Major HBV splice variant encoding a novel protein important for infection.

BACKGROUND & AIMS: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection. METHODS: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed. RESULTS: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid. CONCLUSIONS: Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection. IMPACT AND IMPLICATIONS: HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles.

SRPKIN-1 as an inhibitor against hepatitis B virus blocking the viral particle formation and the early step of the viral infection.

New antiviral agents are needed for the treatment of hepatitis B virus (HBV) infection because currently available drugs do not completely eradicate chronic HBV in patients. Phosphorylation dynamics of the HBV core protein (HBc) regulate several processes in the HBV life cycle, including nucleocapsid formation, cell trafficking, and virus uncoating after entry. In this study, the SRPK inhibitors SPHINX31, SRPIN340, and SRPKIN-1 showed concentration-dependent anti-HBV activity. Detailed analysis of the effects of SRPKIN-1, which exhibited the strongest inhibitory activity, on the HBV replication process showed that it inhibits the formation of infectious particles by inhibiting pregenomic RNA packaging into capsids and nucleocapsid envelopment. Mass spectrometry analysis combined with cell-free translation system experiments revealed that hyperphosphorylation of the C-terminal domain of HBc is inhibited by SRPKIN-1. Further, SRPKIN-1 exhibited concentration-dependent inhibition of HBV infection not only in HepG2-hNTCP-C4 cells but also in fresh human hepatocytes (PXB cells) and in the single-round infection system. Treatment with SRPKIN-1 at the time of infection reduced the nuclease sensitivity of HBV DNA in the nuclear fraction. These results suggest that SRPKIN-1 has the potential to not only inhibit the HBV particle formation process but also impair the early stages of viral infection.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

Current status and challenges of neurosurgical procedures for patients with myelomeningocele in real-world Japan.

BACKGROUND: Little is known about the real-world status of neurosurgical treatment of myelomeningocele patients. OBJECTIVE: To investigate the real-world status of neurosurgical treatment of myelomeningocele patients, medical claims data provided by the Japan Medical Data Center (JMDC) were analyzed. METHODS: The health claims data of 556 patients with myelomeningoceles from January 2005 to March 2020 were examined. The number of neurosurgical procedures, including myelomeningocele repair, tethered cord release, cerebrospinal fluid (CSF) shunt, CSF drainage, and endoscopic third ventriculostomy (ETV), was determined. RESULTS: A total of 313 neurosurgical procedures were performed for 135 patients in 74 institutions during the study period. The shunt survival rate was most affected by shunts that were revised when the patient was less than 1 year old, which had a significantly lower survival rate than all of the initial shunts performed when the patient was less than on1 year old; the 1-year shunt survival rate was 35 vs 64% (P = 0.0102). The survival rate was significantly lower in patients younger than 1 year who had CSF drainage before shunting compared to those younger than 1 year who did not have CSF drainage before shunting; the 1-year shunt survival rate was 27 vs 59% (P = 0.0196), and 81% of patients remained free of tethered cord release 10 years later. CONCLUSIONS: In this study, a revised shunt of less than 1 year of age and CSF drainage before shunting were the factors that lowered the shunt survival rate in the real world for CSF shunts for hydrocephalus associated with myelomeningocele.

Number of surgeries performed during the lifetime of patients with myelomeningocele.

OBJECTIVE: Patients with myelomeningocele often require multiple surgeries, but no study has clarified the kind of treatment given to these patients throughout their lives. The authors analyzed the type of surgery that was performed and at what age for Japanese patients with myelomeningoceles. METHODS: The Japanese health claims data of 556 patients with myelomeningocele for the period from January 2005 to March 2020 provided by the Japan Medical Data Center Co., Ltd., were examined to investigate the number of surgeries performed and the patient age at surgery for each specialty. The patients were divided into two groups (those ≤ 18 years old [group A] and those > 18 years old [group B]), and the way in which the types of surgery and the percentage of surgeries changed between these two groups was examined. RESULTS: The mean follow-up period was 4.4 years. The mean age at the end of the overall follow-up was 18.6 years (range 0-70.5 years), and 1033 surgeries were performed on 294 patients (0.42 surgeries performed per patient per year) during this period. The number of surgeries for patients in group A was 818 in 192 patients, with 0.62 surgeries per patient per year, and for patients in group B it was 215 in 102 patients, with 0.19 surgeries per patient per year. The number of surgeries and the mean age at the time of surgery were as follows: 313 neurosurgeries, 5.16 years; 280 orthopedic surgeries, 11.36 years; 70 urological surgeries, 14.57 years; and 202 dermatological/plastic surgeries, 16.19 years. In the surgeries related to myelomeningocele, the rates of CSF shunt placement, tethered cord release, muscle and tendon surgery, and other bone and joint surgery decreased significantly in group B, but they continued to undergo these surgeries. In group B, the rates of skin surgery, nephrostomy, ureterostomy, and cystostomy were significantly higher. CONCLUSIONS: A significant number of surgeries in multiple specialties related to myelomeningocele continue to be performed in adulthood, indicating that these patients require continuous care throughout their lives.

