV) against glioblastoma. The peculiar features of this virus offer the opportunity to use an agent already tested in vivo through natural transmission, with minimal effects on adults, to specifically target a tumor such as glioblastoma. This remarkable specificity prompted us to explore the potential use of ZIKV oncolytic action against other tumor types. In particular, we focused on the subgroup of pancreatic tumors with a neuroendocrine origin known as neuroendocrine tumors (NETs). We found that ZIKV exerts its oncolytic activity by specifically infecting NET cells, leading to growth inhibition and cell death. We also assessed whether the oncolytic action could be extended to pancreatic tumors different from NETs. However, as expected, the viral specificity is limited to NETs and is not applicable to adenocarcinoma tumors, indicating a narrow spectrum of action for this virus. These findings support the potential use of ZIKV in therapeutic approaches not only in glioblastoma, but also against other tumors, such as neuroendocrine pancreatic tumors.

The creation of an engineered trachea with robust phenotype and sufficient mechanical properties for clinical application remains a challenge. In their work, Tang et al. 1 propose a stacked approach of alternating cartilaginous and fibrous rings to form a tracheal segment, which integrated and retain patency in rabbits for 8 weeks., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cigarette smoking impairs the lung innate immune response making smokers more susceptible to infections and severe symptoms. Dysregulation of cell death is emerging as a key player in chronic inflammatory conditions. We have recently reported that short exposure of human monocyte-derived macrophages (hMDMs) to cigarette smoke extract (CSE) altered the TLR4-dependent response to lipopolysaccharide (LPS). CSE caused inhibition of the MyD88-dependent inflammatory response and activation of TRIF/caspase-8/caspase-1 pathway leading to Gasdermin D (GSDMD) cleavage and increased cell permeability. Herein, we tested the hypothesis that activation of caspase-8 by CSE increased pro-inflammatory cell death of LPS-stimulated macrophages. To this purpose, we measured apoptotic and pyroptotic markers as well as the expression/release of pro-inflammatory mediators in hMDMs exposed to LPS and CSE, alone or in combination, for 6 and 24 h. We show that LPS/CSE-treated hMDMs, but not cells treated with CSE or LPS alone, underwent lytic cell death (LDH release) and displayed apoptotic features (activation of caspase-8 and -3/7, nuclear condensation, and mitochondrial membrane depolarization). Moreover, the negative regulator of caspase-8, coded by CFLAR gene, was downregulated by CSE. Activation of caspase-3 led to Gasdermin E (GSDME) cleavage. Notably, lytic cell death caused the release of the damage-associated molecular patterns (DAMPs) heat shock protein-60 (HSP60) and S100A8/A9. This was accompanied by an impaired inflammatory response resulting in inhibited and delayed release of IL6 and TNF. Of note, increased cleaved caspase-3, higher levels of GSDME and altered expression of cell death-associated genes were found in alveolar macrophages of smoker subjects compared to non-smoking controls. Overall, our findings show that CSE sensitizes human macrophages to cell death by promoting pyroptotic and apoptotic pathways upon encountering LPS. We propose that while the delayed inflammatory response may result in ineffective defenses against infections, the observed cell death associated with DAMP release may contribute to establish chronic inflammation. CS exposure sensitizes human macrophages to pro-inflammatory cell death. Upon exposure to LPS, CS inhibits the TLR4/MyD88 inflammatory response, downregulating the pro-inflammatory genes TNF and IL6 and the anti-apoptotic gene CFLAR, known to counteract caspase-8 activity. CS enhances caspase-8 activation through TLR4/TRIF, with a partial involvement of RIPK1, resulting on the activation of caspase-1/GSDMD axis leading to increased cell permeability and DAMP release through gasdermin pores [19]. At later timepoints caspase-3 becomes strongly activated by caspase-8 triggering apoptotic events which are associated with mitochondrial membrane depolarization, gasdermin E cleavage and secondary necrosis with consequent massive DAMP release., (© 2023. The Author(s).)

