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Background and Purpose: Only limited therapeutic agents have been developed for non-alcoholic steatohepatitis (NASH). Glabridin, a promising anti-obesity candidate, has only limited druggability due to its low in vivo chemical stability and bioavailability. Therefore, we developed vutiglabridin (VUTI), which is based on a glabridin backbone, and investigated its mechanism of action in treating NASH in animal models., Experimental Approach: Anti-NASH effects of VUTI were determined in in vitro fatty liver models, spheroids of primary human hepatocytes and L02 normal liver cell lines. To identify VUTI possible cellular target/s, biotin-labelled VUTI was synthesized and underwent chemical proteomic analysis. Further, the evaluation of VUTI therapeutic efficacy was carried out using an amylin-NASH and high-fat (HF) diet-induced obese (DIO) mouse models. This was carried out using transcriptomic, lipidomic and proteomic analyses of the livers from the amylin-NASH mouse model., Key Results: VUTI treatment markedly reduces hepatic steatosis, fibrosis and inflammation by promoting lipid catabolism, activating autophagy and improving mitochondrial dysfunction, all of which are hallmarks of effective NASH treatment. The cellular target of VUTI was identified as paraoxonase 2 (PON2), a newly proposed protein target for the treatment of NASH, VUTI enhanced PON2 activity. The results using PON2 knockdown cells demonstrated that PON2 is important for VUTI- activation of autophagy, promoting mitochondrial function, decreasing oxidative stress and alleviating lipid accumulation under lipotoxic condition., Conclusion and Implications: Our data demonstrated that VUTI is a promising therapeutic for NASH. Targeting PON2 may be important for improving liver function in various immune-metabolic diseases including NASH., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Tumor necrosis factor superfamily (TNFSF) resistance contributes to the development and progression of tumors and resistance to various cancer therapies. Tumor-intrinsic alterations involved in the adaptation to the TNFSF response remain largely unknown. Here, we demonstrate that protein kinase C substrate 80K-H (PRKCSH) abundance in lung cancers boosts oncogenic IGF1R activation, leading to TNFSF resistance. PRKCSH abundance is correlated with IGF1R upregulation in lung cancer tissues. Specifically, PRKCSH interacts with IGF1R and extends its half-life. The PRKCSH-IGF1R axis in tumor cells impairs caspase-8 activation, increases Mcl-1 expression, and inhibits caspase-9, leading to an imbalance between cell death and survival. PRKCSH deficiency augmented the antitumor effects of natural killer (NK) cells, representative TNFSF effector cells, in a tumor xenograft IL-2Rg-deficient NOD/SCID (NIG) mouse model. Our data suggest that PRKCSH plays a critical role in TNFSF resistance and may be a potential target to improve the efficacy of NK cell-based cancer therapy., (© 2024. The Author(s).)

Purpose: Septoturbinoplasty is frequently performed to correct nasal obstruction; however, there is still a lack of research on changes in nasal and nose-related symptoms early after septoturbinoplasty. Therefore, we aimed to investigate changes in subjective outcomes within 6 months after septoturbinoplasty., Materials and Methods: The medical records of patients who underwent septoturbinoplasty at Gangnam Severance Hospital were retrospectively analyzed. Symptom scores were evaluated using the Sino-nasal Outcome Test (SNOT-22) and obstruction scores. The SNOT-22 and obstruction scores were investigated before surgery and at 1, 3, and 6 months after surgery., Results: We noted significant decreases in both SNOT-22 and obstruction scores at 1 month after surgery, compared to those before surgery ( p <0.001). However, there were no significant changes at 3 and 6 months after surgery, compared to scores at 1 month after surgery. Using multivariate logistic regression analysis, a larger difference between SNOT-22 scores preoperatively and 1 month after surgery was significantly associated with a significant improvement in symptoms at 3 or 6 months after septoturbinoplasty ( p =0.029)., Conclusion: These results imply that subjective outcomes and degree of improvement in the first month after septoturbinoplasty can be used as a predictor of the results thereof and for counseling patients about its progress., Competing Interests: The authors have no potential conflicts of interest to disclose., (© Copyright: Yonsei University College of Medicine 2023.)

