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Hepatitis B virus (HBV) contains a partially double-stranded, relaxed circular (RC) DNA genome synthesized within a nucleocapsid (NC) in the host cell cytoplasm. The release of RC DNA from the NC, in an ill-defined process called uncoating, to the nucleus is required for its conversion to the covalently closed circular (CCC) DNA, the viral episome serving as the transcriptional template for all viral RNAs necessary for replication and, thus, essential for establishing and sustaining viral infection. In efforts to better understand uncoating, we analyzed HBV core (HBc) mutants that show various levels of nuclear CCC DNA but little to no cytoplasmic RC DNA. We found that RC DNA could be synthesized by these mutants outside the cell, but in contrast to the wild type (wt), the mutant NCs were unable to protect RC DNA from digestion by the endogenous nuclease(s) in cellular lysates or exogenous DNase. Subcellular fractionation suggested that the major RC DNA-degrading activity was membrane associated. Digestion with sequence-specific and nonspecific DNases revealed the exposure of specific regions of RC DNA from the mutant NC. Similarly, treatment of wt NCs with a core inhibitor known to increase CCC DNA by affecting uncoating also led to region-specific exposure of RC DNA. Furthermore, a subpopulation of untreated wild type (wt) mature NCs showed site-specific exposure of RC DNA as well. Competition between RC DNA degradation and its conversion to CCC DNA during NC uncoating thus likely plays an important role in the establishment and persistence of HBV infection and has implications for the development of capsid-targeted antivirals. IMPORTANCE Disassembly of the hepatitis B virus (HBV) nucleocapsid (NC) to release its genomic DNA, in an ill-understood process called uncoating, is required to form the viral nuclear episome in the host cell nucleus, a viral DNA essential for establishing and sustaining HBV infection. The elimination of the HBV nuclear episome remains the holy grail for the development of an HBV cure. We report here that the HBV genomic DNA is exposed in a region-specific manner during uncoating, which is enhanced by mutations of the capsid protein and a capsid-targeted antiviral compound. The exposure of the viral genome can result in its rapid degradation or, alternatively, can enhance the formation of the nuclear episome, thus having a major impact on HBV infection and persistence. These results are thus important for understanding fundamental mechanisms of HBV replication and persistence and for the ongoing pursuit of an HBV cure.

Interactions between the N-terminal (assembly) domain (NTD) and the linker region of the hepatitis B virus (HBV) capsid protein and the large (L) envelope protein are required for virion formation, which occurs via budding of cytoplasmic mature nucleocapsids (NCs) containing the relaxed circular (RC) DNA genome into an intracellular membrane compartment containing viral envelope proteins. L-capsid interactions also negatively regulates covalently closed circular (CCC) DNA formation, which occurs after RC DNA release from mature NCs and nuclear import. We have now found that L could increase RC DNA in cytoplasmic mature NCs that are destabilized due to mutations in the NTD or the linker, even in those that apparently fail to support secretion of complete virions extracellularly. Other mutations in the capsid linker could block the effects of L on both cytoplasmic NC DNA and nuclear CCC DNA. Furthermore, the maturity of RC DNA in cytoplasmic NCs that was enhanced by L or found in secreted virions was modulated by the capsid linker sequence. The level and maturity of the cytoplasmic RC DNA was further influenced by the efficiency of extracellular virion secretion dependent on viral genotype-specific envelope proteins. These results suggest that interactions between the capsid and envelope proteins regulate one or more steps during virion secretion beyond initial capsid envelopment, and highlights the critical role of the capsid linker in regulating capsid-envelope interaction, including the timing of envelopment during NC maturation. Importance Hepatitis B virus (HBV) is a major human pathogen causing serious liver diseases including cancer. The interactions between the HBV capsid and the large (L) envelope protein is required for formation of infectious viral particles and also negatively regulate formation of an HBV DNA episome in the host cell nucleus, which serves as the sole transcriptional template capable of supporting all viral gene expression to sustain HBV replication and therefore, is the molecular basis of HBV persistence. Here, we report evidence indicating that L-capsid interactions modulate the timing of formation of infectious HBV particles during replication and facilitate extracellular release following their formation. Furthermore, a short linker sequence in the capsid protein plays a critical role in these processes as well as controls the amplification of the nuclear episome. These findings inform fundamental mechanisms of HBV replication as well as antiviral development targeting the HBV capsid and DNA episome.

