Nicotinamide N-Methyltransferase inhibits HBV replication by suppressing NR5A1 expression invitro.

Chronic hepatitis B virus (HBV) infection can lead to fibrosis, liver cirrhosis, and primary hepatocellular carcinoma. Investigating host factors that regulate HBV replication helps to identify antiviral targets. In the current study, we identified Nicotinamide N-Methyltransferase gene (NNMT) as a novel factor that regulates HBV transcription. NNMT is up-regulated at both the mRNA and protein levels in HepG2.2.15 cells compared to HepG2 cells. Overexpression of NNMT reduces HBV replication in several cell models, while knockdown of NNMT enhances HBV DNA levels. Mechanistically, NNMT suppresses HBV DNA replication by inhibiting HBV RNA transcription. The region required for the inhibitory effect of NNMT was narrowed to nt 1672-1708 in enhancer II by luciferase assays. On the other hand, ChIP assays and EMSA results showed that NNMT does not bind to this region substantially, either directly or indirectly. Next, a collection of hepatic nuclear receptor transcription factors was screened to determine whether they were affected by NNMT overexpression. NR5A1, a positive regulator of HBV replication, decreased significantly after NNMT overexpression. Collectively, the findings of this study shed light on the regulation of HBV transcription.

Dicoumarol, an NQO1 inhibitor, blocks cccDNA transcription by promoting degradation of HBx.

BACKGROUND & AIMS: Current antiviral therapies help keep HBV under control, but they are not curative, as they are unable to eliminate the intracellular viral replication intermediate termed covalently closed circular DNA (cccDNA). Therefore, there remains an urgent need to develop strategies to cure CHB. Functional silencing of cccDNA is a crucial curative strategy that may be achieved by targeting the viral protein HBx. METHODS: We screened 2,000 small-molecule compounds for their ability to inhibit HiBiT-tagged HBx (HiBiT-HBx) expression by using a HiBiT lytic detection system. The antiviral activity of a candidate compound and underlying mechanism of its effect on cccDNA transcription were evaluated in HBV-infected cells and a humanised liver mouse model. RESULTS: Dicoumarol, an inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), significantly reduced HBx expression. Moreover, dicoumarol showed potent antiviral activity against HBV RNAs, HBV DNA, HBsAg and HBc protein in HBV-infected cells and a humanised liver mouse model. Mechanistic studies demonstrated that endogenous NQO1 binds to and protects HBx protein from 20S proteasome-mediated degradation. NQO1 knockdown or dicoumarol treatment significantly reduced the recruitment of HBx to cccDNA and inhibited the transcriptional activity of cccDNA, which was associated with the establishment of a repressive chromatin state. The absence of HBx markedly blocked the antiviral effect induced by NQO1 knockdown or dicoumarol treatment in HBV-infected cells. CONCLUSIONS: Herein, we report on a novel small molecule that targets HBx to combat chronic HBV infection; we also reveal that NQO1 has a role in HBV replication through the regulation of HBx protein stability. LAY SUMMARY: Current antiviral therapies for hepatitis B are not curative because of their inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nuclei of infected cells. HBV X (HBx) protein has an important role in regulating cccDNA transcription. Thus, targeting HBx to silence cccDNA transcription could be an important curative strategy. We identified that the small molecule dicoumarol could block cccDNA transcription by promoting HBx degradation; this is a promising therapeutic strategy for the treatment of chronic hepatitis B.

SIRT7 restricts HBV transcription and replication through catalyzing desuccinylation of histone H3 associated with cccDNA minichromosome.

Chronic hepatitis B virus (HBV) infection is a significant public health burden worldwide. HBV covalently closed circular DNA (cccDNA) organized as a minichromosome in nucleus is responsible for viral persistence and is the key obstacle for a cure of chronic hepatitis B (CHB). Recent studies suggest cccDNA transcription is epigenetically regulated by histone modifications, especially histone acetylation and methylation. In the present study, we identified transcriptionally active histone succinylation (H3K122succ) as a new histone modification on cccDNA minichromosome by using cccDNA ChIP-Seq approach. Silent mating type information regulation 2 homolog 7 (SIRT7), as an NAD+-dependent histone desuccinylase, could bind to cccDNA through interaction with HBV core protein where it catalyzed histone 3 lysine 122 (H3K122) desuccinylation. Moreover, SIRT7 acts cooperatively with histone methyltransferase, suppressor of variegation 3-9 homolog 1 (SUV39H1) and SET domain containing 2 (SETD2) to induce silencing of HBV transcription through modulation of chromatin structure. Our data improved the understanding of histone modifications of the cccDNA minichromosome, thus transcriptional silencing of cccDNA may represent a novel antiviral strategy for the prevention or treatment of HBV infection.

