Mitochondrial DNA is a target of HBV integration.

Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular carcinoma (HCC) development remains unclear. In this study, we apply a high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identify 3339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with HCC. We detect 2107 clonally expanded integrations (1817 in tumour and 290 in non-tumour tissues), and a significant enrichment of clonal HBV integrations in mitochondrial DNA (mtDNA) preferentially occurring in the oxidative phosphorylation genes (OXPHOS) and D-loop region. We also find that HBV RNA sequences are imported into the mitochondria of hepatoma cells with the involvement of polynucleotide phosphorylase (PNPASE), and that HBV RNA might have a role in the process of HBV integration into mtDNA. Our results suggest a potential mechanism by which HBV integration may contribute to HCC development.

Assessing Genomic Mutations in SARS-CoV-2: Potential Resistance to Antiviral Drugs in Viral Populations from Untreated COVID-19 Patients.

Naturally occurring SARS-CoV-2 variants mutated in genomic regions targeted by antiviral drugs have not been extensively studied. This study investigated the potential of the RNA-dependent RNA polymerase (RdRp) complex subunits and non-structural protein (Nsp)5 of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to accumulate natural mutations that could affect the efficacy of antiviral drugs. To this aim, SARS-CoV-2 genomic sequences isolated from 4155 drug-naive individuals from southern Italy were analyzed using the Illumina MiSeq platform. Sequencing of the 4155 samples showed the following viral variant distribution: 71.2% Delta, 22.2% Omicron, and 6.4% Alpha. In the Nsp12 sequences, we found 84 amino acid substitutions. The most common one was P323L, detected in 3777/4155 (91%) samples, with 2906/3777 (69.9%) also showing the G671S substitution in combination. Additionally, we identified 28, 14, and 24 different amino acid substitutions in the Nsp5, Nsp7, and Nsp8 genomic regions, respectively. Of note, the V186F and A191V substitutions, affecting residues adjacent to the active site of Nsp5 (the target of the antiviral drug Paxlovid), were found in 157/4155 (3.8%) and 3/4155 (0.07%) samples, respectively. In conclusion, the RdRp complex subunits and the Nsp5 genomic region exhibit susceptibility to accumulating natural mutations. This susceptibility poses a potential risk to the efficacy of antiviral drugs, as these mutations may compromise the drug ability to inhibit viral replication.

Frequency of somatic mutations in TERT promoter, TP53 and CTNNB1 genes in patients with hepatocellular carcinoma from Southern Italy.

Somatic mutations in the TERT promoter and in the TP53 and CTNNB1 genes are considered drivers for hepatocellular carcinoma (HCC) development. They show variable frequencies in different geographic areas, possibly depending on liver disease etiology and environmental factors. TP53, CTNNB1 and TERT genetic mutations were investigated in tumor and non-tumor liver tissues from 67 patients with HCC and liver tissue specimens from 41 control obese subjects from Southern Italy. Furthermore, TERT expression was assessed by reverse transcription-quantitative PCR. Neither CTNNB1 mutations or TP53 R249S substitution were detected in any case. The TP53 R72P polymorphism was found in 10/67 (14.9%) tumors, but was not found in either non-tumor tissues (P=0.001) or controls (P=0.009). TERT gene promoter mutations were found in 29/67 (43.3%) tumor tissues but were not found in either non-tumor (P<0.0001) or control liver specimens (P<0.0001). The most frequent mutation in the tumors was the known hot spot at -124 bp from the TERT ATG start site (-124G>A, 28 cases, 41.8%; P<0.0001). A new previously never reported TERT promoter mutation (at -297 bp from the ATG, -297C>T) was found in 5/67 (7.5%) tumors, in 0/67 (0%) non-tumor (P<0.0001), and in 0/41 (0%) controls (P=0.07). This mutation creates an AP2 consensus sequence, and was found alone (1 case) or in combination (4 cases) with the -124 bp mutation. The mutation at -124 and -297 bp induced a 33-fold (P<0.0001) and 40-fold increase of TERT expression levels, respectively. When both mutations were present, TERT expression levels were increased >300-fold (P=0.001). A new TERT promoter mutation was identified, which generates a de novo binding motif for AP2 transcription factors, and which significantly increases TERT promoter transcriptional activity.

Glutamine-induced filamentous cells of Pseudomonas mediterranea CFBP-5447T as producers of PHAs.

Polyhydroxyalkanoates (PHAs) are considerable biopolymers that have gained an increasing biotechnological interest in different applications, although their industrial production presents several limitations. Filamentous bacterial cells could represent a possible strategy to increase PHA yield, since more abundant PHA inclusions can be stored in elongated than in rod-shaped cells. At first, we determined the optimal batch culture conditions to induce filamentation in Pseudomonas mediterranea CFBP-5447T, using glutamine, glycerol, glucose, and sodium octanoate, as the sole carbon source, at low- (100 rpm) or high- (250 rpm) shaking speeds. Successively, a fermentative process was set up using glutamine in a co-metabolic strategy with glycerol, and the PHAs production was compared in rod-shaped and filamentous cells. High glutamine concentrations (from 28 to 56 mM) were able to induce alone filamentation, whereas at lower glutamine concentrations (5-10 mM), the shaking speeds became critical to allow or not filamentous phenotype. PHA granule production was higher in filamentous than in rod-shaped cells, when glycerol (46.6 mM) was added to glutamine (5 mM) in co-metabolism, and fermentation was performed at a low-shaking speed. After extraction and precipitation, PHA yield was about two times higher in filamentous than that rod-shaped cells. Our results provide new insights into filament-inducing conditions and indicate a potential use of filamentous P. mediterranea CFBP-5447T cells to increase PHA yield. These findings could have great advantages in PHAs recovering during downstream processes, since the harvesting of elongated cells is much less time-consuming and energy expensive than required with rod-shaped cells.

The role of glutamine in Pseudomonas mediterranea in biotechnological processes.

In this work, in order to study the effect of glutamine as co-feeder on growth kinetics, biomass and PHA production in Pseudomonas mediterranea, different co-metabolic strategies were employed. Unrelated (glycerol and glucose) and related (sodium octanoate) carbon sources both in presence and absence of glutamine have been tested. For each cultural condition, we (i) evaluated growth kinetics and measured the cell metabolic activity by MTT assay, (ii) monitored PHA production and (iii) estimated the expression of phaC1 and phaC2 genes through RT-PCR. Our results show that the use of glutamine as co-feeder in P. mediterranea led to an improvement of the specific growth rate and cell metabolic activity and enhanced the uptake of all the carbon sources assayed. Moreover, the use of glutamine reduced significantly the time required for PHA production and increased biopolymer yield, as consequence of an early activation of phaC1 and phaC2.