STUDY QUESTION Is a dual ovulation trigger with a combination of GnRH agonist (GnRHa) and hCG superior to single hCG and/or single GnRHa trigger in improving treatment outcomes in advanced-age women (aged ≥ 35 years) undergoing IVF/ICSI treatment? SUMMARY ANSWER Co-administration of GnRHa and hCG as a dual trigger increases the number of good-quality embryos but it is not associated with a higher number of oocytes retrieved, compared with single hCG or GnRHa trigger. WHAT IS KNOWN ALREADY Many studies have demonstrated that a dual trigger has positive impact on oocyte maturation, retrieval rate and pregnancy rate without increasing the risk of ovarian hyperstimulation syndrome (OHSS) in some groups of IVF patients, when compared with single hCG trigger. Few studies have however been conducted to compare a dual trigger with a single GnRHa trigger, and insufficient evidence exists to support which trigger can achieve the best outcomes in IVF patients aged ≥35 years. STUDY DESIGN, SIZE, DURATION This was an open-label randomized controlled trial of 510 participants conducted at single reproductive medical center from January 2019 to December 2021. After a sample size calculation performed by retrospectively analyzing our previous clinical data, we planned to recruit 170 patients in each group and 510 patients in total for the study. PARTICIPANTS/MATERIALS, SETTING, METHODS Women aged ≥35 years undergoing IVF/ICSI treatment, receiving a non-pituitary down-regulation protocol, and with low risk of OHSS, were enrolled in this trial. On the trigger day, patients were randomized into three groups: hCG alone (who received 6000 IU of hCG), GnRHa alone (who received 0.2 mg of triptorelin) and dual trigger (who received 0.2 mg of triptorelin plus 2000 IU of hCG) groups. The primary outcome parameter was the number of retrieved oocytes. The secondary outcome parameters included, among others, the number and rates of mature oocytes, two pronuclei (2PN) embryos and good-quality embryos, as the rates of OHSS, clinical pregnancy, miscarriage and live birth. MAIN RESULTS AND THE ROLE OF CHANCE There were no significant differences in the baseline demographic characteristics among the three groups. The dual trigger was associated with a higher retrieval rate (87.9% vs 84.1% in the hCG group, P = 0.031; 87.9% vs 83.6% in the GnRHa group, P = 0.014). However, the number of retrieved oocytes in the dual trigger group was comparable with those in the hCG group (4.08 ± 2.79 vs 3.60 ± 2.71, P = 0.080) and the GnRHa group (4.08 ± 2.79 vs 3.81 ± 3.38, P = 0.101); comparable data between the groups were also found when analyzing the number of 2PN embryos and the 2PN rate. In the dual trigger group, the numbers of good-quality embryos and viable embryos were both significantly higher than in the hCG group (1.74 ± 1.90 vs 1.19 ± 1.45, P = 0.016 and 2.19 ± 2.11 vs 1.56 ± 1.66, P = 0.008, respectively) and the GnRHa group (1.74 ± 1.90 vs 1.20 ± 1.67, P = 0.003 and 2.19 ± 2.11 vs 1.45 ± 1.75, P = 0.001, respectively). Pregnancy outcomes after fresh embryo transfer (ET) were comparable between the groups. The live birth rate and ongoing pregnancy rate after frozen ET in the dual trigger group were significantly higher than those in the GnRHa group (32.6% vs 14.1%, P = 0.007 and 34.8% vs 17.6%, P = 0.013, respectively), but not superior to those in the hCG group (32.6% vs 27.9%, P = 0.537 and 34.8% vs 27.9%, P = 0.358, respectively). LIMITATIONS, REASONS FOR CAUTION Women of advanced age are quite a heterogeneous population and overlap with poor ovarian responders or patients with diminished ovarian reserve. We therefore could not entirely exclude selection biases or confounding factors. This study was also not a double-blinded trial; the patients in the GnRHa and dual trigger groups could have been affected by the placebo effect. WIDER IMPLICATIONS OF THE FINDINGS The results of this study suggest that in advanced-age women with low risk of OHSS, a dual trigger or even a single hCG trigger may be a better choice than a single GnRHa trigger. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Shanghai Municipal Health Commission of Science and Research Fund (20184Y0289). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER This trial was registered in the Chinese Clinical Trial Registry (ChiCTR-1800016285). TRIAL REGISTRATION DATE 24 May 2018 DATE OF FIRST PATIENT’S ENROLMENT 2 January 2019

