Ferroptosis resistance cooperates with cellular senescence in the overt stage of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis

Cellular senescence and ferroptosis are the two main, fine-tuned processes in tissue damage restraint; however, they can be overactivated in pathologies such as nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH), becoming dangerous stimuli. Senescence is characterized by a decline in cell division and an abnormal release of reactive oxygen species (ROS), and ferroptosis is represented by iron deposition associated with an excessive accumulation of ROS. ROS and cellular stress pathways are also drivers of NAFLD/NASH development. The etiology of NAFLD/NASH lies in poor diets enriched in fat and sugar. This food regimen leads to liver steatosis, resulting in progressive degeneration of the organ, with a late onset of irreversible fibrosis and cirrhosis. Few studies have investigated the possible connection between senescence and ferroptosis in NAFLD/NASH progression, despite the two events sharing some molecular players. We hypothesized a possible link between senescence and ferroptosis in a NAFLD background. To thoroughly investigate this in the context of “Western-style” diet (WSD) abuse, we used an amylin-modified liver NASH mouse model. The main NASH hallmarks have been confirmed in this model, as well as an increase in apoptosis, and Ki-67 and p53 expression in the liver. Senescent beta-galactosidase-positive cells were elevated, as well as the expression of the related secretory molecules Il-6 and MMP-1. Features of DNA damage and iron-overload were found in the livers of NASH mice. Gpx4 (glutathione peroxidase 4) expression, counteracting ferroptotic cell death, was increased. Notably, an increased number of senescent cells showing overexpression of gpx4 was also found. Our data seem to suggest that senescent cells acquire a gpx4-mediated mechanism of ferroptosis resistance and thus remain in the liver, fostering the deterioration of liver fitness.Key words: NAFLD/NASH, senescence, ferroptosis, beta-galactosidase, gpx4     

Stereotactic radiotherapy for liver oligometastases

The liver is the first metastatic site in 15–25% of colorectal cancer patients and one of the first metastatic sites for lung and breast cancer patients.A computed tomography (CT ) scan with contrast medium is a standard procedure for assessing liver lesions but magnetic resonance imaging (MRI) characterizes small lesions better thanks to its high soft-tissue contrast. Positron emission tomography with computed tomography (PET-CT ) plays a complementary role in the diagnosis of liver metastases. Triphasic (arterial, venous and time-delayed) acquisition of contrast-medium CT images is the first step in treatment planning. Since the liver exhibits a relatively wide mobility due to respiratory movements and bowel filling, appropriate techniques are needed for target identification and motion management. Contouring requires precise recognition of target lesion edges. Information from contrast MRI and/or PET-CT is crucial as they best visualize metastatic disease in the parenchyma. Even though different fractionation schedules were reported, doses and fractionation schedules for liver stereotactic radiotherapy (SRT ) have not yet been established. The best local control rates were obtained with BED₁₀ values over 100 Gy. Local control rates from most retrospective studies, which were limited by short follow-ups and included different primary tumors with intrinsic heterogeneity, ranged from 60% to 90% at 1 and 2 years. The most common SRT-related toxicities are increases in liver enzymes, hyperbilirubinemia and hypoalbuminemia. Overall, late toxicity is mild even in long-term follow-ups.     

Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

The actin motor protein myosin VI is a multivalent protein with diverse functions. Here, we identified and characterised a myosin VI ubiquitous interactor, the oral‐facial‐digital syndrome 1 (OFD1) protein, whose mutations cause malformations of the face, oral cavity, digits and polycystic kidney disease. We found that myosin VI regulates the localisation of OFD1 at the centrioles and, as a consequence, the recruitment of the distal appendage protein Cep164. Myosin VI depletion in non‐tumoural cell lines causes an aberrant localisation of OFD1 along the centriolar walls, which is due to a reduction in the OFD1 mobile fraction. Finally, loss of myosin VI triggers a severe defect in ciliogenesis that could be, at least partially, ascribed to an impairment in the autophagic removal of OFD1 from satellites. Altogether, our results highlight an unprecedent layer of regulation of OFD1 and a pivotal role of myosin VI in coordinating the formation of the distal appendages and primary cilium with important implications for the genetic disorders known as ciliopathies.     

B7-H1 Blockade Increases Survival of Dysfunctional CD8+ T Cells and Confers Protection against Leishmania donovani Infections

Experimental visceral leishmaniasis (VL) represents an exquisite model to study CD8⁺ T cell responses in a context of chronic inflammation and antigen persistence, since it is characterized by chronic infection in the spleen and CD8⁺ T cells are required for the development of protective immunity. However, antigen-specific CD8⁺ T cell responses in VL have so far not been studied, due to the absence of any defined Leishmania-specific CD8⁺ T cell epitopes. In this study, transgenic Leishmania donovani parasites expressing ovalbumin were used to characterize the development, function, and fate of Leishmania-specific CD8⁺ T cell responses. Here we show that L. donovani parasites evade CD8⁺ T cell responses by limiting their expansion and inducing functional exhaustion and cell death. Dysfunctional CD8⁺ T cells could be partially rescued by in vivo B7-H1 blockade, which increased CD8⁺ T cell survival but failed to restore cytokine production. Nevertheless, B7-H1 blockade significantly reduced the splenic parasite burden. These findings could be exploited for the design of new strategies for immunotherapeutic interventions against VL.     

Soil-Atmosphere Methane Exchange in Undisturbed and Burned Mediterranean Shrubland of Southern Italy

Soils represent the primary biotic sink for atmospheric methane (CH₄). Uncertainty is associated, however, with global soil CH₄ consumption because of the few data available from many areas and, in particular, from Mediterranean-type ecosystems. In this study, soil-atmosphere CH₄ exchange was measured for one year in a coastal Italian shrubland (maquis), from both undisturbed areas and areas treated with experimental fire. Although fire represents one of the most frequent disturbance factors in seasonally dry environments, very few studies in these ecosystems have focused on its effect on soil CH₄ fluxes. Significant differences in soil ammonium content, water content, and temperature were measured between burned and unburned plots, however, no statistical differences were observed for CH₄ fluxes. CH₄ fluxes varied between −0.39 and −16.1 mg CH₄ m⁻² day⁻¹ and temporal variations were mainly driven by variations in soil water content and temperature. The highest CH₄ oxidation rates were measured during the driest and warmest period. Low gravimetric soil water content in the top 10 cm, as well as high NH₄⁺ concentration, did not seem to reduce methanotrophic activity, suggesting that maximal CH₄ oxidation activity might take place deeper in the soil profile, at least during part of the year.     

Identification of genes involved in growth inhibition of breast cancer cells transduced with estrogen receptor

Estrogen receptor alpha (ERalpha)-negative breast cancer cells display an aggressive phenotype. We previously showed that adenoviral expression of ERalpha in ER-negative breast cancer cells leads to an estrogen-dependent down-regulation of the proliferation, which could be of interest to control the growth of such cells. In this study, we observed an increase in protein levels of p21 and p27 cyclin-dependent kinase inhibitors, whereas pRb phosphorylation is strongly decreased. Flow cytometry experiments showed a slower transit of cells in G1 (hormone-independent), a hormone-induced accelerated transit through S phase and a possible arrest in G2/M phase. In addition, ERalpha-expressing cells were undergoing apoptosis. By using cDNA macroarrays, we identified a novel collection of genes regulated by liganded ERalpha potentially regulating cell cycle, apoptosis, cell signalling, stress response and DNA repair.     

Inhibition of protein phosphatase 2A activity by selective electrophile alkylation damage

Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.