Reanimating facial structures following paralysis and muscle loss is a surgical objective that would benefit from improved options for harvesting appropriately sized muscle flaps. The objective of this study is to apply electrohydrodynamic processing to generate a cellularized, elastic, biocomposite scaffold that could develop and mature as muscle in a prepared donor site in vivo, and then be transferred as a thin muscle flap with a vascular and neural pedicle. First, an effective extracellular matrix (ECM) gel type is selected for the biocomposite scaffold from three types of ECM combined with poly(ester urethane)urea microfibers and evaluated in rat abdominal wall defects. Next, two types of precursor cells (muscle-derived and adipose-derived) are compared in constructs placed in rat hind limb defects for muscle regeneration capacity. Finally, with a construct made from dermal ECM and muscle-derived stem cells, protoflaps are implanted in one hindlimb for development and then microsurgically transferred as a free flap to the contralateral limb where stimulated muscle function is confirmed. This construct generation and in vivo incubation procedure may allow the generation of small-scale muscle flaps appropriate for transfer to the face, offering a new strategy for facial reanimation., (© 2023 Wiley-VCH GmbH.)

Development of tissue engineered scaffolds for cardiac valve replacement is nearing clinical translation. While much work has been done to characterize mechanical behavior of native and bioprosthetic valves, and incorporate those data into models improving valve design, similar work for degradable valve scaffolds is lacking. This is particularly important given the implications mechanics have on short-term survival and long-term remodeling. As such, this study aimed to characterize spatially-resolved strain profiles on the leaflets of degradable polymeric valve scaffolds, manipulating common design features such as material stiffness by blending poly(carbonate urethane)urea with stiffer polymers, and geometric configuration, modeled after either a clinically-used valve design (Mk1 design) or an anatomically "optimized" design (Mk2 design). It was shown that material stiffness plays a significant role in overall valve performance, with the stiffest valve groups showing asymmetric and incomplete opening during systole. However, the geometric configuration had a significantly greater effect on valve performance as well as strain magnitude and distribution. Major findings in the strain maps included systolic strains having overall higher strain magnitudes than diastole, and peak radial-direction strain concentrations in the base region of Mk1 valves during systole, with a significant mitigation of radial strain in Mk2 valves. The high tunability of tissue engineered scaffolds is a great advantage for valve design, and the results reported here indicate that design parameters have significant and unequal impact on valve performance and mechanics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: W.R. Wagner, A. D'Amore are inventors on patent #US11129711B2 issued to RiMED Foundation and University of Pittsburgh and licensed to Neoolife, Inc. W.R. Wagner reports a relationship with Neoolife that includes: equity or stocks. A. D'Amore reports a relationship with Neoolife that includes: equity or stocks, chief technical officer, board member., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The genome of SARS-CoV-2 coronavirus is made up of a single-stranded RNA fragment that can assume a specific secondary structure, whose stability can influence the virus's ability to reproduce. Recent studies have identified putative guanine quadruplex sequences in SARS-CoV-2 genome fragments that are involved in coding for both structural and non-structural proteins. In this contribution, we focus on a specific G-rich sequence referred to as RG-2, which codes for the non-structural protein 10 (Nsp10) and assumes a guanine-quadruplex (G4) arrangement. We provide the secondary structure of RG-2 G4 at atomistic resolution by molecular modeling and simulation, validated by the superposition of experimental and calculated electronic circular dichroism spectra. Through both experimental and simulation approaches, we have demonstrated that pyridostatin (PDS), a widely recognized G4 binder, can bind to and stabilize RG-2 G4 more strongly than RG-1, another G4 forming sequence that was previously proposed as a potential target for antiviral drug candidates. Overall, this study highlights RG-2 as a valuable target to inhibit the translation and replication of SARS-CoV-2, paving the way towards original therapeutic approaches against emerging RNA viruses., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Neural stem cells (NSCs) were described for the first time more than two decades ago for their ability to differentiate into all neural cell lineages. The isolation of NSCs from adults and embryos was carried out by various laboratories and in different species, from mice to humans. Similarly, no more than two decades ago, cancer stem cells were described. Cancer stem cells, previously identified in hematological malignancies, have now been isolated from several solid tumors (breast, brain, and gastrointestinal compartment). Though the origin of these cells is still unknown, there is a wide consensus about their role in tumor onset, propagation and, in particular, resistance to treatments. Normal and neoplastic neural stem cells share common characteristics, and can thus be considered as two sides of the same coin. This is particularly true in the case of the Zika virus (ZIKV), which has been described as an inhibitor of neural development by specifically targeting NSCs. This understanding prompted us and other groups to evaluate ZIKV action in glioblastoma stem cells (GSCs). The results indicate an oncolytic activity of this virus vs. GSCs, opening potentially new possibilities in glioblastoma treatment.

