Increased expression of long non-coding RNA FIRRE promotes hepatocellular carcinoma by HuR-CyclinD1 axis signaling.

There is a critical need to understand the disease processes and identify improved therapeutic strategies for hepatocellular carcinoma (HCC). The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. The aim of this study was to identify a lncRNA as a potential biomarker of HCC and investigate the mechanisms by which the lncRNA promotes HCC progression using human cell lines and in vivo. Using RNA-Seq analysis, we found that lncRNA FIRRE was significantly upregulated in hepatitis C virus (HCV) associated liver tissue and identified that lncRNA FIRRE is significantly upregulated in HCV-associated HCC compared to adjacent non-tumor liver tissue. Further, we observed that FIRRE is significantly upregulated in HCC specimens with other etiologies, suggesting this lncRNA has the potential to serve as an additional biomarker for HCC. Overexpression of FIRRE in hepatocytes induced cell proliferation, colony formation, and xenograft tumor formation as compared to vector-transfected control cells. Using RNA pull-down proteomics, we identified HuR as an interacting partner of FIRRE. We further showed that the FIRRE-HuR axis regulates cyclin D1 expression. Our mechanistic investigation uncovered that FIRRE is associated with an RNA-binding protein HuR for enhancing hepatocyte growth. Together, these findings provide molecular insights into the role of FIRRE in HCC progression.

Hepatitis C virus modified sE2F442NYT as an antigen in candidate vaccine facilitates human immune cell activation.

The rational selection of hepatitis C virus (HCV) vaccine antigen will aid in the prevention of future chronic liver disease burden and associated healthcare costs. We have previously shown that HCV E2 glycoprotein is not highly immunogenic, and the modification of E2 reduced CD81 binding and displayed altered cytokine and protective immune responses in vitro and in a surrogate mouse model. Here, we compared the influence of a parental and a modified sE2F442NYT glycoprotein region from HCV genotype 1a for the activation of peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), CD4+T cells, and B cells. Modified sE2F442NYT, when incubated with DCs, induced a higher number of CD86-positive cells. The sE2F442NYT or parental sE2 encapsulated as mRNA-lipid nanoparticle (sE2F442NYT mRNA-LNP) primed DCs co-cultured with autologous CD4+T cells did not induce CD25 or forkhead box P3 expression. PBMC-derived CD4+T cells treated with sE2F442NYT exhibited enhanced signal transducer and activator of transcription (Stat)1/Stat4 phosphorylation in response to anti-CD3/CD28 stimulation in comparison to parental sE2 treatment and facilitated isotype switching in B cells, leading to the generation of a broader subclass of antibodies. Cells treated with modified sE2F442NYT displayed an increase in activated Stat3 and extracellular signal-regulated kinase (ERK). Likewise, PBMC-derived naïve B cells upon in vitro stimulation with sE2F442NYT induced an increased proliferation, Stat3 and ERK activation, and protein kinase B (Akt) suppression. Thus, the modified sE2F442NYT antigen from HCV facilitates improved DC, CD4+T, and B cell activation compared to parental sE2 to better induce a robust protective immune response, supporting its selection as an HCV candidate vaccine antigen for preclinical and clinical HCV vaccine trials.IMPORTANCEThe nature of an enhanced immune response induced by sE2F442NYT will help in the selection of a broad cross-protective antigen from hepatitis C virus genotypes, and the inclusion of relatively conserved sE1 with sE2F442NYT may further strengthen the efficacy of the candidate vaccine in evaluating it for human use.

Clinical effects and emerging issues of atezolizumab plus bevacizumab in patients with advanced hepatocellular carcinoma from Japanese real-world practice.