SIRT3 restricts hepatitis B virus transcription and replication through epigenetic regulation of covalently closed circular DNA involving suppressor of variegation 3-9 homolog 1 and SET domain containing 1A histone methyltransferases.

Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA), which serves as a template for HBV RNA transcription, is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified silent mating type information regulation 2 homolog 3 (SIRT3) as a host factor restricting HBV transcription and replication by screening seven members of the sirtuin family, which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA, as well as replicative intermediate DNA in HBV-infected HepG2-Na+ /taurocholate cotransporting polypeptide cells and primary human hepatocytes. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. A mechanistic study found that nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9. Importantly, occupancy of SIRT3 on cccDNA could increase the recruitment of histone methyltransferase suppressor of variegation 3-9 homolog 1 to cccDNA and decrease recruitment of SET domain containing 1A, leading to a marked increase of trimethyl-histone H3 (Lys9) and a decrease of trimethyl-histone H3 (Lys4) on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor Yin Yang 1 to cccDNA. Finally, hepatitis B viral X protein could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. CONCLUSION: SIRT3 is a host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase; these data provide a rationale for the use of SIRT3 activators in the prevention or treatment of HBV infection. (Hepatology 2018).

Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay.

The capsid of the hepatitis B virus is an attractive antiviral target for developing therapies against chronic hepatitis B infection. Currently available core protein allosteric modulators (CpAMs) mainly affect one of the two major types of protein-protein interactions involved in the process of capsid assembly, namely, the interaction between the core dimers. Compounds targeting the interaction between two core monomers have not been rigorously screened due to the lack of screening models. We report here a cell-based assay in which the formation of core dimers is indicated by split luciferase complementation (SLC). Making use of this model, 2 compounds, Arbidol (umifenovir) and 20-deoxyingenol, were identified from a library containing 672 compounds as core dimerization regulators. Arbidol and 20-deoxyingenol inhibit the hepatitis B virus (HBV) DNA replication in vitro by decreasing and increasing the formation of core dimer and capsid, respectively. Our results provided a proof of concept for the cell model to be used to screen new agents targeting the step of core dimer and capsid formation.

The nano-artificial periosteum made of PCL/MgO/AS-IV enhances MC3T3-E1 cell osteogenic differentiation and promotes bone defect repair via the EphB4/EphrinB2 signaling pathway.

Bone regeneration plays a pivotal role in periodontal tissue repair. With advancements in biotechnology materials, the utilization of nanotechnology offers a reliable platform for bone restoration in periodontitis. In this study, we successfully established a long-term bacterial infection model using Porphyromonas gingivalis (P. gingivalis) with MOI = 50. CCK-8 and ROS immunofluorescence results demonstrated that the combined effect of Mg2+ and AS-IV significantly enhanced cell proliferation and effectively suppressed the inflammatory response during bacterial infection. Alkaline phosphatase and alizarin red staining revealed that the synergistic action of Mg2+ and AS-IV notably promoted osteogenic differentiation of MC3T3-E1 cells under P. gingivalis-infected conditions. Considering the properties of these two biomaterials, we fabricated polycaprolactone (PCL) artificial periosteum loaded with MgO and AS-IV using an electrostatic spinning technique. The findings indicated that PCL/MgO/AS-IV artificial periosteum exhibited excellent biocompatibility and hydrophilicity, thereby substantially enhancing cellular adhesion to its surface as well as augmenting cellular value-added rate. Moreover, efficient drug release from the PCL/MgO/AS-IV artificial bone membrane conferred remarkable antimicrobial activity along with in vitro osteogenic potentiality. The in vivo experiments conducted on animals further substantiated the exceptional properties exhibited by PCL/MgO/AS-IV artificial periosteum in bone defect repair. Additionally, it was observed that PCL/MgO/AS-IV artificial periosteum could modulate EphB4-EphrinB2 signaling to enhance osteogenic differentiation under P.gingivalis-infected conditions.This exciting outcome suggests that PCL/MgO/AS-IV artificial periosteum holds great promise as a biomaterial for treating periodontal bone loss.