Virus-like particles with FLAG-tagged envelope protein as a tetravalent dengue vaccine candidate.

The global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future.

BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow.

The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation of proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 µL sample of human serum.

Prevalence and viral loads of polyomaviruses BKPyV, JCPyV, MCPyV, TSPyV and NJPyV and hepatitis viruses HBV, HCV and HEV in HIV-infected patients in China.

Human polyomaviruses (PyVs) and hepatitis viruses are often more prevalent or persistent in human immunodeficiency virus (HIV)-infected persons and the associated diseases are more abundant than in immunocompetent individuals. Here, we evaluated seroreactivities and viral loads of human PyVs and hepatitis viruses in HIV/AIDS patients and the general population in China in the combination antiretroviral therapy (cART) era. A total of 810 HIV-1-infected patients and age- and sex-matched HIV-negative individuals were enrolled to assess seroprevalence of PyVs BKPyV, JCPyV, MCPyV, TSPyV, and NJPyV and hepatitis viruses HBV, HCV, and HEV. 583 (72%) patients received cART, and among them, 31.2% had undetectable HIV RNA. While no significant difference was observed in prevalence of anti-PyV antibodies between HIV-positive and -negative groups, serum DNA positivity and DNA copy level of MCPyV were higher in the HIV-positive group. Among HIV-infected patients, BKPyV DNA positivity was significantly higher in patients with CD4 + cell counts < 200 cells/mm3 compared to those with CD4 + cell counts > 500 cells/mm3, suggesting possible reactivation caused by HIV-induced immune suppression. Higher HBV and HCV seropositivities but not HEV seropositivity were also observed in the HIV-positive group. Further correlation analyses demonstrated that HBV and HEV are potential risk factors for increased prevalence of PyV infection.

Dynamics of host and graft after cell sheet transplantation: Basic study for the application of amyotrophic lateral sclerosis.

Stem cells offer great hope for the therapy of neurological disorders. Using a human artificial chromosome (HAC), we generated modified mesenchymal stem cells (MSCs), termed HAC-MSC that express 3 growth factors and 2 marker proteins including luciferase, and previously demonstrated that intrathecal administration of HAC-MSCs extended the lifespan in a mouse model of amyotrophic lateral sclerosis (ALS). However, donor cells disappeared rapidly after transplantation. To overcome this poor survival, we transplanted the HAC-MSCs as a sheet structure which retained the extracellular matrix. We investigated, here, whether cell sheet showed a longer survival than intrathecal administration. Also, the therapeutic effects on ALS model mice were examined. In vivo imaging showed that luciferase signals increased immediately after transplantation up to 7 days, and these signals were sustained for up to 14 days. In contrast, following intrathecal administration, signals were drastically decreased by day 3. Moreover, cell sheet transplantation successfully prolonged the survival of donor HAC-MSCs. Cell sheet transplantation increased the level of p-Akt at the graft area. Pathologically, none of the donor cells differentiated into neurons, astrocytes or microglial cells. When the cell sheet was transplanted into ALS model mice, there was an encouraging trend in the delayed onset of symptoms and increased lifespan. If each group was subdivided into rapid and slow progressors based on cut-off values for respective median survival, the survival of rapid progressors differed significantly between groups (treated vs. sham-operated = 145.4 ±â€¯1.4 vs. 139.2 ±â€¯1.2). The effect of HAC-MSC sheet transplantation still has a temporally narrow therapeutic window. Further improvement could be achieved by optimization of the transplantation conditions, e.g. co-transplantation of HAC-MSCs with endothelial progenitor cells.

Cell Type Diversity in Hepatitis B Virus RNA Splicing and Its Regulation.

Although RNA splicing of hepatitis B virus (HBV) is a commonly observed in livers of hepatitis B patients as well as in the cultured cells replicating the viral genome, its biological significance in the HBV life cycle and the detailed regulatory mechanisms are still largely unclear. In this study, we found cell-type dependency of HBV splicing of the 3.5 kb pregenomic RNA, which is efficiently spliced in human hepatoma cells but not in cells derived from human hepatic stellate, mouse hepatoma and human non-hepatic cells. It may be likely that RNA splicing is one of the determinants of host range restriction of HBV. Given the finding indicating the difference in cell-type dependency of the splicing efficiency between HBV and simian virus 40, we carried out intron-swapping experiments. The results suggest the presence of putative exonic splicing enhancer that possibly works in the cell-type dependent fashion. Together with further mutational analyses, a novel 50-nt intronic splicing silencer, whose secondary structure is well conserved among the HBV strains, was identified. It appears that this intronic silencer functions effectively independent of cell backgrounds.