Mesenchymal stromal/stem cells (MSCs) have emerged as a therapeutic tool in regenerative medicine. Recent studies have shown that exosome (EXO)-derived microRNAs (miRNAs) play a crucial role in mediating MSC functions. Additionally, intracellular miRNAs have been found to regulate MSC therapeutic capacities. However, the molecular mechanisms underlying miRNA-mediated MSC effects are not fully understood. We used 3D culture and IFN-γ to prime/enhance the MSC therapeutic effects in terms of functional miRNAs. After priming, our analysis revealed stable variations in intracellular miRNA among the MSC biological replicates. Conversely, a significant variability of miRNA was observed among EXOs released from biological replicates of the priming treatment. For each priming, we observed distinct miRNA expression profiles between the MSCs and their EXOs. Moreover, in both types of priming, gene ontology (GO) analysis of deregulated miRNAs highlighted their involvement in tissue repair/regeneration pathways. In particular, the 3D culture enhanced angiogenic properties in both MSCs and EXOs, while IFN-γ treatment enriched miRNAs associated with immunomodulatory pathways. These findings suggest that 3D culture and IFN-γ treatment are promising strategies for enhancing the therapeutic potential of MSCs by modulating miRNA expression. Additionally, the identified miRNAs may contribute to understanding the molecular mechanisms underlying the miRNA-mediated therapeutic effects of MSCs.

Glypican-3 (GPC-3) is a heparin sulfate proteoglycan located extracellularly and anchored to the cell membrane of transformed hepatocytes. GPC-3 is not expressed in normal or cirrhotic liver tissue but is overexpressed in hepatocellular carcinoma (HCC). Because of this, GPC-3 is one of the most important emerging immunotargets for treatment and as an early detection marker of HCC. To determine if GPC-3 domains associated with serum small extracellular vesicles (sEVs) could be used as an HCC diagnostic marker, we predicted in silico GPC-3 structural properties and tested for the presence of its full-length form and/or cleaved domains in serum sEVs isolated from patients with HCC. Structural analysis revealed that the Furin cleavage site of GPC-3 is exposed and readily accessible, suggesting the facilitation of GPC-3 cleavage events. Upon isolation of sEVs from both hepatocytes, culture media and serum of patients with HCC were studied for GPC-3 content. This data suggests that Furin-dependent GPC-3 cleaved domains could be a powerful tool for detection of initial stages of HCC and serve as a predictor for disease prognosis.

Immunoproteasome inhibition is a promising strategy for the treatment of hematological malignancies, autoimmune diseases, and inflammatory diseases. The design of non-covalent inhibitors of the immunoproteasome β1i/β5i catalytic subunits could be a novel approach to avoid the drawbacks of the known covalent inhibitors, such as toxicity due to off-target binding. In this work, we report the biological evaluation of thirty-four compounds selected from a commercially available collection. These hit compounds are the outcomes of a virtual screening strategy including a dynamic pharmacophore modeling approach onto the β1i subunit and a pharmacophore/docking approach onto the β5i subunit. The computational studies were first followed by in vitro enzymatic assays at 100 μM. Only compounds capable of inhibiting the enzymatic activity by more than 50% were characterized in detail using Tian continuous assays, determining the dissociation constant ( K i ) of the non-covalent complex where K i is also the measure of the binding affinity. Seven out of thirty-four hits showed to inhibit β1i and/or β5i subunit. Compound 3 is the most active on the β1i subunit with K i = 11.84 ± 1.63 µM, and compound 17 showed K i = 12.50 ± 0.77 µM on the β5i subunit. Compound 2 showed inhibitory activity on both subunits ( K i = 12.53 ± 0.18 and K i = 31.95 ± 0.81 on the β1i subunit and β5i subunit, respectively). The induced fit docking analysis revealed interactions with Thr1 and Phe31 of β1i subunit and that represent new key residues as reported in our previous work. Onto β5i subunit, it interacts with the key residues Thr1, Thr21, and Tyr169. This last hit compound identified represents an interesting starting point for further optimization of β1i/β5i dual inhibitors of the immunoproteasome.