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent immuno-metabolic disease that can progress to hepatic cirrhosis and cancer. NAFLD pathogenesis is extremely complex and is characterized by oxidative stress, impaired mitochondrial function and lipid metabolism, and cellular inflammation. Thus, in-depth research on its underlying mechanisms and subsequent investigation into a potential drug target that has overarching effects on these features will help in the discovery of effective treatments for NAFLD. Our study examines the role of endogenous paraoxonase-2 (PON2), a membrane protein with reported antioxidant activity, in an in vitro cell model of NAFLD. We found that the hepatic loss of PON2 activity aggravated steatosis and oxidative stress under lipotoxic conditions, and our transcriptome analysis revealed that the loss of PON2 disrupts the activation of numerous functional pathways closely related to NAFLD pathogenesis, including mitochondrial respiratory capacity, lipid metabolism, and hepatic fibrosis and inflammation. We found that PON2 promoted the activation of the autophagy pathway, specifically the mitophagy cargo sequestration, which could potentially aid PON2 in alleviating oxidative stress, mitochondrial dysfunction, lipid accumulation, and inflammation. These results provide a mechanistic foundation for the prospect of PON2 as a drug target, leading to the development of novel therapeutics for NAFLD., (© 2022. The Author(s).)

By affecting the tongue position and oropharyngeal airway volume, torus mandibularis is an anatomical factor associated with obstructive sleep apnea (OSA). This study aimed to investigate the influence of torus mandibularis on the surgical outcomes of multilevel upper airway surgery with tongue base resection (TBR) in patients with OSA. Patients with OSA who underwent palatal surgery and TBR were retrospectively analyzed. The patients were divided into two groups according to the presence or absence of torus mandibularis upon physical examination or on computed tomography images. The anatomical characteristics of the upper airway and pre/postoperative polysomnography were analyzed. The control and torus mandibularis groups comprised 69 and 35 patients, respectively, with all of them showing improved sleep quality after surgery. Apnea-hypopnea index (AHI) scores decreased from 42.1 ± 22.2 preoperatively to 23.9 ± 21.4 postoperatively in the control group (p < 0.001), and from 45.2 ± 19.9 to 22.5 ± 13.5 in the torus mandibularis group (p < 0.001). Comparing the postoperative changes in AHI, the AHI of the torus mandibularis group improved by 22.7 ± 23.4, whereas that of the control group improved by 18.1 ± 19.6 (p = 0.296). Sleep efficiency improved from 90.0 ± 7.5 to 92.8 ± 6.8 in the control group, and from 90.3 ± 8.7 to 93.6 ± 6.5 in the torus mandibularis group; however, there was no statistical difference between the two groups (p = 0.816). The presence of torus mandibularis did not appear to significantly affect the surgical results in OSA patients, but it did elicit significant changes in polysomnographic parameters compared with the control group. Therefore, following the identification of torus mandibularis in OSA patients, TBR should be considered as part of planning, as it may help to predict surgical outcomes., Competing Interests: Declaration of competing interest The authors have no financial or personal conflicts of interest to declare., (Copyright © 2021 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Autosomal recessive nonsyndromic hearing loss 3 (DFNB3) mainly leads to congenital and severe-to-profound hearing impairment, which is caused by variants in MYO15A. However, audiological heterogeneity in patients with DFNB3 hinders precision medicine in hearing rehabilitation. Here, we aimed to elucidate the heterogeneity of the auditory phenotypes of MYO15A variants according to the affected domain and the feasibilities for acoustic stimulation. We conducted whole-exome sequencing for 10 unrelated individuals from seven multiplex families with DFNB3; 11 MYO15A variants, including the novel frameshift c.900delT (p.Pro301Argfs*143) and nonsense c.4879G > T (p.Glu1627*) variants, were identified. In seven probands, residual hearing at low frequencies was significantly higher in the groups with one or two N-terminal frameshift variants in trans conformation compared to that in the group without these variants. This is consistent with the 56 individuals from the previously published reports that carried a varying number of N-terminal truncating variants in MYO15A. In addition, patients with missense variants in the second FERM domain had better hearing at low frequencies than patients without these variants. Subsequently, acoustic stimulation provided by devices such as hearing aids or cochlear implants was feasible in patients with one or two N-terminal truncating variants or a second FERM missense variant. In conclusion, N-terminal or second FERM variants in MYO15A allow the practical use of acoustic stimulation through hearing aids or electroacoustic stimulation for aural rehabilitation., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021. Published by Elsevier B.V.)