[Establishment of a stable cell line replicating hepatitis B virus DNA carrying the reverse transcriptase region derived from a clinical isolate].

OBJECTIVE: To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate. METHODS: Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis. RESULTS: Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained. CONCLUSIONS: A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.

Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy.

Phenotypic assays of hepatitis B virus (HBV) play an important role in research related to the problem of drug resistance that emerges during long-term nucleot(s)ide therapy in patients with chronic hepatitis B. Most of the phenotypic assay systems that are available currently rely on the transfection of recombinant replication-competent HBV DNA into hepatoma cell lines. Cloning clinical HBV isolates using conventional digestion-and-ligation techniques to generate replication-competent recombinants can be very difficult because of the sequence heterogeneity and unique structure of the HBV genome. In this study, a new strategy for constructing an HBV 1.1× recombinant was developed. The core of this strategy is the "fragment substitution reaction" (FSR). FSR allows PCR fragments to be cloned without digestion or ligation, providing a new tool for cloning fragments or genomes amplified from serum HBV DNA, and therefore making the assay of HBV phenotypes more convenient. Using this strategy, a phenotypic assay was performed on an HBV strain carrying an rtS246T variant isolated from a patient with chronic hepatitis B that was only responsive partially to entecavir therapy. The results indicated that this strain is sensitive to entecavir in vitro.

Etoposide phosphate enhances the acetylation level of translation elongation factor 1A in PLC5 cells.

Translation elongation factor 1A (eEF1A) is a factor critically involved in the process of protein synthesis. The activity of eEF1A has been shown by several studies to be regulated by post-translational modifications such as phosphorylation and dephosphorylation. However, until now less research has focused on other post-translational modifications of eEF1A, especially acetylation. In this report, we provide new evidence for the existence of eEF1A acetylation in PLC5 cells by immunoprecipitation and Western blotting. Using the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), we found that the deacetylation of eEF1A is mainly attributable to classes I and II HDAC rather than class III HDAC, and, furthermore, that the antitumour agent etoposide phosphate (VP 16) enhances the acetylation of eEF1A in a synergistic way with TSA. Our data suggest the possibility that the increased acetylation of eEF1A could be a new mechanism for the antitumour effect of etoposide.

Establishment of a human cell line with a surface display system for screening and optimizing Na+-taurocholate cotransporting polypeptide-binding peptides.

One of the most desirable targets for HBV medications is the sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for the hepatitis B virus (HBV). N-myristoylated preS1 2-48 (Myrcludex B or Hepcludex), an NTCP-binding peptide from the large surface protein of HBV, has been developed as the first-in-class entry inhibitor. However, its relatively large molecular weight contributes to increased immunogenicity and antibody production. As a result, it is preferable to look for an NTCP-binding peptide with a smaller size. To do this, we developed a human cell surface display strategy and screened peptides based on preS1-21. PreS1-21 (genotype D) was extended by 7 random amino acids and fused with mCherry and FasL transmembrane domain. The pooled constructs were transfected into HEK293 cells by using the transposon/transposase system to create a library displaying various peptides on the cell surface with red fluorescence. On the other hand, we expressed NTCP protein fused with EGFP on HEK293 and used the membrane lysate containing NTCP-GFP as the bait protein to select peptides with increased NTCP affinity. After 7 cycles of selection, the deep sequencing results revealed that some polypeptides were more than 1,000 times enriched. Further screening of the mostly enriched 10 peptides yields the peptide preS1-21-pep3. Replacing the preS1-21 sequence of preS1-21-pep3 with those from different genotypes demonstrated that the consensus sequence of genotype A-F had the best performance. The peptide (Myr-preS1-21-pep3) was synthesized and tested on the HepG2-NTCP cell model. The results showed that Myr-preS1-21-pep3 is approximately 10 times more potent than the initial peptide Myr-preS1-21 in preventing HBV infection. In conclusion, we developed a new strategy for screening peptides binding to membrane proteins and identified a new NTCP-binding peptide with a much smaller size than Hepcludex.

Identification of STAU1 as a regulator of HBV replication by TurboID-based proximity labeling.

