Marialuisa Casella

MUS81 is a structure-specific endonuclease that processes DNA intermediates during mitosis and in S-phase following replication stress. It plays a crucial role in cleaving deprotected reversed forks in BRCA2-deficient cells. However, how MUS81 is regulated during replication stress in human cells remains unknown. Our study reveals that CHK2 binds to the MUS8–EME2 complex in S-phase through the FHA domain and positively regulates the formation of DSBs in response to persistent or pathological replication stress. Mechanistically, our cellular and biochemical data identify a dual-site phosphorylation regulatory mechanism involving priming at the CDK2 site S95 followed by CHK2-dependent modification of the activatory site S97. Phosphorylation of MUS81 does not affect recruitment to the stalled forks but is crucial for the replication stress-dependent association with SLX4 in S-phase. At deprotected forks, in BRCA2-depleted cells, the CHK2–MUS81 complex assembles downstream of fork reversal and degradation, and CHK2-dependent phosphorylation is essential for replication fork recovery and viability. Together, our findings elucidate a novel regulatory mechanism of the MUS81 complex in S-phase and reveal a previously unrecognized role of the ATM–CHK2 axis in responding to persistent replication fork arrest or fork deprotection in the absence of BRCA2.

Research on viper venom has expanded into diverse medical applications, including cancer treatment. This study investigates the potential of Vipera ammodytes venom in oncology, evaluating its cytotoxicity and chemosensitising effects on malignant melanoma cells. Proteomic analysis identified 125 proteins in the venom, with Phospholipases A2, C-type lectins, and metalloproteinases among the most abundant components. These proteins are associated with cytotoxic, anti-proliferative, and tumor-inhibiting properties. Three melanoma cell lines (M001, Me501, and A375) were used to assess venom cytotoxicity. The IC50 values demonstrated consistent venom sensitivity across cell lines (approximately 1.1 µg/mL). Combined treatment with venom and cisplatin significantly increased the cytotoxicity compared to single-agent treatments. Notably, venom enhanced the sensitivity of cisplatin in resistant cell lines (M001 and Me501), increasing cell mortality by up to 40%. The A375 cell line, inherently more sensitive to cisplatin, exhibited additional cytotoxic effects only at higher venom doses. The morphological changes observed under microscopy confirmed venom-induced cellular changes, further supporting its potential as an anti-cancer agent. The selective targeting of melanoma cells by venom components, particularly in muscle-associated metastases, suggests a unique therapeutic niche. While cisplatin was chosen for this pilot study due to its established cytotoxicity, future research will explore venom combinations with contemporary treatments such as immunotherapy and targeted therapies. Although preliminary, these findings provide a foundation for integrating venom-based strategies into advanced melanoma protocols, aiming to improve outcomes in resistant or metastatic cases.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial–mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFβ receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFβ signaling in regulating this process.

Polybrominated diphenyl ethers (PBDEs) are persistent organic chemicals implied as flame retardants. Humans are mainly exposed to BDE-47, -99, and -209 congeners by diet. PBDEs are metabolic disruptors with the liver as the main target organ. To investigate their mode of action at a human-relevant concentration, we exposed HepG2 cells to these congeners and their mixture at 1 nM, analyzing their transcriptomic and proteomic profiles. KEGG pathways and GSEA Hallmarks enrichment analyses evidenced that BDE-47 disrupted the glucose metabolism and hypoxia pathway; all the congeners and the MIX affected lipid metabolism and signaling Hallmarks regulating metabolism as mTORC1 and PI3K/AKT/MTOR. These results were confirmed by glucose secretion depletion and increased lipid accumulation, especially in BDE-47 and -209 treated cells. These congeners also affected the EGFR/MAPK signaling; further, BDE-47 enriched the estrogen pathway. Interestingly, BDE-209 and the MIX increased ERα gene expression, whereas all the congeners and the MIX induced ERβ and PPARα. We also found that PBDEs modulated several lncRNAs and that HNRNAP1 represented a central hub in all the four interaction networks. Overall, the PBDEs investigated affected glucose and lipid metabolism with different underlying modes of action, as highlighted by the integrated omics analysis, at a dietary relevant concentration. These results may support the mechanism-based risk assessment of these compounds in relation to liver metabolism disruption.

This work describes the activity of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and of its newly identified carboxylic acid metabolite on the human malaria parasite Plasmodium falciparum. NBDHEX has been previously identified as a potent cytotoxic agent against murine and human cancer cells as well as towards the protozoan parasite Giardia duodenalis. We show here that NBDHEX is active in vitro against all blood stages of P. falciparum, with the rare feature of killing the parasite stages transmissible to mosquitoes, the gametocytes, with a 4-fold higher potency than that on the pathogenic asexual stages. This activity importantly translates into blocking parasite transmission through the Anopheles vector in mosquito experimental infections. A mass spectrometry analysis identified covalent NBDHEX modifications in specific cysteine residues of five gametocyte proteins, possibly associated with its antiparasitic effect. The carboxylic acid metabolite of NBDHEX retains the gametocyte preferential inhibitory activity of the parent compound, making this novel P. falciparum transmission-blocking chemotype at least as a new tool to uncover biological processes targetable by gametocyte selective drugs. Both NBDHEX and its carboxylic acid metabolite show very limited in vitro cytotoxicity on VERO cells. This result and previous evidence that NBDHEX shows an excellent in vivo safety profile in mice and is orally active against human cancer xenografts make these molecules potential starting points to develop new P. falciparum transmission-blocking agents, enriching the repertoire of drugs needed to eliminate malaria.

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II–IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0–I, II and III–IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0–I) from late (III–IV) stages’ CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III–IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV’ FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.

Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/−2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.