With the climate change problems being strongly emphasized by the international communities, sharing electric bicycle is promoted to reduce air pollution in China. However, the consumer attitude, intention, and behavior on the electric bicycle is not well verified. We thus propose a theoretical framework that hypothesizes service quality, attitude, subject norm, perceived behavioral control, and residual effects of past behavior on the intention and behavior toward the shared electric bicycles. An online survey was conducted with 503 users of the bicycles in China at year 2018, and the data were analyzed with the partial least sequenced structural equation modeling (PLS-SEM). We find that shared electric bicycle's service quality has a positive effect on attitude toward shared electric bicycle and behavior intention, which, in turn, influence low-carbon commuting of shared electric bicycle's behavior. However, the residual effects of past behavior has no effect on further behavior of shared electric bicycles. Moreover, we also find that perceived behavioral control positively affect shared electric bicycle' intention and positively moderate the relationship between attitude and behavioral intention. Based on our findings, we discuss the policy implications and further studies.

Unfolded protein response (UPR) is an adaptive mechanism that aims at restoring ER homeostasis under severe environmental stress. Malignant cells are resistant to environmental stress, which is largely due to an activated UPR. However, the molecular mechanisms by which different UPR branches are selectively controlled in tumor cells are not clearly understood. Here, we provide evidence that PRKCSH, previously known as glucosidase II beta subunit, functions as a regulator for selective activation of the IRE1α branch of UPR. PRKCSH boosts ER stress-mediated autophosphorylation and oligomerization of IRE1α through mutual interaction. PRKCSH contributes to the induction of tumor-promoting factors and to tumor resistance to ER stress. Increased levels of PRKCSH in various tumor tissues are positively correlated with the expression of XBP1-target genes. Taken together, our data provide a molecular rationale for selective activation of the IRE1α branch in tumors and adaptation of tumor cells to severe environmental stress.

Objective: Fungal rhinosinusitis occurs in different forms depending on race and region. While allergic fungal rhinosinusitis is common in Caucasians, fungal ball (FB) is more common in Asians. However, most cases are reported as unilateral, and clinical data on bilateral FB (BFB) are rare. Therefore, the purpose of this study was to analyze and to compare the clinical characteristics of BFB and unilateral FB (UFB) in Koreans., Methods: We retrospectively analyzed medical records and computed tomography (CT) images of 434 patients diagnosed with FB. The patients were divided into two groups: BFB and UFB. Demographic data, multiple allergen simultaneous test including total or specific immunoglobulin E (IgE) levels, symptoms, CT findings, treatment, and outcomes were analyzed., Results: Among the patients, 26 had BFB and 408 had UFB. Hypertension was noted in 61.5% of the BFB and 39% of the UBF individuals (p = 0.023). While total IgE levels were similar between the two groups, Dermatophagoides pteronyssinus (p = 0.004), Cladosporium (p = 0.017), and Aspergillus-specific IgE positivity (p = 0.025) were significantly higher in the BFB than in the UFB group. Not only symptoms such as postnasal drip (p = 0.013), mucopurulent rhinorrhea (p = 0.009), and foul odor (p = 0.037), but also sphenoid sinus involvement on CT images were more common in the BFB than in the UFB group (p = 0.011)., Conclusion: Patients with BFB in Korea showed more common hypertension and symptoms of foul odor, mucopurulent rhinorrhea, and postnasal drip with allergy positivity compared to those with UFB. Therefore, understanding clinical characteristics of BFB will allow clinicians to approach BFB more appropriately.

Background & Aims: Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance., Methods: Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis., Results: Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC 50 value for wild-type HBV was 3.8 ± 0.6 µM, whereas the IC 50 values for CYE and CYEI mutants were 14.1 ± 1.8 and 58.1 ± 0.9 µM, respectively. The IC 90 value for wild-type HBV was 30 ± 0.5 µM, whereas the IC 90 values for CYE and CYEI mutants were 185 ± 0.5 and 790 ± 0.2 µM, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3-778 (IC 50  <0.4 µM vs. IC 50  = 0.4 µM, respectively)., Conclusions: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC 50 and 26.3-fold in IC 90 . These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high., Lay Summary: Tenofovir is the most potent nucleotide analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers >10-fold resistance to tenofovir up to 8 years. Herein, we identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC 50 ) and 26.3-fold (IC 90 ) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment., (Copyright © 2019. Published by Elsevier B.V.)

