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BACKGROUND & AIMS Early determination of the prognosis of patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is important to guide clinical management and decrease mortality. The aim of this study was to develop a new simplified prognostic score to accurately predict outcomes in patients with HBV-ACLF. METHODS Prospective clinical data from 2,409 hospitalized patients with acute deterioration of HBV-related chronic liver disease were used to develop a new prognostic score that was validated in an external group. RESULTS A total of 954 enrolled patients with HBV-ACLF were diagnosed based on the Chinese Group on the Study of Severe Hepatitis B-ACLF (COSSH-ACLF) criteria. Six predictive factors were significantly related to 28-day mortality and constituted a new prognostic score (=1.649×ln(international normalized ratio)+0.457×hepatic encephalopathy score+0.425×ln(neutrophil)+0.396×ln(total bilirubin)+0.576×ln(serum urea)+0.033×age). The C-indices of the new score for 28-/90-day mortality (0.826/0.809) were significantly higher than those of 4 other scores (COSSH-ACLF, 0.793/0.784; CLIF-C ACLF, 0.792/0.770; MELD, 0.731/0.727; MELD-Na, 0.730/0.726; all p

The unique flavor of Allium tuberosum is primarily associated with the hydrolysis of a series of organosulfur compounds, S-alk(en)yl cysteine sulphoxides (CSOs), upon tissue bruising or maceration. To obtain the tissue-specific transcriptomes, 18 RNA-Seq libraries representing leaf, root, stem, mature flower, inflorescence, and seed tissues of A. tuberosum were sequenced, finally yielding 133.7 Gb clean reads. The de novo assembled transcriptomes enabled the identification of 223,529 unigenes, which were functionally annotated and analyzed for the gene ontology and metabolic pathways. Furthermore, to reveal the flavor metabolic pathways, a total of 205 unigenes involved in the sulfur assimilation and CSO biosynthesis were identified, and their expression profiles were analyzed by RNA-Seq and qRT-PCR. Collectively, this study provides a valuable resource for in-depth molecular and functional researches especially on flavor formation, as well as for the development of molecular markers, and other genetic studies in A. tuberosum.

When Saccharomyces cerevisiae divides, a structure composed of different septin proteins arranged according to a certain rule is formed at the cell division site. The structure undergoes multiple remodeling stages during the cell cycle, thus guiding the yeast cells to complete the entire division process. Although the higher-order structure of septins can be determined using electron microscopy, the septin’s dynamic processes are poorly understood because of limitations in living cell super-resolution imaging technology. Herein, we describe a high lateral resolution and temporal resolution technique, known as fast fluctuation-enhanced structured illumination microscopy (fFE-SIM), which more than doubles the SIM resolution at a frame rate of 38 Hz in living cells. This allows a highly dynamic and sparse septin structure to be observed in Saccharomyces cerevisiae.

Background: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. Method: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. Findings: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts but increases in neutrophil counts than 27 mild cases. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8 + T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-CD8+ T cell ratio (N8R) were identified as the most powerful prognostic factor affecting the prognosis for severe COVID-19. Interpretation: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R may serve as a useful prognostic factor for early identification of severe COVID-19 cases. Funding Statement: This work is supported by the National Natural Science Foundation of China (81861138044, 91742114 and 91642118), the National Science and Technology Major Project (2018ZX10723203, 2018ZX10302206, 2017ZX10202201, 2017ZX10202202 and 2017ZX10202203), the Innovation Team Project of Health Commission of Hubei Province (WJ2019C003), the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College and the “Double-First Class” Project for the International Cooperation Center on Infection and Immunity, HUST, and a special joint project of University Hospital Essen, University of Duisburg-Essen. Declaration of Interests: The authors disclose no conflicts of interest. Ethics Approval Statement: A written informed consent was regularly obtained from all patients upon admission into Wuhan Union Hospital, China. The study was approved by the Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology.

Nucleic acid polymers (NAPs) block the release of HBsAg from infected hepatocytes. These compounds have been previously shown to have the unique ability to eliminate serum surface antigen in DHBV-infected Pekin ducks and achieve multilog reduction of HBsAg or HBsAg loss in patients with chronic HBV infection and HBV/HDV coinfection. In ducks and humans, the blockage of HBsAg release by NAPs occurs by the selective targeting of the assembly and/or secretion of subviral particles (SVPs). The clinically active NAP species REP 2055 and REP 2139 were investigated in other relevant animal models of HBV infection including woodchucks chronically infected with WHV, HBV transgenic mice and HBV infected SCID-Hu mice. The liver accumulation of REP 2139 in woodchucks following subcutaneous administration was examined and was found to be similar to that observed in mice and ducks. However, in woodchucks, NAP treatment was associated with only mild (36–79% relative to baseline) reductions in WHsAg (4/10 animals) after 3–5 weeks of treatment without changes in serum WHV DNA. In HBV infected SCID-Hu mice, REP 2055 treatment was not associated with any reduction of HBsAg, HBeAg or HBV DNA in the serum after 28 days of treatment. In HBV transgenic mice, no reductions in serum HBsAg were observed with REP 2139 with up to 12 weeks of treatment. In conclusion, the antiviral effects of NAPs in DHBV infected ducks and patients with chronic HBV infection were weak or absent in woodchuck and mouse models despite similar liver accumulation of NAPs in all these species, suggesting that the mechanisms of SVP assembly and or secretion present in rodent models differs from that in DHBV and chronic HBV infections.