Hepatitis B virus (HBV) capsid or core protein (HBc) consists of an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. Dynamic phosphorylation and dephosphorylation of HBc regulate its multiple functions in capsid assembly and viral replication. The cellular cyclin-dependent kinase 2 (CDK2) plays a major role in HBc phosphorylation and, furthermore, is incorporated into the viral capsid, accounting for most of the “endogenous kinase” activity associated with the capsid. The packaged CDK2 is thought to play a role in phosphorylating HBc to trigger nucleocapsid disassembly (uncoating), an essential step during viral infection. However, little is currently known on how CDK2 is recruited and packaged into the capsid. We have now identified three RXL motifs in the HBc NTD known as cyclin docking motifs (CDMs), which mediate the interactions of various CDK substrates/regulators with CDK/cyclin complexes. Mutations of the CDMs in the HBc NTD reduced CTD phosphorylation and diminished CDK2 packaging into the capsid. Also, the CDM mutations showed little effects on capsid assembly and pregenomic RNA (pgRNA) packaging but impaired the integrity of mature nucleocapsids. Furthermore, the CDM mutations blocked covalently closed circular DNA (CCC DNA) formation during infection while having no effect on or enhancing CCC DNA formation via intracellular amplification. These results indicate that the HBc NTD CDMs play a role in CDK2 recruitment and packaging, which, in turn, is important for productive infection. IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and persistently infects hundreds of millions of people, who are at high risk of cirrhosis and liver cancer. HBV capsid packages a host cell protein kinase, the cyclin-dependent kinase 2 (CDK2), which is thought to be required to trigger disassembly of the viral nucleocapsid during infection by phosphorylating the capsid protein, a prerequisite for successful infection. We have identified docking sites on the capsid protein for recruiting CDK2, in complex with its cyclin partner, to facilitate capsid protein phosphorylation and CDK2 packaging. Mutations of these docking sites reduced capsid protein phosphorylation, impaired CDK2 packaging into HBV capsids, and blocked HBV infection. These results provide novel insights regarding CDK2 packaging into HBV capsids and the role of CDK2 in HBV infection and should facilitate the development of antiviral drugs that target the HBV capsid protein.