The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, toward proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degradation. Consistent with intrinsic protein instability as basis for this gain-of-function, internal structural disorder is associated with the unique cytotoxic sequences of the 1918 PB1-F2 protein. Infusing mice with recombinant DDX3 protein completely rescued them from lethal infection with the 1918 PB1-F2-producing virus. Alongside NS1 protein, 1918 PB1-F2 therefore constitutes a potent IFN antagonist causative for the severe pathogenicity of the 1918 influenza strain. Our identification of molecular determinants of pathogenesis should be useful for the future design of new antiviral strategies against influenza pandemics., (© 2019 The Authors.)

Hepatitis B virus (HBV) infection is a leading cause of liver diseases; however, the host factors which facilitate the replication and persistence of HBV are largely unidentified. Cellular FLICE inhibitory protein (c-FLIP) is a typical antiapoptotic protein. In many cases of liver diseases, the expression level of c-FLIP is altered, which affects the fate of hepatocytes. We previously found that c-FLIP and its cleaved form interact with HBV X protein (HBx), which is essential for HBV replication, and regulate diverse cellular signals. In this study, we investigated the role of endogenous c-FLIP in HBV replication and its underlying mechanisms. The knockdown of endogenous c-FLIP revealed that this protein regulates HBV replication through two different mechanisms. (i) c-FLIP interacts with HBx and protects it from ubiquitin-dependent degradation. The N-terminal DED1 domain of c-FLIP is required for HBx stabilization. (ii) c-FLIP regulates the expression or stability of hepatocyte nuclear factors (HNFs), which have critical roles in HBV transcription and maintenance of hepatocytes. c-FLIP regulates the stability of HNFs through physical interactions. We verified our findings in three HBV infection systems: HepG2-NTCP cells, differentiated HepaRG cells, and primary human hepatocytes. In conclusion, our results identify c-FLIP as an essential factor in HBV replication. c-FLIP regulates viral replication through its multiple effects on viral and host proteins that have critical roles in HBV replication. IMPORTANCE Although the chronic hepatitis B virus (HBV) infection still poses a major health concern, the host factors which are required for the replication of HBV are largely uncharacterized. Our studies identify cellular FLICE inhibitory protein (c-FLIP) as an essential factor in HBV replication. We found the dual roles of c-FLIP in regulation of HBV replication: c-FLIP interacts with HBx and enhances its stability and regulates the expression or stability of hepatocyte nuclear factors which are essential for transcription of HBV genome. Our findings may provide a new target for intervention in persistent HBV infection., (Copyright © 2018 American Society for Microbiology.)

Background: The orientation of the pelvis is of particular importance to the sagittal curve of the spine and has geometric relationships with the top of the spine and lumbar lordosis. Changes in spinal shape or disruptions of sagittal balance in the spine, such as a flattened lumbar spine, have significant negative effects., Objective: The aim of this study was to investigate the effects of the posterior superior iliac spine support device (PSD) on upper trunk acceleration during gait in individuals with flat lumbar back posture., Methods: In total, 10 young male subjects with reduced lumbar lordosis (global lumbar lordosis angle (T10-S2): <-20∘) were recruited for this study. Participants walked 7 m with and without wearing a PSD at a self-selected speed while fitted with an accelerometer attached over the T7 spinous process., Results: The normalized AP acceleration of T7 with PSD (40.57 ± 11.22%) was significantly higher than those without PSD (37.10 ± 10.46%, p= 0.035)., Conclusions: We found that wearing the PSD immediately improved pelvic physiological movement during walking in individuals with lumbar flat back posture. Our findings may help healthcare professionals manage flat back posture in asymptomatic younger individuals.

