Hepatitis B surface antigen (HBsAg) loss or functional cure (FC), is considered the desirable therapeutic outcome for chronic hepatitis B (CHB) patients. However, the immune-pathological biomarkers and underlying mechanisms remain unclear. In this study we comprehensively interrogate disease-associated cell states (DACS) identified within intra-hepatic tissue and matched PBMCs from either CHB or FC patients, at the resolution of single cells, to provide novel insights into putative mechanisms underlying FC. We combined single cell transcriptomics (scRNA-seq) with multiparametric flow cytometry-based immune phenotyping, and multiplexed immunofluorescence to elucidate the immunopathological cell states associated with CHB vs FC. We find that the intra-hepatic environment of CHB and FC patients displays specific cell identities and molecular signatures that are distinct from those found in matched PBMCs. FC is associated with emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes (CD4-CTLs), and an activated innate response represented by liver-resident natural killer (LR-NK) cells, specific Kupffer cell (KC) subtypes and marginated neutrophils. Surprisingly, we find that FC patients are also characterized by the presence of MHC class II-expressing hepatocytes and low but persistent levels of cccDNA and pgRNA, which may play an important role in achieving functional cure in HBV patients. Our study provides conceptually novel insights into the immuno-pathological control of HBV cure, and opens exciting new avenues for clinical management, biomarker discovery and therapeutic interventions. We believe that the discoveries from this study, as it relates to the activation of an innate and altered immune response that may facilitate sustained, low-grade inflammation, may have broader implications in the resolution of chronic viral hepatitis.

Superficial pT1 bladder tumors are characterized by a high risk of recurrence and progression in grade and stage. Few studies provided evidence that loss of adipocyte‐fatty acid binding protein (A‐FABP) expression was associated with bladder cancer progression. A‐FABP is a lipid binding protein playing a role in intracellular lipid transport and metabolism, as well as in signal transduction. We reported from bladder tumors that decrease of A‐FABP transcript level significantly correlated to tumor stage and to histologic grade (p < 0.05). Namely, in poor prognosis high grade pT1 tumors there was a loss of A‐FABP expression compared to good prognosis tumors suggesting that re‐expression of A‐FABP could be a therapeutic approach in early stage bladder cancer to prevent disease progression. We demonstrated for the first time that this marker is upregulated by Peroxisome Proliferator‐Activated Receptor (PPAR) α, β and γ in T24 cells (derived from an undifferentiated grade III carcinoma) and only by PPARβ in RT4 cells (derived from a well differentiated grade I papillary tumor). This effect occurred through a PPAR‐dependent transcriptional mechanism without modifying mRNA stability and interestingly required de novo protein synthesis. Data as a whole suggest a prognostic significance of A‐FABP in bladder cancer outcome and the potential utility of overexpression of this protein by PPAR agonists open up new perspectives in the treatment of bladder cancer.

Objective Inflammation and oxidative stress drive disease progression in chronic hepatitis C (CHC) towards hepatocellular carcinoma. HCV is known to increase intracellular levels of reactive oxygen species (ROS), but how it eliminates ROS is less well known. The role of the ROS scavenger glutathione peroxidase 4 (GPx4), induced by HCV, in the viral life cycle was analysed. Design The study was performed using a replicative in vitro HCV infection model and liver biopsies derived from two different CHC patient cohorts. Results A screen for HCV-induced peroxide scavengers identified GPx4 as a host factor required for HCV infection. The physiological role of GPx4 is the elimination of lipid peroxides from membranes or lipoproteins. GPx4-silencing reduced the specific infectivity of HCV by up to 10-fold. Loss of infectivity correlated with 70% reduced fusogenic activity of virions in liposome fusion assays. NS5A was identified as the protein that mediates GPx4 induction in a phosphatidylinositol-3-kinase-dependent manner. Levels of GPx4 mRNA were found increased in vitro and in CHC compared with control liver biopsies. Upon successful viral eradication, GPx4 transcript levels returned to baseline in vitro and also in the liver of patients. Conclusions HCV induces oxidative stress but controls it tightly by inducing ROS scavengers. Among these, GPx4 plays an essential role in the HCV life cycle. Modulating oxidative stress in CHC by specifically targeting GPx4 may lower specific infectivity of virions and prevent hepatocarcinogenesis, especially in patients who remain difficult to be treated in the new era of interferon-free regimens.