Hepatitis B virus (HBV) capsid or core protein (HBc) contains an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. HBc plays a critical role in virtually every step of viral replication, which is further modulated by dynamic phosphorylation and dephosphorylation of its CTD. While several cellular kinases have been identified that mediate HBc CTD phosphorylation, there is little information on the cellular phosphatases that mediate CTD dephosphorylation. Herein, a consensus binding motif for the protein phosphatase 2A (PP2A) regulatory subunit B56 was recognized within the HBc linker peptide. Mutations within this motif designed to block or enhance B56 binding showed pleiotropic effects on CTD phosphorylation state as well as on viral RNA packaging, reverse transcription, and virion secretion. Furthermore, linker mutations affected the HBV nuclear episome (the covalently closed circular or CCC DNA) differentially during intracellular amplification vs. infection. The effects of linker mutations on CTD phosphorylation state varied with different phosphorylation sites and were only partially consistent with the linker motif serving to recruit PP2A-B56, specifically, to dephosphorylate CTD, suggesting that multiple phosphatases and/or kinases may be recruited to modulate CTD (de)phosphorylation. Furthermore, pharmacological inhibition of PP2A could decrease HBc CTD dephosphorylation and increase the nuclear HBV episome. These results thus strongly implicate the HBc linker in recruiting PP2A and other host factors to regulate multiple stages of HBV replication., Author summary Hepatitis B virus (HBV) causes acute and chronic viral hepatitis, liver fibrosis, cirrhosis and cancer. The dynamic phosphorylation and dephosphorylation of the viral capsid protein (HBc), which are controlled by host cell protein kinases and phosphatases, play a critical role in regulating multiple stages of HBV replication. While a number of cellular kinases have been identified that mediate HBc phosphorylation, there is little information on cellular phosphatases that mediate its dephosphorylation. Herein we have identified a consensus binding motif in HBc for one of the major cellular phosphatases, the protein phosphatase 2A (PP2A). Genetic analysis of this motif revealed that it played multiple roles in regulating CTD phosphorylation state, as well as viral RNA packaging, reverse transcription, virion secretion, and formation of the nuclear HBV episome responsible for viral persistence. Furthermore, pharmacological inhibition of PP2A decreased HBc dephosphorylation and increased the nuclear episome, further supporting a role of PP2A in HBc dephosphorylation and HBV persistence. These results thus suggest that HBc recruits PP2A, among other host factors, to regulate HBc phosphorylation and dephosphorylation dynamics and HBV replication and persistence.

To investigate the effect of basal hormone levels including follicle stimulating hormone (FSH) and estradiol (E2) and age on the pregnancy outcomes in women undergoing in vitro fertilization and embryo transfer (IVF-ET), we designed a retrospective cohort analysis and collected information of patients undergoing IVF between January 2014 and April 2017. We found that young women had much better pregnancy outcomes than older women regardless of the hormone levels. The pregnancy outcomes declined in young women with high basal E2, even though they had normal FSH. Older women with higher FSH had a worse outcome of pregnancy, especially with increased E2 level. The results suggest that a combination of FSH, E2 and age could effectively predict the pregnancy outcome for women undergoing IVF-ET, and we should encourage infertile women with diminished ovarian reserve, especially young infertile women, to try the assisted reproduction as early as possible. After 40-yearold, the cost-benefit ratio should be considered according to the ovarian function.

The aim of the present study was to investigate the associations between adiponectin receptor 2 (AdipoR2) gene polymorphisms, AdipoR2 protein expression levels and the risk of colorectal cancer (CRC). From April 2012 to May 2015, 281 CRC patients (case group) admitted to the China-Japan Friendship Hospital and 325 healthy control subjects (control group) were recruited for the study. Peripheral venous blood samples were collected and the DNA was extracted. Genotyping was performed using denaturing high-performance liquid chromatography in the condition of partial degeneration. Linkage disequilibrium and haplotype were analyzed using SHEsis analysis software. AdipoR2 protein expression levels were detected by immunohistochemistry and logistic regression analysis was performed to determine the risk factors of CRC. The distribution of the TT genotype of AdipoR2 rs10773989 and the CC genotype of AdipoR2 rs1044471 was higher in the case group than in the control group (P