Death receptors of TNFSF10/TRAIL (tumor necrosis factor superfamily member 10) contribute to immune surveillance against virus-infected or transformed cells by promoting apoptosis. Many viruses evade antiviral immunity by modulating TNFSF10 receptor signaling, leading to persistent infection. Here, we report that hepatitis B virus (HBV) X protein (HBx) restricts TNFSF10 receptor signaling via macroautophagy/autophagy-mediated degradation of TNFRSF10B/DR5, a TNFSF10 death receptor, and thus permits survival of virus-infected cells. We demonstrate that the expression of the TNFRSF10B protein is dramatically reduced both in liver tissues of chronic hepatitis B patients and in cell lines transfected with HBV or HBx. HBx-mediated downregulation of TNFRSF10B is caused by the lysosomal, but not proteasomal, degradation pathway. Immunoblotting analysis of LC3B and SQSTM1, and microscopy analysis of tandem-fluorescence-tagged LC3B revealed that HBx promotes complete autophagy. Inhibition of autophagy with a pharmacological inhibitor and LC3B knockdown revealed that HBx-induced autophagy is crucial for TNFRSF10B degradation. Immunoprecipitation and GST affinity isolation assays showed that HBx directly interacts with TNFRSF10B and recruits it to phagophores, the precursors to autophagosomes. We confirmed that autophagy activation is related to the downregulation of the TNFRSF10B protein in liver tissues of chronic hepatitis B patients. Inhibition of autophagy enhanced the susceptibility of HBx-infected hepatocytes to TNFSF10. These results identify the dual function of HBx in TNFRSF10B degradation: HBx plays a role as an autophagy receptor-like molecule, which promotes the association of TNFRSF10B with LC3B; HBx is also an autophagy inducer. Our data suggest a molecular mechanism for HBV evasion from TNFSF10-mediated antiviral immunity, which may contribute to chronic HBV infection.

The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

Hepatocystin/80K-H is known as a causative gene for autosomal dominant polycystic liver disease. However, the role of hepatocystin in hepatitis B virus-related liver disease remains unknown. Here, we investigated the role of hepatocystin on the cytokine-mediated antiviral response against hepatitis B virus infection. We investigated the antiviral effect and mechanism of hepatocystin by ectopic expression and RNAi knockdown in cell culture and mouse livers. Hepatocystin suppressed the replication of hepatitis B virus both in vitro and in vivo. This inhibitory effect was HBx-independent and mediated by the transcriptional regulation of viral genome via the activation of exogenous signal-regulated kinase 1/2 and the reduced expression of hepatocyte nuclear factor 4α, a transcription factor essential for hepatitis B virus replication. The amino-terminal region of hepatocystin was essential for regulation of this antiviral signaling pathway. We also found that hepatocystin acts as a critical component in interferon-mediated mitogen-activated protein kinase signaling pathway, and the interferon-induced antiviral activity against hepatitis B virus is associated with the expression levels of hepatocystin. We demonstrated that hepatocystin plays a critical role in modulating the susceptibility of hepatitis B virus to interferon, suggesting that the modulation of hepatocystin expression is important for cytokine-mediated viral clearance during hepatitis B virus infection., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV replication as well as HBV-induced hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, a combination of affinity purification and mass spectrometry was applied to identify the host factors interacting with HBx in hepatoma cells. Thirteen proteins were identified as HBx binding partners. Among them, we first focused on determining the functional significance of the interaction between HBx and hepatocystin. A physical interaction between HBx and hepatocystin was confirmed by co-immunoprecipitation and Western blotting. Immunocytochemistry demonstrated that HBx and hepatocystin colocalized in the hepatoma cells. Domain mapping of both proteins revealed that the HBx C-terminus (amino acids 110-154) was responsible for binding to the mannose 6-phosphate receptor homology domain (amino acids, 419-525) of hepatocystin. Using translation and proteasome inhibitors, we found that hepatocystin overexpression accelerated HBx degradation via a ubiquitin-independent proteasome pathway. We demonstrated that this effect was mediated by an interaction between both proteins using a HBx deletion mutant. Hepatocystin overexpression significantly inhibited HBV DNA replication and expression of HBs antigen concomitant with HBx degradation. Using the hepatocystin mutant constructs that bind HBx, we also confirmed that hepatocystin inhibited HBx-dependent HBV replication. In conclusion, we demonstrated for the first time that hepatocystin functions as a chaperon-like molecule by accelerating HBx degradation, and thereby inhibits HBV replication. Our results suggest that inducing hepatocystin may provide a novel therapeutic approach to control HBV infection., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Sustained activation of NF-κB is one of the causative factors for various liver diseases, including liver inflammation and hepatocellular carcinoma (HCC). It has been known that activating the NF-κB signal by hepatitis B virus X protein (HBx) is implicated in the development of HCC. However, despite numerous studies on HBx-induced NF-κB activation, the detailed mechanisms still remain unsolved. Recently, p22-FLIP, a cleavage product of c-FLIPL, has been reported to induce NF-κB activation through interaction with the IκB kinase (IKK) complex in primary immune cells. Since our previous report on the interaction of HBx with c-FLIPL, we explored whether p22-FLIP is involved in the modulation of HBx function. First, we identified the expression of endogenous p22-FLIP in liver cells. NF-κB reporter assay and electrophoretic mobility shift assay (EMSA) revealed that the expression of p22-FLIP synergistically enhances HBx-induced NF-κB activation. Moreover, we found that HBx physically interacts with p22-FLIP and NEMO and potentially forms a ternary complex. Knock-down of c-FLIP leading to the downregulation of p22-FLIP showed that endogenous p22-FLIP is involved in HBx-induced NF-κB activation, and the formation of a ternary complex is necessary to activate NF-κB signaling. In conclusion, we showed a novel mechanism of HBx-induced NF-κB activation in which ternary complex formation is involved among HBx, p22-FLIP and NEMO. Our findings will extend the understanding of HBx-induced NF-κB activation and provide a new target for intervention in HBV-associated liver diseases and in the development of HCC.