ADAM15 is a member of the disintegrin-metalloproteinase family of sheddases, which plays a role in several biological processes including cartilage homeostasis. In contrast with well-characterized ADAMs, such as the canonical sheddases ADAM17 and ADAM10, little is known about substrates of ADAM15 or how the enzyme exerts its biological functions. Herein, we used "surface-spanning enrichment with click-sugars (SUSPECS)" proteomics to identify ADAM15 substrates and/or proteins regulated by the proteinase at the cell surface of chondrocyte-like cells. Silencing of ADAM15 by siRNAs significantly altered membrane levels of 13 proteins, all previously not known to be regulated by ADAM15. We used orthogonal techniques to validate ADAM15 effects on 3 of these proteins which have known roles in cartilage homeostasis. This confirmed that ADAM15-silencing increased cell surface levels of the programmed cell death 1 ligand 2 (PDCD1LG2) and reduced cell surface levels of vasorin and the sulfate transporter SLC26A2 through an unknown post-translational mechanism. The increase of PDCD1LG2 by ADAM15 knockdown, a single-pass type I transmembrane protein, suggested it could be a proteinase substrate. However, shed PDCD1LG2 could not be detected even by a data-independent acquisition mass spectrometry, a highly sensitive method for identification and quantification of proteins in complex protein samples, suggesting that ADAM15 regulates PDCD1LG2 membrane levels by a mechanism different from ectodomain shedding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Calligaris, Yang, Bonelli, Spanò, Müller, Lichtenthaler, Troeberg and Scilabra.)

SARS-CoV-2, which causes COVID-19, has altered human activities all over the world and has become a global hazard to public health. Despite considerable advancements in pandemic containment techniques, in which vaccination played a key role, COVID-19 remains a global threat, particularly for frail patients and unvaccinated individuals, who may be more susceptible to developing ARDS. Several studies reported that patients with COVID-19-related ARDS who were treated with ECMO had a similar survival rate to those with COVID-19-unrelated ARDS. In order to shed light on the potential mechanisms underlying the COVID-19 infection, we conducted this proof-of-concept study using single-cell V(D)J and gene expression sequencing of B cells to examine the dynamic changes in the transcriptomic BCR repertoire present in patients with COVID-19 at various stages. We compared a recovered and a deceased COVID-19 patient supported by ECMO with one COVID-19-recovered patient who did not receive ECMO treatment and one healthy subject who had never been infected previously. Our analysis revealed a downregulation of FXYD, HLA-DRB1, and RPS20 in memory B cells; MTATP8 and HLA-DQA1 in naïve cells; RPS4Y1 in activated B cells; and IGHV3-73 in plasma cells in COVID-19 patients. We further described an increased ratio of IgA + IgG to IgD + IgM, suggestive of an intensive memory antibody response, in the COVID ECMO D patient. Finally, we assessed a V(D)J rearrangement of heavy chain IgHV3, IGHJ4, and IGHD3/IGHD2 families in COVID-19 patients regardless of the severity of the disease.

Background: The current curative approaches for ischemia/reperfusion injury on liver transplantation are still under debate for their safety and efficacy in patients with end-stage liver disease. We present the SIMVA statin donor treatment before Liver Transplants study., Methods: SIMVA statin donor treatment before Liver Transplants is a monocentric, double-blind, randomized, prospective tial aiming to compare the safety and efficacy of preoperative brain-dead donors' treatment with the intragastric administration of 80 mg of simvastatin on liver transplant recipient outcomes in a real-life setting. Primary aim was incidence of patient and graft survival at 90 and 180 d posttransplant; secondary end-points were severe complications., Results: The trial enrolled 58 adult patients (18-65 y old). The minimum follow-up was 6 mo. No patient or graft was lost at 90 or 180 d in the experimental group (n = 28), whereas patient/graft survival were 93.1% ( P = 0.016) and 89.66% ( P = 0.080) at 90 d and 86.21% ( P = 0.041) and 86.2% ( P = 0.041) at 180 d in the control group (n = 29). The percentage of patients with severe complications (Clavien-Dindo ≥IIIb) was higher in the control group, 55.2% versus 25.0% in the experimental group ( P = 0.0307). The only significant difference in liver tests was a significantly higher gamma-glutamyl transferase and alkaline phosphatase at 15 d ( P = 0.017), ( P = 0.015) in the simvastatin group., Conclusions: Donor simvastatin treatment is safe, and may significantly improve early graft and patient survival after liver transplantation, although further research is mandatory., Competing Interests: D.P. and E.O. reported receiving grants from Italian government research funding. The other authors declare no conflicts of interest. The authors hereby certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or nonfinancial interest (such as personal or professional relationships, affiliations, knowledge, or beliefs) in the subject matter or materials discussed in this manuscript., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