Hepatitis B virus (HBV) delivers a partially double-stranded, relaxed circular (RC) DNA genome in complete virions to the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, which establishes and sustains viral infection. An overlength pregenomic RNA (pgRNA) is then transcribed from CCC DNA and packaged into immature nucleocapsids (NCs) by the viral core (HBc) protein. pgRNA is reverse transcribed to produce RC DNA in mature NCs, which are then enveloped and secreted as complete virions, or delivered to the nucleus to replenish the nuclear CCC DNA pool. RC DNA, whether originating from extracellular virions or intracellular mature NCs, must be released upon NC disassembly (uncoating) for CCC DNA formation. HBc is known to undergo dynamic phosphorylation and dephosphorylation at its C-terminal domain (CTD) to facilitate pgRNA packaging and reverse transcription. Here, two putative phosphorylation sites in the HBc N-terminal domain (NTD), S44 and S49, were targeted for genetic and biochemical analysis to assess their potential roles in viral replication. The NTD mutant that mimics the non-phosphorylated state (N2A) was competent in all steps of viral replication tested from capsid assembly, pgRNA packaging, reverse transcription, to virion secretion, except for a decrease in CCC DNA formation. On the other hand, the phosphor-mimetic mutant N2E showed a defect in the early step of pgRNA packaging but enhanced the late step of mature NC uncoating and consequently, increased CCC DNA formation. N2E also enhanced phosphorylation in CTD and possibly elsewhere in HBc. Furthermore, inhibition of the cyclin-dependent kinase 2 (CDK2), which is packaged into viral capsids, could block CCC DNA formation. These results prompted us to propose a model whereby rephosphorylation of HBc at both NTD and CTD by the packaged CDK2, following CTD dephosphorylation during NC maturation, facilitates uncoating and CCC DNA formation by destabilizing mature NCs., Author summary Hepatitis B virus (HBV) persistently infects hundreds of millions of people worldwide, causing viral hepatitis, cirrhosis and liver cancer. The basis of HBV persistence is the viral covalently closed circular (CCC) DNA, a nuclear episome, that drives all viral gene expression to sustain viral replication. CCC DNA is derived from the relaxed circular (RC) DNA, which is formed inside a proteinaceous shell, the viral capsid, but has to be released from the capsid in order to be converted to CCC DNA by host cell factors. We report here that the phosphorylation state of the capsid protein, regulated by host cell enzymes including one that is packaged inside the viral capsid, plays a critical role in regulating the release of RC DNA and thus controlling CCC DNA formation. Intense ongoing efforts are being directed at developing novel antiviral strategies to eliminate the HBV CCC DNA for curing persistence HBV infection, including those targeting the capsid protein. Our results should inspire novel approaches to targeting the HBV capsid and CCC DNA. Furthermore, uncoating is an essential step in the infection process for virtually all viruses that remains ill-understood. Thus, our results have broad implications for understanding viral infection in general.

AIM To detect abnormal microRNA (miRNA) expression in type 1 gastric neuroendocrine neoplasms (g-NENs) and find potential target genes. METHODS Tumour tissues from patients with type 1 g-NENs were used as experimental samples, and gastric mucosal tissues from the same patients obtained during gastroscopy review after several months were used as control samples. miRNA expression was examined with Agilent human miRNA chips and validated via RT-PCR. Three types of target gene prediction software (TargetScan, PITA, and microRNAorg) were used to predict potential target genes of the differentially expressed miRNAs, and a dual-luciferase reporter assay system was used for verification. RESULTS Six miRNAs were significantly upregulated or downregulated in the tumours compared to the control samples. Among them, miR-202-3p was extraordinarily upregulated. RT-PCR of seven sample sets confirmed that miR-202-3p was upregulated in tumour tissues. In total, 215 target genes were predicted to be associated with miR-202-3p. Among them, dual-specificity phosphatase 1 (DUSP1) was reported to be closely related to tumour occurrence and development. The dual-luciferase reporter assay showed that miR-202-3p directly regulated DUSP1 in 293T cells. CONCLUSION miR-202-3p is upregulated in type 1 g-NEN lesions and might play important roles in the pathogenesis of type 1 g-NENs by targeting DUSP1.

Background The goal of this study was to assess the association between endometrial thickness on the chorionic gonadotropin (hCG) day and in vitro fertilization and embryo transfer (IVF-ET) outcome in normal responders after GnRH antagonist administration. Methods A retrospective cohort study was performed in normal responders with GnRH antagonist administration from January 2011–December 2013. Patients were divided into four groups according to endometrial thickness, as follows: = 7- = 8- =14 mm (group 4). Results A total of 2106 embryo transfer cycles were analyzed. The pregnancy rate (PR) was 44.87%. The clinical pregnancy rate, ongoing pregnancy rate and the implantation rate (17.28%, 13.79%, 10.17%, respectively) were significantly lower in group 1 compared to the other three groups (p