BACKGROUND: Although the efficacy of atezolizumab has been demonstrated in randomized controlled trials, its long-term efficacy and association with adverse events in real-world practice are unknown. This study was designed to shed light on these issues. METHODS: In this multicenter retrospective study, data were collected from patients with advanced hepatocellular carcinoma treated with atezolizumab plus bevacizumab in seven institutions in Japan. The authors focused on the efficacy and adverse events related to vascular endothelial growth factor (VEGF) inhibition. RESULTS: A total of 123 patients were enrolled in this study. The median progression-free survival (PFS) for the first-line treatment group was 8.0 months (95% confidence interval [CI], 6.1-9.9), whereas the median PFS for the second- or later-line treatment group was 4.1 months (95% CI, 2.6-5.7), which was significantly worse than that of the first-line treatment group (p = .005). Twenty-seven patients had interrupted bevacizumab treatment. Proteinuria accounted for the largest proportion of bevacizumab treatment interruptions. The cumulative incidence rate of bevacizumab interruption due to anti-VEGF-related adverse events was significantly higher in patients with hypertension and/or diabetes mellitus than in those without (p = .026). The landmark analysis showed that patients experienced bevacizumab interruption by 24 weeks from treatment initiation had poorer PFS than those who did not (p = .013). CONCLUSIONS: The PFS of atezolizumab plus bevacizumab as first-line treatment mostly replicates that of a global phase 3 trial. Interrupted bevacizumab treatment was more common in patients with hypertension and/or diabetes mellitus, which may be associated with worsening long-term PFS. PLAIN LANGUAGE SUMMARY: Atezolizumab plus bevacizumab has been the standard front line systemic therapy for advanced hepatocellular carcinoma. With the growing incidence of fatty liver due to metabolic syndrome as a background liver disease for hepatocellular carcinoma, the rate of comorbid hypertension and diabetes mellitus has been increasing accordingly. The present study demonstrated the cumulative incidence rate of bevacizumab interruption due to anti-VEGF-related adverse events was significantly higher in patients with hypertension and/or diabetes mellitus. The landmark analysis clarified that interruption of bevacizumab might be a risk of impaired efficacy of atezolizumab plus bevacizumab over the long term in patients with advanced hepatocellular carcinoma.

Hyperprogressive disease during atezolizumab plus bevacizumab treatment in patients with advanced hepatocellular carcinoma from Japanese real-world practice.

BACKGROUND: Hyperprogressive disease (HPD) is a phenomenon with greatly accelerated tumor growth and clinical deterioration rates compared to pre-therapy, in patients treated with immune checkpoint inhibitors (ICI). The aim of this study is to clarify the reality of HPD in patients with advanced hepatocellular carcinoma (HCC) who were treated with atezolizumab plus bevacizumab (Atez/Bev) using tumor dynamics. METHODS: Medical records of consecutive patients with advanced HCC who were treated with Atez/Bev were retrospectively reviewed. HPD was defined as a more than two- or fourfold increase in tumor growth rate (TGR) or tumor growth kinetics rate (TGKR) before and after treatment. Overall survival (OS) and baseline characteristics with or without HPD were analyzed. RESULTS: A total of 85 patients were included in the analysis. When HPD was defined as a twofold of TGR or TGKR, 8 patients (8/85, 9.4%) had HPD and 11 had PD without HPD. A total of 5 patients (5/85, 5.9%) were diagnosed with HPD and 14 with PD without HPD when HPD was defined as a fourfold of TGR or TGKR. No significant difference was observed in the baseline characteristics between HPD and non-HPD. CONCLUSION: The prevalence of HPD in patients with advanced HCC treated with Atez/Bev was lower than those treated with nivolumab monotherapy. The HPD mechanism in ICI combined with antibodies targeting vascular endothelial growth factor (VEGF) remains to be elucidated.

Differences in Enantioselective Hydroxylation of 2,2',3,6-Tetrachlorobiphenyl (CB45) and 2,2',3,4',6-Pentachlorobiphenyl (CB91) by Human and Rat CYP2B Subfamilies.