Polyphenol oxidases catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms. They are key enzymes playing a significant role during the synthesis of lignin. The inhibition of the synthesis of lignin in grapevine can cause seedless grapevine berry development. In this study, grapevine PPO (Vv-PPO) was predicted as the target gene of Vv-miR058 by bioinformatics analysis, and it was further cloned and its homologous conservation in various plants was analyzed. The expression profiles of miR058 and its target Vv-PPO were detected by qRT-PCR in peel, pulp and seeds of three grapevine cultivars and Vv-PPO was expressed in an opposite variation way with Vv-miR058 where both of them could be detected, suggesting that Vv-miR058 can play an important role by regulating the expression of Vv-PPO. In addition, the potential target gene Vv-PPO for Vv-miR058 was verified by RLM-RACE. This result would be helpful in theoretical basis for further research and seedless grapevine berry production.

The Cytochrome P450 (CYP) superfamily is a large group of ancient proteins with enzymatic activities involved in various physiological processes. The rice striped stem borer, Chilo suppressalis, is an important insect pest in rice production. Here, we report the identification and characterization of 77 CYP genes from rice striped stem borer (SSB) through genome and transcriptome sequence analyses. All these CYP genes were confirmed by RT-PCR and direct sequencing. Twenty-eight CYP transcripts have full open reading frame (ORF) and four additional transcripts have a nearly full length coding region. The SSB CYP genes were classified into four clans, the mitochondrial, CYP2, CYP3, and CYP4. Phylogenetic analysis indicated that there was an apparent expansion of the CYP3 clan in insects. The CYP6AB subfamily of the CYP3 clan had nine members in SSB. Evolutionary analysis showed that this subfamily was expanded only in lepidopteran insects. In this study, we identified a new P450 subfamily, CYP321F, which is unique to SSB and located in the genome as tandem repeats. Our work provided a foundation for future studies on the functions and mechanism of P450s in the destructive rice pest.

Previous studies have shown that expression of the interferon-sensitive gene (ISG)15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.

The interferon-alpha (IFN-alpha) gene family is an important part of the immune system. Recombinant interferon-alpha is widely used to treat viral hepatitis and malignant diseases. Marmota himalayana has been found to be susceptible to woodchuck hepatitis virus, a virus genetically related to hepatitis B virus (HBV), and is suitable as an animal model for studies on HBV infection. Here, the IFN-alpha gene family of M. himalayana (cwIFN-alpha) was characterized. Sequence data indicate that the cwIFN-alpha family consists of at least 8 functional sequences and 6 pseudogenes with high homology within the family and to IFN-alpha of Marmota monax, a related species and well-established animal model. The recombinant cwIFN-alpha subtypes were expressed and tested to be active in viral protection assay and to induce expression of MxA in a species-specific manner. This work provides essential information for future work on testing new therapeutic approaches of HBV infection based on IFN-alpha in M. himalayana.

To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation, we constructed an expression plasmid which contained 1.3 copies of the HBV genome, and measured the level of viral replication after transient transfection in Huh7 cells. We then observed the effect of antiviral drug administration. 1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion. The recombinant plasmid was transient transfected into Huh7 cells, HBsAg, HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively. The antiviral effect of adefovir, a novel anti-HBV nucleotide analogue, was evaluated in this cellular model system. The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells, adefovir could inhibit HBV replication in this cellular model, and the inhibition was dosage-dependent. The conclusion is HBV replicon, which can initiate viral replication efficiently in hepatoma cells, may be a useful tool in the study of HBV replication and antiviral drug.