Influenza is a serious global public health problem and an economic burden. With the continual emergence of new influenza A virus strains, new antiviral drugs are needed urgently. In this study, an improved embryonated chicken egg model for evaluating antiviral activity against Influenza A virus was developed. In the model, the influenza A virus was injected into the allantoic cavity and ribavirin was injected into the albumen of the egg. The levels of influenza A virus in the allantoic fluid was titrated by the hemagglutination test after incubation for 72 h at 35.5 degrees C and 12 h at 4 degrees C. Ribavirin treatment at a dose of 25 mg/kg to 100 mg/kg decreased significantly the hemagglutination titers both of Influenza virus A/FM1, H1N1 (IVA1) (p < 0.01) and influenza virus A/Wuhan/359/95, H3N2 (IVA3) (p < 0.01). In a time-dependent drug addition assay, significant efficacy of ribavirin against both IVA1 and IVA3 was observed when the drug was administered before and shortly after viral inoculation (p < 0.01 or p < 0.05). In conclusion, ribavirin treatment showed significant antiviral activity against IVA1 and IVA3 in this model, suggesting that the improved model would be useful for evaluating the anti-influenza virus activity of potential inhibitors.

Based on a photoelectron transfer (PET) mechanism, vicinal diaminobenzoacridine (VDABA), a fluorescent probe for the determination of trace amounts of nitric oxide radical in biological sample, was synthesized and characterized by elemental analysis, IR, 1H NMR and 13C NMR spectrum. Combining a flow injection with spectrofluorimetry, a high-throughput method for detecting NO was obtained, which was successfully applied to the determination of NO in the human serum. The proposed method was simple, rapid, precise and automatic. Under optimum conditions, the linear calibration range was from 1.1 × 10−7 to 5.0 × 10−6 M and the detection limit was 3.1 × 10−8 M. Furthermore, the probe could make cell-derived NO “visible” by using confocal laser scanning microscope.

Zinc finger protein, one of the most important transcription factors, plays an essential role in regulating gene expression. C(2)H(2) type zinc protein with KRAB domain contains two parts, one is C(2)H(2) zinc finger motif which is use to binding to the DNA, while the other is KRAB associated box which mostly performs as a transcription repressor. In this study, we report the cloning and characterization of a novel splice variant of the human ZNF300 gene (ZNF300-B). The ZNF300-B gene cDNA is 2293 bp in length, encoding a putative protein with 619 amino acid residues. ZNF300-B gene is mapped to chromosome 5q32-5q33 with seven exons. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ZNF300-B and ZNF-300 were expressed highly in human testis.

Bim proteins are essential factors of apoptosis. Nine isoforms of Bim have been submitted to GenBank database. In order to improve the understanding of the regulation of Bims' proapoptotic activity, we screened a multiple tissue cDNA panels for Bim isoforms and tested their proapoptotic activity by over-expression. Two novel cDNA isoforms of Bim family are generated by alternative splicing. One isoform encodes a 79 residue putative protein with a BH3 domain, named Bim alpha3. There is not any significant ORF found in another isoform, named Bim beta5. Subcellular localized analysis of EGFP-Bim fusion protein suggests Bim alpha3 distributed to both plasma and nucleus of HEK 293 cell. HEK 293 cells transfected with pcDNA-Bim alpha3 presented a similar proapoptotic activity (32.05 +/- 6.42%) with Bim alpha2 (30.14 +/- 2.66%). The proapoptotic activity of Bim alpha3 was obviously weaker than that of Bim S (46.52 +/- 5.07%) and Bim L (55.53 +/- 1.99%). Anti-sense over-expression of Bim in HEK 293 cells results in a weak down-regulated proapoptotic level. Expression pattern analysis reveals that both the novel cDNAs are expressed widely in normal tissue just like the other reported isoforms. The expression pattern of Bim isoforms shows tissue specific obviously. The results suggest that BH3 domain is sufficiency for proapoptotic activity of Bim proteins. The functional state of Bims might be regulated both in the transcript and post transcript process.