Although polychlorinated biphenyls (PCBs) were commercially banned half a century ago, contamination of the environment and organisms by PCBs is still observed. PCBs show high persistence and bioaccumulation, resulting in toxicity. Among PCBs, chiral PCBs with more than three chlorine atoms at the ortho-position exhibit developmental and neurodevelopmental toxicity. Because toxicity is dependent on the atropisomer, atropisomer-specific metabolism is vital in determining toxicity. However, structural information on enantioselective metabolism remains elusive. Cytochrome P450 (CYP, P450) monooxygenases, particularly human CYP2B6 and rat CYP2B1, metabolize separated atropisomers of 2,2',3,6-tetrachlorobiphenyl (CB45) and 2,2',3,4',6-pentachlorobiphenyl (CB91) to dechlorinated and hydroxylated metabolites. Docking studies using human CYP2B6 predict 4'-hydroxy (OH)-CB45 from (aR)-CB45 as a major metabolite of CB45. Di-OH- and dechlorinated OH-metabolites from human CYP2B6 and rat CYP2B1 are also detected. Several hydroxylated metabolites are derived from CB91 by both P450s; 5-OH-CB91 is predicted as a major metabolite. CB91 dechlorination is also detected by identifying 3-OH-CB51. A stable conformation of PCBs in the substrate-binding cavity and close distance to P450 heme are responsible for high metabolizing activities. As hydroxylation and dechlorination change PCB toxicity, this approach helps understand the possible toxicity of chiral PCBs in mammals.

Sequential therapy with sorafenib and regorafenib for advanced hepatocellular carcinoma: a multicenter retrospective study in Japan.

Background Conversion from sorafenib to regorafenib is primarily an evidence-based treatment strategy in patients with advanced hepatocellular carcinoma (HCC). This study aimed to assess the safety and efficacy of sequential therapy with sorafenib and regorafenib in patients with advanced HCC by analysis of outcomes in clinical practice with the aim to complement phase III findings. Methods The medical records of patients with advanced HCC receiving regorafenib were retrieved to collect data on sorafenib administration at seven Japanese institutions. Radiological responses and adverse events were evaluated using the Response Evaluation Criteria in Solid Tumors version 1.1 and the Common Terminology Criteria for Adverse Events version 4.0, respectively. Results Before March 2018, 44 patients were administered regorafenib for advanced HCC. The median sorafenib treatment duration was 8.4 months. The most common adverse events were similar to those reported by the RESORCE trial. The median overall survival (OS) was 17.3 months (95% confidence interval [CI] 11.4-22.9), and 17 of 37 patients (45.9%) discontinued regorafenib and received sequential systemic therapy after regorafenib. These patients had significantly longer OS than those who were treated by the best supportive care or sub-optimal therapy (not reached versus 8.7 months [95% CI 5.8-11.7]; P < 0.001). Conclusion The results based on Japanese clinical practices verified the tolerability of regorafenib in advanced HCC. Major regorafenib-associated adverse events were similar to those related to sorafenib. OS was significantly longer than expected, which might be associated with the sequential systemic therapies after regorafenib, mainly lenvatinib.

Superinfection of hepatitis A virus in hepatocytes infected with hepatitis B virus.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis including acute liver failure. Hepatitis B infection (HBV) occurs worldwide, with the highest rates in Asian and African countries, and there are several reports that HAV infection may have a more severe clinical course in patients with chronic HBV infection. We previously demonstrated that Japanese miso extracts have inhibitory effects on HAV replication. In the present study, we examined the replication of HAV and HBV in a hepatocyte superinfection model and the inhibitory effects of Japanese miso extracts on both viruses. According to the results, HAV infection inhibited HBV replication in superinfected hepatocytes, and Japanese rice-koji miso extracts had inhibitory effects on HAV replication. Our findings provide useful information for clinicians in managing HAV infection in patients with chronic HBV infection.

Interferon induces interleukin 8 and bone marrow stromal cell antigen 2 expression, inhibiting the production of hepatitis B virus surface antigen from human hepatocytes.