The family of protein kinases comprises more than 500 genes involved in numerous functions. Hence, their physiological dysfunction has paved the way toward drug discovery for cancer, cardiovascular, and inflammatory diseases. As a matter of fact, Kinase binding sites high similarity has a double role. On the one hand it is a critical issue for selectivity, on the other hand, according to poly-pharmacology, a synergistic controlled effect on more than one target could be of great pharmacological interest. Another important aspect of binding similarity is the possibility of exploit it for repositioning of drugs on targets of the same family. In this study, we propose our approach called Kinase drUgs mAchine Learning frAmework (KUALA) to automatically identify kinase active ligands by using specific sets of molecular descriptors and provide a multi-target priority score and a repurposing threshold to suggest the best repurposable and non-repurposable molecules. The comprehensive list of all kinase-ligand pairs and their scores can be found at https://github.com/molinfrimed/multi-kinases ., (© 2022. The Author(s).)

Theoretically, Aspergillus spp. grow in culture media, but frequently, blood cultures of patients with invasive Aspergillosis are negative, even if until now, the reasons are not clear. This aspect underlines the lack of a good strategy for the cultivation and isolation of Aspergillus spp. In order to develop a complete analytical method to detect Aspergillus in clinical and pharmaceutical samples, we investigated the growth performance of two blood culture systems versus the pharmacopeia standard method. At <72 h, all test systems showed comparable sensitivity, about 1−2 conidia. However, the subculture analysis showed a suboptimal recovery for the methods, despite the positive growth and the visualization of the “Aspergillus balls” in the culture media. To investigate this issue, we studied three different subculture approaches: (i) the use of a sterile subculture unit, (ii) the use of a sterile subculture unit and the collection of a larger aliquot (100 µL), following vigorous agitation of the vials, and (iii) to decapsulate the bottle, withdrawing and centrifuging the sample, and aliquot the pellet onto SDA plates. Our results showed that only the third procedure recovered Aspergillus from all positive culture bottles. This work confirmed that our strategy is a valid and faster method to culture and isolate Aspergillus spp. from blood culture bottles.

Although a disease is defined as rare when it has a prevalence of less than 1:2000, the overall prevalence of rare diseases in the population is greater than 1%. Among potential organ donors, a similar frequency is observed. To date, guidelines have not been established, and operational decisions have been made empirically, case- by-case, based on the experience and expertise of clinicians. For this reason, the Italian Superior Health Council (CSS) has appointed a working Group to address "patients with a rare disease as potential organ donors," with the aim of devising recommendations for the management of transplant cases in which the donors have a rare disease. This group evaluated 493 diseases (10% of all rare diseases, including over 95% of patients with a rare disease) to deliver a technical report dealing with the suitability of organ donation and transplantation, with a focus on the organs most frequently used, including kidney, liver, heart, lung, and pancreas. This work has made it clear that a rare disease "per se" does not contraindicate organ donation at all. Indeed, in donors affected by a rare disease, almost 80% of the organs are suitable for transplantation, approximately 7% are unsuitable, and approximately 14% are suitable as non-standard with an acceptable risk., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Klebsiella pneumoniae is a Gram-negative pathogen of clinical relevance, which can provoke serious urinary and blood infections and pneumonia. This bacterium is a major public health threat due to its resistance to several antibiotic classes. Using a reverse vaccinology approach, 7 potential antigens were identified, of which 4 were present in most of the sequences of Italian carbapenem-resistant K. pneumoniae clinical isolates. Bioinformatics tools demonstrated the antigenic potential of these bacterial proteins and allowed for the identification of T and B cell epitopes. This led to a rational design and in silico characterization of a multiepitope vaccine against carbapenem-resistant K. pneumoniae strains. As adjuvant, the mycobacterial heparin-binding hemagglutinin adhesin (HBHA), which is a Toll-like receptor 4 (TLR-4) agonist, was included, to increase the immunogenicity of the construct. The multiepitope vaccine candidate was analyzed by bioinformatics tools to assess its antigenicity, solubility, allergenicity, toxicity, physical and chemical parameters, and secondary and tertiary structures. Molecular docking binding energies to TLR-2 and TLR-4, two important innate immunity receptors involved in the immune response against K. pneumoniae infections, and molecular dynamics simulations of such complexes supported active interactions. A codon optimized multiepitope sequence cloning strategy is proposed, for production of recombinant vaccine in classical bacterial vectors. Finally, a 3 dose-immunization simulation with the multiepitope construct induced both cellular and humoral immune responses. These results suggest that this multiepitope construct has potential as a vaccination strategy against carbapenem-resistant K. pneumoniae and deserves further validation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Repetitive sequences represent about half of the human genome. They are actively transcribed and play a role during development and in epigenetic regulation. The altered activity of repetitive sequences can lead to genomic instability and they can contribute to the establishment or the progression of degenerative diseases and cancer transformation. In this work, we analyzed the expression profiles of DNA repetitive sequences in the breast cancer specimens of the HMUCC cohort. Satellite expression is generally upregulated in breast cancers, with specific families upregulated per histotype: in HER2-enriched cancers, they are the human satellite II (HSATII), in luminal A and B, they are part of the ALR family and in triple-negative, they are part of SAR and GSAT families, together with a perturbation in the transcription from endogenous retroviruses and their LTR sequences. We report that the background expression of repetitive sequences in healthy tissues of cancer patients differs from the tissues of non-cancerous controls. To conclude, peculiar patterns of expression of repetitive sequences are reported in each specimen, especially in the case of transcripts arising from satellite repeats.