The core promoter (CP) of hepatitis B virus (HBV) is critical for HBV replication by controlling the transcription of pregenomic RNA (pgRNA). Host factors regulating the activity of the CP can be identified by different methods. Biotin-based proximity labeling, a powerful method with the capability to capture weak or dynamic interactions, has not yet been used to map proteins interacting with the CP. Here, we established a strategy, based on the newly evolved promiscuous enzyme TurboID, for interrogating host factors regulating the activity of HBV CP. Using this strategy, we identified STAU1 as an important factor involved in the regulation of HBV CP. Mechanistically, STAU1 indirectly binds to CP mediated by TARDBP, and recruits the SAGA transcription coactivator complex to the CP to upregulate its activity. Moreover, STAU1 binds to HBx and enhances the level of HBx by stabilizing it in a ubiquitin-independent manner.

Protein sensors combining both on-and-off model for antibody homogeneous assay.

Protein sensors based on allosteric enzymes responding to target binding with rapid changes in enzymatic activity are potential tools for homogeneous assays. However, a high signal-to-noise ratio (S/N) is difficult to achieve in their construction. A high S/N is critical to discriminate signals from the background, a phenomenon that might largely vary among serum samples from different individuals. Herein, based on the modularized luciferase NanoLuc, we designed a novel biosensor called NanoSwitch. This sensor allows direct detection of antibodies in 1 µl serum in 45 min without washing steps. In the detection of Flag and HA antibodies, NanoSwitches respond to antibodies with S/N ratios of 33-fold and 42-fold, respectively. Further, we constructed a NanoSwitch for detecting SARS-CoV-2-specific antibodies, which showed over 200-fold S/N in serum samples. High S/N was achieved by a new working model, combining the turn-off of the sensor with human serum albumin and turn-on with a specific antibody. Also, we constructed NanoSwitches for detecting antibodies against the core protein of hepatitis C virus (HCV) and gp41 of the human immunodeficiency virus (HIV). Interestingly, these sensors demonstrated a high S/N and good performance in the assays of clinical samples; this was partly attributed to the combination of off-and-on models. In summary, we provide a novel type of protein sensor and a working model that potentially guides new sensor design with better performance.

Comparison of a novel real-time PCR assay with sequence analysis, reverse hybridization, and multiplex PCR for hepatitis B virus type B and C genotyping.

We compared a novel real-time genotyping and quantitative PCR (GQ-PCR) assay, direct sequence analysis, reverse hybridization, and multiplex PCR for genotyping hepatitis B virus (HBV) in 127 HBV-infected patients. We found that GQ-PCR had the highest concordance with sequence analysis and the highest detection rate for mixed genotype detecting.

[Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations].

OBJECTIVE: To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system. METHOD: 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively. RESULT: Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/µl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test. CONCLUSION: Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.

DNA Engineering and Hepatitis B Virus Replication.

Recombinant DNA technology is a vital method in human hepatitis B virus (HBV), producing reporter viruses or vectors for gene transferring. Researchers have engineered several genes into the HBV genome for different purposes; however, a systematic analysis of recombinant strategy is lacking. Here, using a 500-bp deletion strategy, we scanned the HBV genome and identified two regions, region I (from nt 2,118 to 2,814) and region II (from nt 99 to 1,198), suitable for engineering. Ten exogenous genes, including puromycin N-acetyl transferase gene (Pac), blasticidin S deaminase gene (BSD), Neomycin-resistance gene (Neo), Gaussia luciferase (Gluc), NanoLuc (Nluc), copGFP, mCherry, UnaG, eGFP, and tTA1, were inserted into these two regions and fused into the open reading frames of hepatitis B core protein (HBC) and hepatitis B surface protein (HBS) via T2A peptide. Recombination of 9 of the 10 genes at region 99-1198 and 5 of the 10 genes at region 2118-2814 supported the formation of relaxed circular (RC) DNA. HBV DNA and HBV RNA assays implied that exogenous genes potentially abrogate RC DNA by inducing the formation of adverse secondary structures. This hypothesis was supported because sequence optimization of the UnaG gene based on HBC sequence rescued RC DNA formation. Findings from this study provide an informative basis and a valuable method for further constructing and optimizing recombinant HBV and imply that DNA sequence might be intrinsically a potential source of selective pressure in the evolution of HBV.

[Detection of HBV resistant mutations related to lamivudine, adefovir and entecavir by reverse hybridization technique].

OBJECTIVES: To establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues. METHODS: According to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively. RESULTS: The specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing. CONCLUSION: Reverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.

Niacin analogue, 6-Aminonicotinamide, a novel inhibitor of hepatitis B virus replication and HBsAg production.