Hepatitis B virus (HBV) surface antigen (HBsAg) loss is one of the treatment goals of chronic HBV infection. Bone marrow stromal cell antigen 2 (BST2) is one of the interferon (IFN)-stimulated genes (ISGs) and inhibits the release of various enveloped viruses. Here we examined the effects of antiviral treatment on HBsAg levels and its intracellular mechanism in HBsAg-producing hepatocytes. In PLC/PRF/5 and Huh1, IFNα-2a treatment decreased HBsAg levels in their conditioned media. Upregulation of interleukin 8 (IL8), toll-like receptor 2 (TLR2) and interferon gamma-induced protein 10 (IP10) mRNAs was associated with the reduction of HBsAg in both PLC/PRF/5 and Huh1. The HBsAg level was upregulated by knockdown of IL8, TLR2 or IP10. Exogenous addition of IL8 enhanced BST2 promoter activity and BST2 mRNA expression. Additionally, knockdown of IL8 could lead to the downregulation of BST2 mRNA. Transfection of poly(I-C) enhanced IL8 and BST2 mRNA expression and inhibited HBsAg secretion from PLC/PRF/5 cells. In conclusion, IL8 might play an important role in the enhancement of BST2 and be involved in HBsAg eradication.

Molecular insights into the role of a distal F240A mutation that alters CYP1A1 activity towards persistent organic pollutants.

BACKGROUND: Cytochromes P450 are major drug-metabolizing enzymes involved in the biotransformation of diverse xenobiotics and endogenous chemicals. Persistent organic pollutants (POPs) are toxic hydrophobic compounds that cause serious environmental problems because of their poor degradability. This calls for rational design of enzymes capable of catalyzing their biotransformation. Cytochrome P450 1A1 isoforms catalyze the biotransformation of some POPs, and constitute good starting points for the design of biocatalysts with tailored substrate specificity. METHODS: We rationalized the activities of wild type and mutant forms of rat cytochrome P450 1A1 towards 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and 3,3',4,4'-tetrachlorobiphenyl (PCB77) using experiments and molecular dynamics simulations. RESULTS: We showed that the enhanced activity of the CYP1A1 mutant towards TCDD was due to more efficient binding of the substrate in the active site even though the mutated site was over 2.5nm away from the catalytic center. Moreover, this mutation reduced activity towards PCB77. GENERAL SIGNIFICANCE: Amino acids that affect substrate access channels can be viable targets for rational enzyme design even if they are located far from the catalytic site.

Real-World Experiences with the Combination Treatment of Ledipasvir plus Sofosbuvir for 12 Weeks in HCV Genotype 1-Infected Japanese Patients: Achievement of a Sustained Virological Response in Previous Users of Peginterferon plus Ribavirin with HCV NS3/4A Inhibitors.

The aim of this study was to characterize the treatment response and serious adverse events of ledipasvir plus sofosbuvir therapies in Japanese patients infected with hepatitis C virus (HCV) genotype 1 (GT1). This retrospective study analyzed 240 Japanese HCV GT1 patients treated for 12 weeks with 90 mg of ledipasvir plus 400 mg of sofosbuvir daily. Sustained virological response at 12 weeks post-treatment (SVR12) was achieved in 236 of 240 (98.3%) patients. Among treatment-naïve patients, SVR12 was achieved in 136 of 138 (98.6%) patients, and among treatment-experienced patients, SVR12 was achieved in 100 of 102 (98.0%) patients. In patients previously treated with peginterferon plus ribavirin with various HCV NS3/4A inhibitors, 100% SVR rates (25/25) were achieved. Two relapsers had HCV NS5A resistance-associated variants (RAVs), but no HCV NS5B-S282 was observed after they relapsed. We experienced two patients with cardiac events during treatment. In conclusion, combination of ledipasvir plus sofosbuvir for 12 weeks is a potential therapy for HCV GT1 patients. Caution is needed for HCV NS5A RAVs, which were selected by HCV NS5A inhibitors and cardiac adverse events.

Successful sofosbuvir treatment with ribavirin dose reduction for chronic hepatitis C virus genotype 2 infection in a patient with ulcerative colitis: a case report.