Protein tyrosine nitration (PTN) is a post-translational modification that is related to several acute or chronic diseases. PTN introduces a nitro group in the ortho position of the phenolic hydroxyl group of tyrosine residues. PTN has been shown to be involved in the pathogenesis of inflammatory responses, cancers, and neurodegenerative and age-related disorders. Furthermore, it has been proposed that PTN regulates signal cascades related to nitric oxide (NO·) production and NO-mediated processes. Although nitrated proteins as markers of oxidative stress are confirmed by immunological assays in various affected cells or tissues, it is not known how many different types of proteins in living cells are nitrated. Since protein nitration is a low-abundance post-translational modification, development of an effective enrichment method for nitrated proteins is needed to detect nitrated peptides or proteins from the limited amount of pathophysiological samples. In the present study, we developed an enrichment method using specific chemical tagging. Nitroproteome profiling using chemical tagging and mass spectrometry was validated by model proteins. Furthermore, we successfully identified numerous nitrated proteins from the Huh7 human hepatoma cell line.

Clevudine (CLV) is a nucleoside analog with potent antiviral activity against chronic hepatitis B virus (HBV) infection. Viral resistance to CLV in patients receiving CLV therapy has not been reported. The aim of this study was to characterize CLV-resistant HBV in patients with viral breakthrough (BT) during long-term CLV therapy. The gene encoding HBV reverse transcriptase (RT) was analyzed from chronic hepatitis B patients with viral BT during CLV therapy. Sera collected from the patients at baseline and at the time of viral BT were studied. To characterize the mutations of HBV isolated from the patients, we subjected the HBV mutants to in vitro drug susceptibility assays. Several conserved mutations were identified in the RT domain during viral BT, with M204I being the most common. In vitro phenotypic analysis showed that the mutation M204I was predominantly associated with CLV resistance, whereas L229V was a compensatory mutation for the impaired replication of the M204I mutant. A quadruple mutant (L129M, V173L, M204I, and H337N) was identified that conferred greater replicative ability and strong resistance to both CLV and lamivudine. All of the CLV-resistant clones were lamivudine resistant. They were susceptible to adefovir, entecavir, and tenofovir, except for one mutant clone. In conclusion, the mutation M204I in HBV RT plays a major role in CLV resistance and leads to viral BT during long-term CLV treatment. Several conserved mutations may have a compensatory role in replication. Drug susceptibility assays reveal that adefovir and tenofovir are the most effective compounds against CLV-resistant mutants. These data may provide additional therapeutic options for CLV-resistant patients.

Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.

The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is involved in the pathological reaction to SARS and is a key antigen for the development of a sensitive diagnostic assay. However, the antigenic properties of this N protein are largely unknown. To facilitate the studies on the function and antigenicity of the SARS-CoV N protein, 6x histidine-tagged recombinant SARS-CoV N (rSARS-N) with a molecular mass of 46 and 48kDa was successfully produced using the recombinant baculovirus system in insect cells. The rSARS-N expressed in insect cells (BrSARS-N) showed remarkably higher specificity and immunoreactivity than rSARS-N expressed in E. coli (ErSARS-N). Most of all, BrSARS-N proteins were expressed as a highly phosphorylated form with a molecular mass of 48kDa, but ErSARS-N was a nonphosphorylated protein. In further analysis to determine the correlation between the phosphorylation and the antigenicity of SARS-N protein, dephosphorylated SARS-N protein treated with protein phosphatase 1 (PP1) remarkably enhanced the cross-reactivity against SARS negative serum and considerably reduced immunoreactivity with SARS-N mAb. These results suggest that the phosphorylation plays an important role in the immunoreactivity and specificity of SARS-N protein. Therefore, the BrSARS-N protein may be useful for the development of highly sensitive and specific assays to determine SARS infection and for further research of SARS-N pathology.