Tissue inhibitors of metalloproteases (TIMPs) belong to a fascinating protein family expressed in all Metazoa. They act as regulators of the turnover of the extracellular matrix, and they are consistently involved in essential processes. Herein, we recapitulate the main activities of mammalian TIMPs (TIMP1-4) in the control of extracellular-matrix degradation and pathologies associated with aberrant proteostasis. We delineate the activity of TIMPs in the control of extracellular matrix (ECM) homeostasis and discuss the diversity of TIMPs across metazoans taking into account the emergence of the components of the ECM during evolution. Thus, the TIMP repertoire herein analysed includes the homologues from cnidarians, which are coeval with the origins of ECM components; protostomes (molluscs, arthropods and nematodes); and deuterostomes (echinoderms and vertebrates). Several questions, including the maintenance of the structure despite low sequence similarity and the strategies for TIMP engineering, shed light on the possibility to use recombinant TIMPs integrating unique features and binding selectivity for therapeutic applications in the treatment of inflammatory pathologies., Competing Interests: The authors declare no conflicts of interest.

Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5β, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5β has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5β is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials., (© 2022. The Author(s).)

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma., Competing Interests: The authors declare no conflicts of interest.

Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and can be activated by several cytokines, including interleukin-2 (IL-2), IL-15, and interferon-alpha (IFN-α). By exploiting the Huh7.5 hepatoma cell line infected with the HCV JFH1 genome, we provide novel insights into the antiviral effector functions of human primary NK cells after cytokine stimulation. NK cells activated with IFN-α (IFNα-NKs) had enhanced contact-dependent and -independent responses as compared with NK cells activated with IL-2/IL-15 (IL2/IL15-NKs) and could inhibit HCV replication both in vitro and in vivo. Importantly, IFN-α, but not IL-2/IL-15, protected NK cells from the functional inhibition exerted by HCV. By performing flow cytometry, multiplex cytokine profiling, and mass-spectrometry-based proteomics, we discovered that IFNα-NKs secreted high levels of galectin-9 and interferon-gamma (IFN-γ), and by conducting neutralization assays, we confirmed the major role of these molecules in HCV suppression. We speculated that galectin-9 might act extracellularly to inhibit HCV binding to host cells and downstream infection. In silico approaches predicted the binding of HCV envelope protein E2 to galectin-9 carbohydrate-recognition domains, and co-immunoprecipitation assays confirmed physical interaction. IFN-γ, on the other hand, triggered the intracellular expressions of two antiviral gate-keepers in target cells, namely, myxovirus-1 (MX1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Collectively, our data add more complexity to the antiviral innate response mediated by NK cells and highlight galectin-9 as a key molecule that might be exploited to neutralize productive viral infection.