BACKGROUND: Ulcerative colitis is a lifelong, immunologically mediated disease. Direct-acting antivirals (DAAs) are now available for the treatment of chronic hepatitis C virus (HCV) infection. An interferon-free regimen appears useful, safe and effective for many patients for whom interferon-based treatment is contraindicated. CASE PRESENTATION: We studied a 56-year-old treatment-naïve Japanese man with chronic HCV genotype 2b infection who had ulcerative colitis. This patient was treated with sofosbuvir and ribavirin for 12 weeks. During treatment, diarrhoea and bloody faeces were frequent. After ribavirin was reduced to 400 mg daily, these symptoms decreased. Finally, the patient achieved a sustained virologic response 12 weeks after the stoppage of the treatment. CONCLUSION: Clinicians should pay careful attention to the ribavirin dose in the treatment of certain HCV patients with inflammatory bowel disease who are receiving sofosbuvir plus ribavirin.

Daclatasvir plus Asunaprevir Treatment for Real-World HCV Genotype 1-Infected Patients in Japan.

Background. All-oral combination of direct-acting antivirals could lead to higher sustained virologic response (SVR) in hepatitis C virus (HCV)-infected patients. In the present study, we examined the efficacy and safety of the dual oral treatment with HCV nonstructural protein (NS) 5A inhibitor daclatasvir (DCV) plus HCV NS3/4A inhibitor asunaprevir (ASV) for 24 weeks in real-world HCV genotype 1-infected Japanese individuals. Methods. After screening for HCV NS5A resistance-associated variants (RAVs) by PCR invader assay, a total of 54 Japanese patients infected with HCV genotype 1 treated with DCV plus ASV were retrospectively analyzed. SVR12 was used for evaluation of the virologic response. Results. Of the total 54 patients, 46 patients (85.2%) were treated with DCV plus ASV for 24 weeks and achieved SVR12. The other 8 patients (14.8%) discontinued this treatment before 24 weeks due to adverse events. Of these 8 patients, 5 and 3 patients did and did not achieve SVR12, respectively. Finally, 51 of 54 (94.4%) patients achieved SVR12. Conclusion. Treatment with DCV and ASV after screening for HCV NS5A RAVs by PCR invader assay is effective and safe in the treatment of real-world HCV genotype 1-infected patients in Japan.

Sustained Virologic Response at 24 Weeks after the End of Treatment Is a Better Predictor for Treatment Outcome in Real-World HCV-Infected Patients Treated by HCV NS3/4A Protease Inhibitors with Peginterferon plus Ribavirin.

BACKGROUND: Direct-acting antiviral agents against HCV with or without peginterferon plus ribavirin result in higher eradication rates of HCV and shorter treatment duration. We examined which is better for predicting persistent virologic response, the assessment of serum HCV RNA at 12 or 24 weeks after the end of treatment for predicting sustained virologic response (SVR12 or SVR24, respectively) in patients treated by HCV NS3/4A protease inhibitors with peginterferon plus ribavirin. METHODS: In all, 149 Japanese patients infected with HCV genotype 1b treated by peginterferon plus ribavirin with telaprevir or simeprevir were retrospectively analyzed: 59 and 90 patients were treated with telaprevir- and simeprevir-including regimens, respectively. HCV RNA was measured by TaqMan HCV Test, version 2.0, real-time PCR assay. SVR12 or SVR24, respectively, was defined as HCV RNA negativity at 12 or 24 weeks after ending treatment. RESULTS: Total SVR rates were 78.0% and 66.7% in the telaprevir and simeprevir groups, respectively. In the telaprevir group, all 46 patients with SVR12 finally achieved SVR24. In the simeprevir group, 60 (93.8%) of the total 64 patients with SVR12 achieved SVR24, with the other 4 patients all being previous-treatment relapsers. CONCLUSIONS: SVR12 was suitable for predicting persistent virologic response in almost all cases. In simeprevir-including regimens, SVR12 could not always predict persistent virologic response. Clinicians should use SVR24 for predicting treatment outcome in the use of HCV NS3/4A protease inhibitors with peginterferon plus ribavirin for any group of real-world patients chronically infected with HCV.

The sirtuin inhibitor sirtinol inhibits hepatitis A virus (HAV) replication by inhibiting HAV internal ribosomal entry site activity.