Tetanus toxin fragments and Bcl-2 fusion proteins: cytoprotection and retrograde axonal migration.

BACKGROUND: Tetanus neurotoxin (TeNT) is taken up at nerve terminals and undergoes retrograde migration. The toxic properties of TeNT reside in the toxin light chain (L), but like complete TeNT, the TeNT heavy chain (TTH) and the C-terminal domain (TTC) alone can bind and enter into neurons. Here, we explored whether atoxic fragments of TeNT could act as drug delivery vehicles in neurons. In this study, we used Bcl-2, a protein known to have anti-apoptotic properties in vivo and in vitro, as a parcel to couple to TeNT fragments. RESULTS: We expressed Bcl-2 and the TTC fragments alone, and also attempted to express fusion proteins with the Bcl-2 coupled at the N-terminus of TTH (Bcl2-TTH) and the N- and C-terminus of TTC (TTC-Bcl2 and Bcl2-TTC) in mammalian (Cos7 cells) and Escherichia coli systems. TTC and Bcl-2 were efficiently expressed in E. coli and Cos7 cells, respectively, but Bcl-2 and the fusion proteins did not express well in E. coli. The fusion proteins were also not expressed in Cos7 cells. To improve the yield and purity of the fusion protein, we genetically deleted the N-terminal half of TTC from the Bcl2-TTC fusion to yield Bcl2-hTTC. Purified Bcl2-hTTC exhibited neuronal binding and prevented cell death of neuronal PC12 cells induced by serum and NGF deprivation, as evidenced by the inhibition of cytochrome C release from the mitochondria. For in vivo assays, Bcl2-hTTC was injected into the tongues of mice and was seen to selectively migrate to hypoglossal nuclei mouse brain stems via retrograde axonal transport. CONCLUSIONS: These results indicate that Bcl2-hTTC retains both Bcl-2 and TTC functions and therefore could be a potent therapeutic agent for various neurological conditions.

Office-Based Screening for Dementia in Parkinson Disease: The Montreal Parkinson Risk of Dementia Scale in 4 Longitudinal Cohorts.

Importance: Parkinson disease dementia dramatically increases mortality rates, patient expenditures, hospitalization risk, and caregiver burden. Currently, predicting Parkinson disease dementia risk is difficult, particularly in an office-based setting, without extensive biomarker testing. Objective: To appraise the predictive validity of the Montreal Parkinson Risk of Dementia Scale, an office-based screening tool consisting of 8 items that are simply assessed. Design, Setting, and Participants: This multicenter study (Montreal, Canada; Tottori, Japan; and Parkinson Progression Markers Initiative sites) used 4 diverse Parkinson disease cohorts with a prospective 4.4-year follow-up. A total of 717 patients with Parkinson disease were recruited between May 2005 and June 2016. Of these, 607 were dementia-free at baseline and followed-up for 1 year or more and so were included. The association of individual baseline scale variables with eventual dementia risk was calculated. Participants were then randomly split into cohorts to investigate weighting and determine the scale's optimal cutoff point. Receiver operating characteristic curves were calculated and correlations with selected biomarkers were investigated. Main Outcomes and Measures: Dementia, as defined by Movement Disorder Society level I criteria. Results: Of the 607 patients (mean [SD] age, 63.4 [10.1]; 376 men [62%]), 70 (11.5%) converted to dementia. All 8 items of the Montreal Parkinson Risk of Dementia Scale independently predicted dementia development at the 5% significance level. The annual conversion rate to dementia in the high-risk group (score, >5) was 14.9% compared with 5.8% in the intermediate group (score, 4-5) and 0.6% in the low-risk group (score, 0-3). The weighting procedure conferred no significant advantage. Overall predictive validity by the area under the receiver operating characteristic curve was 0.877 (95% CI, 0.829-0.924) across all cohorts. A cutoff of 4 or greater yielded a sensitivity of 77.1% (95% CI, 65.6-86.3) and a specificity of 87.2% (95% CI, 84.1-89.9), with a positive predictive value (as of 4.4 years) of 43.90% (95% CI, 37.76-50.24) and a negative predictive value of 96.70% (95% CI, 95.01-97.85). Positive and negative likelihood ratios were 5.94 (95% CI, 4.08-8.65) and 0.26 (95% CI, 0.17-0.40), respectively. Scale results correlated with markers of Alzheimer pathology and neuropsychological test results. Conclusions and Relevance: Despite its simplicity, the Montreal Parkinson Risk of Dementia Scale demonstrated predictive validity equal or greater to previously described algorithms using biomarker assessments. Future studies using head-to-head comparisons or refinement of weighting would be of interest.