Chronic hepatitis C virus (HCV) infection is the principal etiology of cirrhosis and, ultimately, hepatocellular carcinoma (HCC). At present, approximately 71 million people are chronically infected with HCV, and 10%-20% of these are expected to develop severe liver complications throughout their lifetime. Scientific evidence has clearly shown the causal association between miRNAs, HCV infection and HCC. Although it is not completely clear whether miRNA dysregulation in HCC is the cause or the consequence of its development, variations in miRNA patterns have been described in different liver diseases, including HCC. Many studies have analyzed the importance of circulating miRNAs and their effect on cell proliferation and apoptosis. In this Review, we aim to summarize current knowledge on the association between miRNA, HCV and HCC from a diagnostic point of view, and also the potential implications for therapeutic approaches., Competing Interests: Conflict-of-interest statement: The authors declare that they have no conflict of interest., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.

Statistical tests of differential expression usually suffer from two problems. Firstly, their statistical power is often limited when applied to small and skewed data sets. Secondly, gene expression data are usually discretized by applying arbitrary criteria to limit the number of false positives. In this work, a new statistical test obtained from a convolution of multivariate hypergeometric distributions, the Hy-test, is proposed to address these issues. Hy-test has been carried out on transcriptomic data from breast and kidney cancer tissues, and it has been compared with other differential expression analysis methods. Hy-test allows implicit discretization of the expression profiles and is more selective in retrieving both differential expressed genes and terms of Gene Ontology. Hy-test can be adopted together with other tests to retrieve information that would remain hidden otherwise, e.g., terms of (1) cell cycle deregulation for breast cancer and (2) "programmed cell death" for kidney cancer., (© 2022. The Author(s).)

Immunohistochemistry for vascular network analysis plays a fundamental role in basic science, translational research and clinical practice. However, identifying vascularization in histological tissue images is time consuming and markedly depends on the operator's experience. In this study, we present "blood vessel detection-BVD", an automatic algorithm for quantitative analysis of blood vessels in immunohistochemical images. BVD is based on extraction and analysis of low-level image features and spatial filtering techniques, which do not require a training phase. BVD algorithm performance was comparatively evaluated on histological sections from three different in vivo experiments. Collectively, 173 independent images were analyzed, and the algorithm's results were compared to those obtained by human operators. The developed BVD algorithm proved to be a robust and versatile tool, being able to quantify number, area, and spatial distribution of blood vessels within all three considered histologic datasets. BVD is provided as an open-source application working on different operating systems. BVD is supported by a user-friendly graphical interface designed to facilitate large-scale analysis., (© 2022. The Author(s).)

The development of high-resolution molecular printing allows the engineering of analytical platforms enabling applications at the interface between chemistry and biology, i.e. in biosensing, electronics, single-cell biology, and point-of-care diagnostics. Their successful implementation stems from the combination of large area printing at resolutions from sub-100 nm up to macroscale, whilst controlling the composition and volume of the ink, and reconfiguring the deposition features in due course. Similar to handwriting pens, the engineering of continuous writing systems tackles the issue of the tedious ink replenishment between different printing steps. To this aim, this review article provides an unprecedented analysis of the latest continuous printing methods for bioanalytical chemistry, focusing on ink deposition systems based on specific sets of technologies that have been developed to this aim, namely nanofountain probes, microcantilever spotting, capillary-based polymer pens and continuous 3D printing. Each approach will be discussed revealing the most important applications in the fields of biosensors, lab-on-chips and diagnostics.

In this work, we assessed and characterized the epidemiological scenario of carbapenem-resistant Klebsiella pneumoniae strains (CR-Kp) at IRCCS-ISMETT, a transplantation hospital in Palermo, Italy, from 2008 to 2017. A total of 288 K. pneumoniae clinical isolates were selected based on their resistance to carbapenems. Molecular characterization was also done in terms of the presence of virulence and resistance genes. All patients were inpatients from our facility and clinical isolates were collected from several sources, either from infection or colonization cases. We observed that, in agreement with the Italian epidemiological scenario, initially only ST258 and ST512 clade II (but not from clade I) were identified from 2008 to 2011. From 2012 onwards, other STs have been observed, including the clinically relevant ST101 and ST307, but also others not previously observed in other Italian health settings, such as ST220 and ST753. The presence of genes involved in resistance and virulence was confirmed, and a heterogeneous genetic resistance profile throughout the years was observed. Our work highlights that resistance genes are rapidly disseminating between different and novel K. pneumoniae clones which, combined with resistance to multiple antibiotics, can derive into more aggressive and pathogenic multidrug-resistant strains of clinical importance. Our results stress the importance of continuous surveillance of CR Enterobacterales in health facilities so that novel STs carrying resistance and virulence genes that may become increasingly pathogenic can be identified and adequate therapies to adopted to avoid their dissemination and derived pathologies., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