Epigenetics plays a role in the regulation of gene expression. Epigenetic changes control gene expression at the transcriptional level. Our previous study suggested that the La protein, which is mainly localized in the nucleus, was associated with hepatitis A virus (HAV) internal ribosomal entry site (IRES)-mediated translation and HAV replication. The aim of this study was to investigate whether epigenetic compounds have effects on HAV IRES-mediated translation and HAV replication. Sirtinol, a sirtuin inhibitor, inhibited HAV IRES-mediated translation in COS7-HAV-IRES cells. Treatment with 10 µM sirtinol resulted in a significant reduction in the intracellular RNA levels of HAV HA11-1299 genotype IIIA in Huh7 cells. Epigenetic treatment with a sirtuin inhibitor may represent a new treatment option for HAV infection. In conclusion, epigenetic control was involved in HAV IRES-dependent translation and HAV replication. Special attention should also be paid to underlying viral diseases in the clinical use of epigenetic treatments for malignancies.

Association between hepatitis B virus and MHC class I polypeptide-related chain A in human hepatocytes derived from human-mouse chimeric mouse liver.

Due to the lack of efficient hepatitis B virus (HBV) infection systems, progress in understanding the role of innate immunity in HBV infection has remained challenging. Here we used human hepatocytes from a humanized severe combined immunodeficiency albumin promoter/enhancer driven-urokinase-type plasminogen activator mouse model for HBV infection. HBV DNA levels in culture medium from these human hepatocytes were 4.8-5.7 log IU/mL between day 16 and day 66 post-infection by HBV genotype C inoculum. HBV surface antigen (HBsAg) was also detected by chemiluminescent immunoassay from day 7 to day 66 post-infection. Western blot analysis revealed that major histocompatibility complex class I-related chain A (MICA), which plays a role in the innate immune system, was induced in HBV-infected human hepatocytes 27 days after infection compared with the uninfected control. MICA was reduced at day 62 and undetectable at day 90. Of interest, MICA expression by human hepatocytes increased after HBV infection and decreased before HBsAg loss. Human hepatocytes derived from chimeric mice with hepatocyte-humanized liver could support HBV genome replication. Further studies of the association between HBV replication and MICA induction should be conducted.

The JAK2 inhibitor AZD1480 inhibits hepatitis A virus replication in Huh7 cells.

The JAK2 inhibitor AZD1480 has been reported to inhibit La protein expression. We previously demonstrated that the inhibition of La expression could inhibit hepatitis A virus (HAV) internal ribosomal entry-site (IRES)-mediated translation and HAV replication in vitro. In this study, we analyzed the effects of AZD1480 on HAV IRES-mediated translation and replication. HAV IRES-mediated translation in COS7-HAV-IRES cells was inhibited by 0.1-1 µM AZD1480, a dosage that did not affect cell viability. Results showed a significant reduction in intracellular HAV HA11-1299 genotype IIIA RNA levels in Huh7 cells treated with AZD1480. Furthermore, AZD1480 inhibited the expression of phosphorylated-(Tyr-705)-signal transducer and activator of transcription 3 (STAT3) and La in Huh7 cells. Therefore, we propose that AZD1480 can inhibit HAV IRES activity and HAV replication through the inhibition of the La protein.

Effect of Hepatitis C Virus Genotype 1b Core and NS5A Mutations on Response to Peginterferon Plus Ribavirin Combination Therapy.