Waterlogged archaeological wood is exposed to a high risk of biological degradation during the post-excavation phases of storage and restoration. For this reason, often biocides must be used to preserve wooden remains. In the present work three essential oils (cinnamon, wild thyme, and common thyme) were tested as possible alternative biocides to use in the preservation of waterlogged archaeological wood. The oils were first tested in vitro to establish the minimum inhibitory concentration (MIC) and to evaluate the biocidal activity on selected fungal strains. Then, the established MIC was applied on waterlogged archaeological wood samples and during an actual restoration treatment. The effectiveness of the oils was evaluated through cultural analyses, ATP quantification, and next-generation sequencing. The results showed that the oils caused a significant decrease in the vitality of fungal mycelia grown in vitro and of the microbiota present in treated wood and storage water. Furthermore, an influence on the composition of the bacterial communities of treated wood samples was observed. Although further tests are needed to evaluate interferences with the materials used during restoration procedures, essential oils could be considered as a possible alternative to the currently used biocide.

We describe a technique of S isotope analysis in sulfate form with the first separation stage involving anion-exchange and the second stage of mass-spectrometric analysis by MC-ICP-MS using standard-sample-standard bracketing. Ammonium in 1 : 1 stoichiometric proportion with sulfate was used to improve transmission and stability and to avoid cone and membrane clogging by condensable species. The working resolution of ∼9000 allowed the main interferences, notably ³²SH on ³³S, to be resolved. The matrix effect caused by phosphorus present in biological samples is negligible for S/P ratios ≥10: our chemical protocol allows S/P ≥ 150 to be routinely achieved. Replicate measurements of S standard solutions give values of isotopic abundances within errors of accepted values and demonstrate a reproducibility of ±0.10‰ for δ³⁴S and ±0.15‰ for δ³³S (2s). The technique is adequate for quantities as small as 10 nanomoles. We investigated the δ³⁴S of 110 samples of cancer patients and 10 samples of rheumatoid arthritis patients. We avoided the use of blood collection tubes with sulfate-containing heparin. Sulfur in serum is transported by albumin and fibrinogen. Most serum and plasma δ³⁴S values fall within a narrow interval of ∼1‰ around a mean δ³⁴S_VCDT of ∼6.0‰. The δ³⁴S values of total blood, serum, and plasma are very similar. Despite the short turnover time of albumin and fibrinogen, S is surprisingly well regulated. Subtle variations of 0.2–0.3‰ around the mean value can be assigned to sex and age, with sulfur in male and adult samples tending to be heavier than in their female and juvenile counterparts. This narrow range of variations across the spectrum of a large number of individuals not selected for controlled dietary habits seems paradoxical. In general, breast and prostate cancer and rheumatoid arthritis have very little effect on the average serum δ³⁴S, but increase the scatter of values. We confirm that the serum of patients affected by liver cancer and other pathologies is depleted of albumin-born sulfur. While sulfur in the serum of patients with non-malignant liver pathologies tends to be isotopically light, the serum δ³⁴S of medicated hepatocellular carcinoma patients tends to be at the high end of control values.

Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species. Viruses and bacteria are implicated in 15%–20% of total cancer occurrences. Hepatitis C virus (HCV) infection is one of the main causative agents of liver cancer. “Dependence receptors” are a class of receptors that auto-activate and trigger apoptosis in the absence of their ligands, and “dependence receptor” ligands such as Netrin-1 are known to be overactivated in cancers, especially in inflammation-driven tumors. In this study, we show that HCV and Netrin-1 are mutual inducers—Netrin-1 expression is increased upon HCV infection and, in turn, the rise in Netrin-1 leads to an increase in the HCV particle infectivity. The effects on HCV infectivity involve the liver cancer-related epidermal growth factor receptor (EGFR), which is known to be a host receptor necessary for HCV entry and which we now show is activated by Netrin-1. Our work, therefore, illustrates a pathogenic positive feedback loop involving HCV, Netrin-1, and EGFR, among other factors, in association with cancer development.