Severe acute respiratory syndrome-coronavirus nucleocapsid (SARS-CoV N) protein has been found to be important to the processes related to viral pathogenesis, such as virus replication, interference of the cell process and modulation of host immune response; detection of the antigen has been used for the early diagnosis of infection. We have used recombinant N protein expressed in insect cells to generate 17 mAbs directed against this protein. We selected five mAbs that could be used in various diagnostic assays, and all of these mAbs recognized linear epitopes. Three IgG(2b) mAbs were recognized within the N-terminus of N protein, whereas the epitope of two IgG(1) mAbs localized within the C-terminus. These mAbs were found to have significant reactivity with both non-phosphorylated and phosphorylated N proteins, which resulted in high reactivity with native N protein in virus-infected cells; however, they did not show cross-reactivity with human coronavirus. Therefore, these results suggested that these mAbs would be useful in the development of various diagnostic kits and in future studies of SARS-CoV pathology.

Development of many organs, including the mammary gland, involves ductal morphogenesis. Mammary ducts are bi-layered tubular structures comprising an outer layer of cap/myoepithelial cells (MECs) and an inner layer of luminal epithelial cells (LECs). Slit2 is expressed by cells in both layers, with secreted SLIT2 broadly distributed throughout the epithelial compartment. By contrast, Robo1 is expressed specifically by cap/MECs. Loss-of-function mutations in Slit2 and Robo1 yield similar phenotypes, characterized by disorganized end buds (EBs) reminiscent of those present in Ntn1(-/-) glands, suggesting that SLIT2 and NTN1 function in concert during mammary development. Analysis of Slit2(-/-);Ntn1(-/-) glands demonstrates an enhanced phenotype that extends through the ducts and is characterized by separated cell layers and occluded lumens. Aggregation assays show that Slit2(-/-);Ntn1(-/-) cells, in contrast to wild-type cells, do not form bi-layered organoids, a defect rescued by addition of SLIT2. NTN1 has no effect alone, but synergistically enhances this rescue. Thus, our data establish a novel role for SLIT2 as an adhesive cue, acting in parallel with NTN1 to generate cell boundaries along ducts during bi-layered tube formation.

Serological and virological studies were carried out of a mumps outbreak which occurred in one region, Yoeju County, Southeast of Seoul in Korea from September to December, 1999. Sera from 736 children at 8-13 years of age of patients with mumps and healthy children were tested for mumps-specific antibodies by enzyme immunoassay. The overall IgM positive rate was 7.6% (56/736), compared with 69.8% (514/736) for IgG. Of the 49 children with both IgG and IgM, 32 were also confirmed by both clinical and serological diagnosis. IgM antibodies were detected even in the samples collected up to 3 months after the onset of symptoms. Although 436 children had been vaccinated before the outbreak, 27 (6.2%) were found to be IgM positive, particularly 6 (4.4%) of 136 were positive serologically despite a second-dose vaccinees. Sequence analysis of the small hydrophobic (SH) gene of 4 mumps viruses isolated from 42 saliva specimens revealed that these were related to the genotype H, but distinguishable from European strains. This is the first study on the outbreak due to mumps virus genotype H and provides information to assess the understanding of recent outbreaks of mumps in Korea., (Copyright 2004 Wiley-Liss, Inc.)

Sequence analyses of the entire small hydrophobic (SH) and hemagglutinin-neuraminidase (HN) genes of mumps viruses circulated in Korea from 1998 to 2001 showed that these isolates were grouped into two genotypes, H and I. While genotype I was predominant throughout the country during this period, genotype H was found in the restricted region, 1999. The nucleotide and deduced amino acid sequences of Korean isolates showed the type-specific changes including the signature motif at positions 28-30 in the SH gene and the neutralizing epitopes in the HN gene. Particularly, Asian strains including Korean isolates and European strains differed from 2.3 to 3.8% at the nucleotide sequence level in the SH gene although they belonged to the same genotype H. Furthermore, none of Korean isolates were genetically related to the vaccine strains used in Korea. The results provide important information to understand the epidemiology of mumps infection and to facilitate the development of more efficient vaccine program in Korea.