In recent years, the debate in the field of applications of Deep Learning to Virtual Screening has focused on the use of neural embeddings with respect to classical descriptors in order to encode both structural and physical properties of ligands and/or targets. The attention on embeddings with the increasing use of Graph Neural Networks aimed at overcoming molecular fingerprints that are short range embeddings for atomic neighborhoods. Here, we present EMBER, a novel molecular embedding made by seven molecular fingerprints arranged as different "spectra" to describe the same molecule, and we prove its effectiveness by using deep convolutional architecture that assesses ligands' bioactivity on a data set containing twenty protein kinases with similar binding sites to CDK1. The data set itself is presented, and the architecture is explained in detail along with its training procedure. We report experimental results and an explainability analysis to assess the contribution of each fingerprint to different targets.

The proteolytical cleavage of transmembrane proteins with subsequent release of their extracellular domain, so-called ectodomain shedding, is a post-translational modification that plays an essential role in several biological processes, such as cell communication, adhesion and migration. Metalloproteases are major proteases in ectodomain shedding, especially the disintegrin metalloproteases (ADAMs) and the membrane-type matrix metalloproteases (MT-MMPs), which are considered to be canonical sheddases for their membrane-anchored topology and for the large number of proteins that they can release. The unique ability of TIMP-3 to inhibit different families of metalloproteases, including the canonical sheddases (ADAMs and MT-MMPs), renders it a master regulator of ectodomain shedding. This review provides an overview of the different functions of TIMP-3 in health and disease, with a major focus on the functional consequences in vivo related to its ability to control ectodomain shedding. Furthermore, herein we describe a collection of mass spectrometry-based approaches that have been used in recent years to identify new functions of sheddases and TIMP-3. These methods may be used in the future to elucidate the pathological mechanisms triggered by the Sorsby's fundus dystrophy variants of TIMP-3 or to identify proteins released by less well characterized TIMP-3 target sheddases whose substrate repertoire is still limited, thus providing novel insights into the physiological and pathological functions of the inhibitor.

The advent of induced pluripotent stem cell (iPSC) technology, which allows to transform one cell type into another, holds the promise to produce therapeutic cells and organs on demand. Realization of this objective is contingent on the ability to demonstrate quality and safety of the cellular product for its intended use. Bottlenecks and backlogs to the clinical use of iPSCs have been fully outlined and a need has emerged for safer and standardized protocols to trigger cell reprogramming and functional differentiation. Amidst great challenges, in particular associated with lengthy culture time and laborious cell characterization, a demand for faster and more accurate methods for the validation of cell identity and function at different stages of the iPSC manufacturing process has risen. Artificial intelligence-based methods are proving helpful for these complex tasks and might revolutionize the way iPSCs are managed to create surrogate cells and organs. Here, we briefly review recent progress in artificial intelligence approaches for evaluation of iPSCs and their derivatives in experimental studies., (© 2021. The Author(s).)

Since its identification, HCV has been considered one of the main causes of hepatitis and liver cancer. Currently, the molecular mechanisms of HCC development induced by HCV infection have not been sufficiently clarified. The recent discovery of novel treatments that inhibit HCV replication gave rise to new questions concerning HCC mechanisms. In particular, the HCV eradication mediated by new direct-acting antiviral (DAAs) drugs does not exclude the possibility of de novo HCC development; this finding opened more questions on the interplay between liver cells and the virus. Different groups have investigated the pathways leading to cancer recurrence in patients treated with DAAs. For this reason, we tried to gain molecular insights into the changes induced by HCV infection in the target liver cells. In particular, we observed an increase in microRNA34a (miR34a) expression following HCV infection of HCC cell line Huh7.5. In addition, Huh7.5 treated with extracellular vesicles (EVs) from the previously HCV-infected Huh7.5 underwent apoptosis. Since miR34 expression was increased in Huh7.5 EVs, we hypothesized a paracrine mechanism of viral infection mediated by miR34a cargo of EVs. The balance between viral infection and cell transformation may raise some questions on the possible use of antiviral drugs in association with antineoplastic treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Badami, Carcione, Chinnici, Tinnirello, Conaldi and Iannolo.)