We examined whether hepatitis C virus (HCV) genotype 1b core- and NS5A-region mutations are associated with response to peginterferon α-2b plus ribavirin combination therapy. A total of 103 patients with high HCV genotype 1b viral loads (≥ 100 KIU/mL) were treated with the combination therapy. Pretreatment mutations in the core region and interferon sensitivity determining region (ISDR) in the NS5A region were analyzed. In univariate analysis, arginine and leucine at positions 70 and 91 in the core region, defined as double wild (DW)-type, were associated with early virologic response (p = 0.002), sustained virologic response (SVR) (p = 0.004), and non-response (p = 0.005). Non-threonine at position 110 was associated with SVR (p = 0.004). Multivariate analysis showed the following pretreatment predictors of SVR: hemoglobin level ≥ 14 g/dL (odds ratio (OR) 6.2, p = 0.04); platelet count ≥ 14 × 104/mm³ (OR 5.2, p = 0.04); aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio < 0.9 (OR 6.17, p = 0.009); DW-type (OR 6.8, p = 0.02); non-threonine at position 110 (OR 14.5, p = 0.03); and ≥ 2 mutations in the ISDR (OR 12.3, p = 0.02). Patients with non-DW-type, non-threonine at position 110, and < 2 ISDR mutations showed significantly lower SVR rates than others (11/45 (24.4%) vs. 27/37 (73.0%), respectively; p < 0.001). SVR can be predicted through core and NS5A region mutations and host factors like hemoglobin, platelet count, and AST/ALT ratio in HCV genotype 1b-infected patients treated with peginterferon and ribavirin combination therapy.

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis.

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the 'fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

Hepatitis C virus E1 and modified E2 delivered from an mRNA vaccine induces protective immunity.

Hepatitis C virus (HCV) is characterized by a high number of chronic cases due to an impairment of protective innate and adaptive immune responses. Here, we examined the contribution of the individual ectodomains of E1, E2, or a modified E2 with reduced CD81 binding and an inserted N-linked glycosylation site in combination as vaccine antigen mRNA-lipid nanoparticles (LNPs). The induction of a protective immune response to surrogate recombinant vaccinia virus (VV) expressing homologous HCV glycoprotein(s) challenge infection in a BALB/c mouse model was observed. Vaccination with a mRNA-LNP expressing soluble E1 (sE1) significantly reduced vv/HCV titer in the mouse ovary. However, the addition of sE2 mRNA-LNP for immunization impaired the efficacy of the sE1 construct. Further analysis showed that Th1 related cytokine responses to the sE1 mRNA-LNP were significantly altered in the presence of sE2 following co-immunization. Evaluation of immunogenicity revealed that the use of modified sE2F442NYT nucleoside mRNA-LNP vaccine results in an improved cellular immune response, IgG2a isotype switching, enhanced total IgG, and an increase in the neutralizing antibody response against HCV pseudotype virus. HCV cross genotype specific reactivity to peptides representing conserved E2 specific linear epitopes were enhanced in modified E2 vaccinated animal sera. In the absence of a suitable immunocompetent small animal model for HCV infection, protection from surrogate HCV vaccinia challenge infection model was observed in the immunized mice as compared to sE1 alone or an unmodified sE2 mRNA-LNP vaccine. Inclusion of sE1 with modified sE2F442NYT as mRNA-LNP vaccine candidate appeared to be beneficial for protection.

Enhanced metabolism of 2,3',4,4',5-pentachlorobiphenyl (CB118) by bacterial cytochrome P450 monooxygenase mutants of Bacillus megaterium.

Bacterial cytochrome P450 monooxygenase P450BM3 is a promising enzyme to provide novel substrate specificity and enhanced enzymatic activity. The wild type (WT) has been shown to metabolize the widely distributed polychlorinated biphenyl (PCB) 2,3',4,4',5-pentachlorobiphenyl (CB118) to hydroxylated metabolites. However, this reaction requires the coexistence of perfluoroalkyl carboxylic acids (PFCAs). To locate P450BM3 mutants metabolizing CB118 without PFCAs, mutations were selected from amino acids comprising the substrate-binding cavity and the substrate entrance. The mutant A264G showed enhanced hydroxylation activities compared to the WT for the production of five hydroxylated metabolites. Perfluorooctanoic acid addition provided the highest activity, as found in the WT. The docking model of A264G and CB118 indicated that the enlargement of the space above the heme brought CB118 close to the heme, resulting in high activity. In contrast, the mutants L188Q, QG, LVQ, and GVQ, which contain the L188Q mutation, showed higher activity than WT even without PFCAs. Docking models revealed that the closed form found in substrate binding was induced by the L188Q mutation in the substrate non-binding state of the mutants. These mutants are promising for bioremediation of PCBs using enhanced metabolizing activities.