Despite the marked reduction in the incidence of measles in Korea by the introduction of measles vaccine, a large measles epidemic occurred during 2000-2001. During the epidemic, more than 55,000 measles cases were reported and at least 7 children were dead. In this study, we analyzed the genetic and antigenic properties of 15 measles viruses that isolated during the epidemic. Sequence analyses of entire hemagglutinin (H) and nucleoprotein (N) genes of the viruses indicated that all Korean isolates had a high degree of homology (>99.8%) when compared with each other. They differed from other wild-type viruses by as much as 6.8% in the H gene and 6.5% in the N gene at the nucleotide level. The deduced amino acid variability was up to 6.4% for the H protein and up to 6.5% for the N protein. Phylogenetic analysis of nucleotide sequences and deduced amino acid sequences of the H and N genes revealed that all Korean viruses were grouped into the genotype H1. This strongly demonstrated that single genotype of measles virus has been circulated in Korea during the 2000-2001 epidemic. Plaque reduction neutralizing antibody titers against vaccine strains, Edmonston and Schwarz, and recently isolated Korean strains were measured using sera from vaccinees and recently infected children. Although sera of recently infected children demonstrated higher neutralizing antibody titers against wild-type strains than against vaccine strains, both sera neutralized both strains and the reciprocal geometric mean titers (GMTs) were not significantly different against both strains., (Copyright 2003 Wiley-Liss, Inc.)

Netrin-1 and its receptors play an essential role patterning the nervous system by guiding neurons and axons to their targets. To explore whether netrin-1 organizes nonneural tissues, we examined its role in mammary gland morphogenesis. Netrin-1 is expressed in prelumenal cells, and its receptor neogenin is expressed in a complementary pattern in adjacent cap cells of terminal end buds (TEBs). We discovered that loss of either gene results in disorganized TEBs characterized by exaggerated subcapsular spaces, breaks in basal lamina, dissociated cap cells, and an increased influx of cap cells into the prelumenal compartment. Cell aggregation assays demonstrate that neogenin mediates netrin-1-dependent cell clustering. Thus, netrin-1 appears to act locally through neogenin to stabilize the multipotent progenitor (cap) cell layer during mammary gland development. Our results suggest that netrin-1 and its receptor neogenin provide an adhesive, rather than a guidance, function during nonneural organogenesis.

A multiplex polymerase chain reaction (PCR) assay that is capable of detecting and typing six serotypes of respiratory adenovirus (Ad) was developed, using multiple sets of type-specific primers. The detection of each different serotype depended on distinguishing different numbers and sizes of amplification products on agarose gels following PCR. The multiplex PCR was tested with 26 clinical Ad isolates and other respiratory viruses including influenza viruses, parainfluenza viruses, and respiratory syncytial viruses as well as respiratory bacterial pathogens such as Chlamydia pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae. The multiplex PCR for the detection and typing of Ads gave an excellent correlation with the results by conventional typing with type-specific antisera. This assay may serve as a rapid means of confirming Ad with simultaneous serotype identification of the isolates. It will also have relevance as an adjunctive tool to conventional serotyping for diagnostic and epidemiological purposes., (Copyright 2002 Wiley-Liss, Inc.)

To characterize the genetic properties of currently circulating measles viruses in Korea, the complete nucleotide sequences of hemagglutinin (H) protein and nucleoprotein (N) genes of Korean viruses were analyzed. The entire genes of H and N were directly amplified by RT-PCR from each clinical specimen and sequenced. Sequence analyses of H and N genes indicated that all Korean viruses had a high degree of homology (>99.8%) when compared with each other. The Korean viruses differed from other wild-type viruses by as much as 6.8% in the H gene and 6.5% in the N gene at the nucleotide level. The deduced amino acid variability was up to 6.4% for the H protein and up to 6.5% for the N protein. Phylogenetic analyses of nucleotide sequences and deduced amino acid sequences of the H and N genes revealed that all Korean viruses were grouped into the clade H1.