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The formation of a tetrameric assembly is essential for the ability of the tumor suppressor protein p53 to act as a transcription factor. Such a quaternary conformation is driven by a specific tetramerization domain, separated from the central DNA-binding domain by a flexible linker. Despite the distance, functional crosstalk between the two domains has been reported. This phenomenon can explain the pathogenicity of some inherited or somatically acquired mutations in the tetramerization domain, including the widespread R337H missense mutation present in the population in south Brazil. In this work, we combined computational predictions through extended all-atom molecular dynamics simulations with functional assays in a genetically defined yeast-based model system to reveal structural features of p53 tetramerization domains and their transactivation capacity and specificity. In addition to the germline and cancer-associated R337H and R337C, other rationally designed missense mutations targeting a significant salt-bridge interaction that stabilizes the p53 tetramerization domain were studied (i.e., R337D, D352R, and the double-mutation R337D plus D352R). The simulations revealed a destabilizing effect of the pathogenic mutations within the p53 tetramerization domain and highlighted the importance of electrostatic interactions between residues 337 and 352. The transactivation assay, performed in yeast by tuning the expression of wild-type and mutant p53 proteins, revealed that p53 tetramerization mutations could decrease the transactivation potential and alter transactivation specificity, in particular by better tolerating negative features in weak DNA-binding sites. These results establish the effect of naturally occurring variations at positions 337 and 352 on p53's conformational stability and function.

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

Among scores and staging systems used for HCC, none showed a good prognostic ability in patients with advanced HCC treated with Sorafenib. We aimed to evaluate predictive factors of overall survival (OS) and drug response in HCC patients undergoing Sorafenib included in the Italian Liver Cancer (ITA.LI.CA.) multicenter cohort. Patients in the ITA.LI.CA database treated with Sorafenib and updated on 30 June 2019 were included. Demographic and clinical data before starting Sorafenib treatment were considered. For the evaluation of predictive factors for OS, a time-dependent Cox proportional hazard model was used. A total of 1107 patients were included in our analysis. The mean age was 64.3 years and 81.7% were male. Most patients were staged as BCLC B (205, 18.9%) or C (706, 65.1%). The median time of Sorafenib administration was 4 months (interquartile range (IQR) 2-12), and the median OS was 10 months (IQR: 4-20). A total of 263 patients (33.8%) out of 780 with available evaluation experienced objective tumoral response to Sorafenib. The Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) (hazard ratio (HR) 1.284), maximum tumoral diameter (HR 1.100), plasma total bilirubin (HR 1.119), aspartate amino transferase assessed as multiple of the upper normal value (HR 1.032), alpha-fetoprotein ≥200 ng/mL (HR 1.342), hemoglobin (HR 0.903) and platelet count (HR 1.002) were associated with OS at multivariate Cox regression analysis. Drug response was predicted by maximum tumoral diameter and platelet count. A novel prognostic nomogram for patients undergoing Sorafenib is hereby proposed. The novelty introduced is the comprehensive patient's assessment using common markers of patient's general status, liver damage and function and HCC biology. Further studies are required to test its accuracy and provide external validation.

Previous studies support the role of natural killer (NK) cells in controlling hepatocellular carcinoma (HCC) progression. However, ambiguity remains about the multiplicity and the role of different NK cell subsets, as a pro-oncogenic function has been suggested. We performed phenotypic and functional characterization of NK cells infiltrating HCC, with the corresponding nontumorous tissue and liver from patients undergoing liver resection for colorectal liver metastasis used as controls. We identified a reduced number of NK cells in tumors with higher frequency of CD56 BRIGHT CD16 - NK cells associated with higher expression of NKG2A, NKp44, and NKp30 and downregulation of NKG2D. Liver-resident (CXCR6 + ) NK cells were reduced in the tumors where T-bet hi Eomes lo expression was predominant. HCCs showed higher expression of CD49a with particular enrichment in CD49a + Eomes + NK cells, a subset typically represented in the decidua and playing a proangiogenic function. Functional analysis showed reduced TNF-α production along with impaired cytotoxic capacity that was inversely related to CXCR6 - , T-bet hi Eomes lo , and CD49a + Eomes + NK cells. In conclusion, we identified a subset of NK cells infiltrating HCC, including non-liver-resident cells that coexpressed CD49a and Eomes and showed reduced cytotoxic potential. This NK cell subset likely plays a regulatory role in proangiogenic function.

Background: Hepatocellular carcinoma (HCC) factors, especially maximum tumor diameter (MTD), tumor multifocality, portal vein thrombosis (PVT), and serum alpha-fetoprotein (AFP), influence survival., Aim: To examine patterns of tumor factors in large HCC patients., Methods: A database of large HCC patients was examined., Results: A multiple Cox proportional hazard model on death identified low serum albumin levels and the presence of PVT and multifocality, with each having a hazard ratio ≥2.0. All combinations of these three parameters were examined in relation to survival. Using univariate Cox analysis, the combination of albumin >3.5 g/dL and the absence of both PVT and multifocality had the best survival rate, while all combinations that included the presence of PVT had poor survival and hazard ratios. We identified four clinical phenotypes, each with a distinct median survival: patients with or without PVT or multifocality plus serum albumin ≥3.5 (g/dL), with each subgroup displaying high (≥100 IU/mL) or low (<100 IU/mL) blood AFP levels. Across a range of MTDs, we identified only two significant trends, blood AFP and platelets., Conclusions: Patients with large HCCs have distinct phenotypes and survival, as identified by the combination of PVT, multifocality, and blood albumin levels.