BACKGROUND: Hepatitis B surface antigen (HBsAg) is one of the important clinical indexes for hepatitis B virus (HBV) infection diagnosis and sustained seroconversion of HBsAg is an indicator for functional cure. However, the level of HBsAg could not be reduced by interferons and nucleoside analogs effectively. Therefore, identification of a new drug targeting HBsAg is urgently needed. METHODS: In this study, 6-AN was screened out from 1500 compounds due to its low cytotoxicity and high antiviral activity. The effect of 6-AN on HBV was examined in HepAD38, HepG2-NTCP and PHHs cells. In addition, the antivirus effect of 6-AN was also identified in mouse model. FINDINGS: 6-AN treatment resulted in a significant decrease of HBsAg and other viral markers both in vitro and in vivo. Furthermore, we found that 6-AN inhibited the activities of HBV SpI, SpII and core promoter by decreasing transcription factor PPARα, subsequently reduced HBV RNAs transcription and HBsAg production. INTERPRETATION: We have identified a novel small molecule to inhibit HBV core DNA, HBV RNAs, HBsAg production, as well as cccDNA to a minor degree both in vitro and in vivo. This study may shed light on the development of a novel class of anti-HBV agent.

NAD(P)H: Quinone oxidoreductase 1 overexpression in hepatocellular carcinoma potentiates apoptosis evasion through regulating stabilization of X-linked inhibitor of apoptosis protein.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is an antioxidant enzyme which is associated with poor prognosis in human breast, colon, lung and liver cancers. However, the molecular mechanisms underlying the pro-tumorigenic function of NQO1 remains unclear. This study investigated the function of NQO1 in the context of hepatocellular carcinoma (HCC) development. We found that NQO1 was frequently up-regulated in human liver cancer, and its high expression level was correlated with the tumor stage and low survival rate of HCC patients. Loss-of-function of NQO1 inhibited growth in HCC cells with increased apoptosis in vitro, and suppressed orthotopic tumorigenicity in vivo. Mechanistically, high level of NQO1 in HCC cells enhanced protein stability of X-linked inhibitor of apoptosis protein (XIAP) by increasing its phosphorylation at Ser 87. Reintroduction of wile type XIAP and the phospho-mimic mutants XIAPS87D significantly reversed NQO1 knock-down/out induced growth inhibition and apoptosis. In mouse model with orthotopically implanted hepatocarcinoma, NQO1 suppression and NQO1 inhibitor suppressed tumor growth and induced apoptosis. NQO1 plays an important role in sustaining HCC cell proliferation and may thus act as a potential therapeutic target in HCC treatment.

Elastic intramedullary nail for treatment of extremity fractures in children.

OBJECTIVE: To assess the clinical therapeutic effects of elastic intramedullary nail on extremity fractures in children. METHODS: From June 2005 to March 2008, 40 children with extremity fractures were treated by elastic intramedullary nail, in whom femoral shaft fractures occurred in 26 cases, tibiofibular fractures in 8 cases, radial capitular fractures in 4 cases, ulnoradial fractures in 2 cases. All patients were treated by closed reduction and elastic intramedullary nail fixation. RESULTS: All the fractures gained satisfactory reduction and healing. The average duration needed for fracture healing was 1-2 months. Postoperative follow-up confirmed a sound functional recovery. CONCLUSIONS: The elastic intramedullary nail is a minimally invasive and effective surgical approach for treatment of extremity fractures in children. It allows early functional exercises after operation and secures a satisfactory bone union and functional recovery.

Fluorescent protein tagged hepatitis B virus capsid protein with long glycine-serine linker that supports nucleocapsid formation.

Fusion core proteins of Hepatitis B virus can be used to study core protein functions or capsid trafficking. A problem in constructing fusion core proteins is functional impairment of the individual domains in these fusion proteins, might due to structural interference. We reported a method to construct fusion proteins of Hepatitis B virus core protein (HBc) in which the functions of fused domains were partially kept. This method follows two principles: (1) fuse heterogeneous proteins at the N terminus of HBc; (2) use long Glycine-serine linkers between the two domains. Using EGFP and RFP as examples, we showed that long flexible G4S linkers can effectively separate the two domains in function. Among these fusion proteins constructed, GFP-G4S186-HBc and RFP-G4S47-HBc showed the best efficiency in rescuing the replication of an HBV replicon deficient in the core protein expression, though both of the two fusion proteins failed to support the formation of the relaxed circular DNA. These fluorescent protein-tagged HBcs might help study related to HBc or capsids tracking in cells.