399 PNPLA3 I148M AFFECTS LIVER STEATOSIS IN PATIENTS WITH CHRONIC HEPATITIS B M. Vigano’, L. Valenti, P. Lampertico, B.M. Motta, R. D’Ambrosio, F. Facchetti, P. Dongiovanni, S. Fargion, M. Colombo. Hepatology, Ospedale San Giuseppe IRCCS Multimedica, Internal Medicine, Universita’ di Milano, Fondazione IRCCS Ca’ Granda Milano, Gastroenterology, Fondazione IRCCS Ca’ Granda Milano, Universita degli Studi di Milano, Internal Medicine, Fondazione IRCCS Ca’ Granda Milano, Milan, Italy E-mail: luca.valenti@unimi.it

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PDL1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients. Citation: Liu J, Zhang E, Ma Z, Wu W, Kosinska A, et al. (2014) Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection. PLoS Pathog 10(1): e1003856. doi:10.1371/journal.ppat.1003856 Editor: Charles M. Rice, The Rockefeller University, United States of America Received April 17, 2013; Accepted November 13, 2013; Published January 2, 2014 Copyright: 2014 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (TRR60 and GK 1045/2), National Major Science and Technology Project for Infectious Diseases of China (2008ZX10002-011, 2012ZX10004503), the National Natural Science Foundation of China (No. 30271170, 30571646, 81101248), and the International Science & Technology Cooperation Program of China (2011DFA31030). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: michael.roggendorf@uni-due.de

Background and Aims: Pruritus is a common and disabling symptom in chronic cholestasis. The pathogenesis of this symptom is unknown but recently it has been associated with increased levels of lysophosphatidic acid. The role of other potential molecules is unknown, and therefore we have evaluated the serum metabolomic profile of patients with cholestatic pruritus. Methods: Serum samples were obtained from 36 female patients with primary biliary cirrhosis (PBC), 12 with severe pruritus, and 24 without pruritus, and from 10 healthy controls. All patients received ursodeoxycholic acid (13–15mg/kg/d) for the duration of the disease which was similar in the two groups (8.3±1.4 years). Metabolite extraction was accomplished by fractionating the serum samples into pools of species with similar physicochemical properties. All samples were randomized prior to the metabolite extraction procedure and three different profiles were used: a) polar lipids, non-esterified fatty acids, and bile acids; b) amino acids, and c) apolar lipids. The analyses were performed by UPLC-ESI-QTOFMS, random forests and multivariate data analysis. Results: Around 400 metabolites were identified in serum from patients with PBC. Patients with pruritus had significantly higher content of ceramides (p < 0.001), lysophosphatidylcholines (p = 0.03), phosphatidylcholines (p = 0.04), and triglycerides (p < 0.05) than patients without pruritus. Moreover, the detailed analysis of the lipid profile identified that patients with pruritus had higher levels of most lysophosphatidylcholines and phosphatidylcholines and lower contents of lysophosphatidylethanolamines and phosphatidylethanolamines. The serum level of ceramides, sphingomyelins and diacylglycerophosphoinositol was higher in patients with pruritus as well. No difference in the amino acid profile was observed between the two groups. The overall content of bile acids was not significantly different between patients with and without pruritus. However, a more detailed analysis observed higher amounts of glycocholic and taurocholic acid (p < 0.03), and lower levels of ursodeoxycholic and hyocholic acid (p < 0.05) in patients with pruritus. Conclusion: The metabolomic profile of patients with cholestatic pruritus is characterized by a marked change in the lipid metabolites, particularly in the ceramides, sphingomyelins and lysophospahtidylcholines. The analysis identified a panel of biomarkers that could effectively participate into the pathogenesis of pruritus. Parallel Session: PRE-CLINICAL HBV & HCV

after 2, 6, or 16 hours. Expression of CA markers (iNOS and TNF-a), AA markers (Fizz-1 and TGF-b), and matrix metalloproteinase-9 (MMP-9) were analyzed by real-time PCR. The role of b1-integrins was investigated by culturing in the presence or absence of integrin b1 blocking antibodies. Results: AA macrophages cultured on type III collagen significantly (P < 0.05) upregulated both the AA associated genes Fizz-1 and TGF-b and the CA associated genes TNF-a and iNOS; in contrast, those on type I collagen only reinforced the AA associated genes without effect on CA associated genes. Furthermore, AA macrophages on type III collagen significantly (P < 0.05) upregulated MMP-9, but this did not occur in any of the CA cultures. On type III collagen, TNF-a was maximally upregulated after 2 hours, whereas iNOS and MMP-9 showed the highest upregulation after 16 hours of culture. Inhibition of b1 integrin signalling resulted in downregulation of TNF-a, iNOS and MMP-9, although levels were still raised compared to culture on type I collagen. Conclusions: Type III collagen results in switching AA macrophages into an intermediate phenotype that has both AA and CA features, but also expresses MMP-9, which is neither a specifically described feature of CA or AA macrophages. The increased type III collagen production generally seen in early fibrosis may provide macrophages with such a phenotype in order to remove cellular and ECM debris. This phenotype is b1-integrin driven.