Involvement of PUF60 in Transcriptional and Post-transcriptional Regulation of Hepatitis B Virus Pregenomic RNA Expression.

Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1-2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence.

Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation.

Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.

Critical role of CREBH-mediated induction of transforming growth factor ß2 by hepatitis C virus infection in fibrogenic responses in hepatic stellate cells.

Mechanisms of hepatic fibrogenesis induced by hepatitis C virus (HCV), one of the leading causes of liver fibrosis, are not fully understood. We studied transcriptional up-regulation of transforming growth factor ß (TGF-ß), especially TGF-ß2, which is mediated by activation of liver-enriched transcription factor cAMP-responsive element-binding protein, hepatocyte specific (CREBH) triggered by HCV infection and its functional significance for induction of profibrogenic phenotypes by interaction of HCV-infected cells with hepatic stellate cells (HSCs). Compared to TGF-ß1, expression of TGF-ß2 mRNA was induced faster and to a higher level upon HCV infection. Serum TGF-ß2 levels in hepatitis C patients were higher compared to those in healthy individuals and were positively correlated with hepatic fibrosis stages F0-F2. TGF-ß2 promoter activity was decreased and increased, respectively, by silencing and overexpression of CREBH. CREBH recognition sites were identified in the TGF-ß2 promoter. CREBH binding to the promoter and its increase in cells expressing HCV Core-NS2 were shown by gel mobility shift and chromatin immunoprecipitation, respectively. The active form of CREBH was detectable in HCV-infected chimeric mice with human livers and cells expressing HCV proteins. Involvement of CREBH in HCV-induced fibrogenic response was further demonstrated in the CREBH null-mutant mouse model. Fibrogenic phenotypes were assessed using co-cultures of HCV-infected cells and HSCs. Expressions of fibrogenic factors and TGF-ß1 increasing in the co-cultures was prevented by TGF-ß2- or CREBH silencing. CONCLUSION: CREBH was identified as a key positive regulator of TGF-ß2 transcription in HCV-infected cells. TGF-ß2 released from infected cells potentially contributes to cross-induction of TGF-ß in an autocrine manner through its own signaling pathway, leading to an increase in fibrogenic responses in adjacent HSCs. (Hepatology 2017;66:1430-1443).

LUC7L3/CROP inhibits replication of hepatitis B virus via suppressing enhancer II/basal core promoter activity.

The core promoter of hepatitis B virus (HBV) genome is a critical region for transcriptional initiation of 3.5 kb, pregenome and precore RNAs and for the viral replication. Although a number of host-cell factors that potentially regulate the viral promoter activities have been identified, the molecular mechanisms of the viral gene expression, in particular, regulatory mechanisms of the transcriptional repression remain elusive. In this study, we identified LUC7 like 3 pre-mRNA splicing factor (LUC7L3, also known as hLuc7A or CROP) as a novel interacting partner of HBV enhancer II and basal core promoter (ENII/BCP), key elements within the core promoter, through the proteomic screening and found that LUC7L3 functions as a negative regulator of ENII/BCP. Gene silencing of LUC7L3 significantly increased expression of the viral genes and antigens as well as the activities of ENII/BCP and core promoter. In contrast, overexpression of LUC7L3 inhibited their activities and HBV replication. In addition, LUC7L3 possibly contributes to promotion of the splicing of 3.5 kb RNA, which may also be involved in negative regulation of the pregenome RNA level. This is the first to demonstrate the involvement of LUC7L3 in regulation of gene transcription and in viral replication.

Atypical Arteritis in Internal Carotid Arteries: A Novel Concept of Isolated Internal Carotid Arteritis.

We presented a 38-year-old woman suffering from acute cerebral infarction due to arteritis limited to bilateral internal carotid arteries without a condition of giant cell arteritis or granulomatosis with polyangitis. Our case is unprecedented and characterized by a young woman with wall enhancement in the internal carotid arteries on contrast-enhanced magnetic resonance imaging (MRI), therapeutic effects of steroids, and positive status for human leucocyte antigen-B39, -B51 and -DR4. These disease characteristics were not in accordance with existing diagnostic criteria of vasculitis, such as Takayasu's arteritis, giant cell arteritis, granulomatosis with polyangiitis, and Behcet's disease. We suggested consideration of a novel "isolated internal carotid arteritis" disease concept.

Acute Motor-dominant Polyneuropathy as Guillain-Barré Syndrome and Multiple Mononeuropathies in a Patient with Sjögren's Syndrome.

A patient with xerostomia and xerophthalmia due to Sjögren's syndrome presented with acute motor-dominant polyneuropathy and multiple mononeuropathy with antiganglioside antibodies. Nerve conduction studies and a sural nerve biopsy revealed the neuropathy as a mixture of segmental demyelination and axonal degeneration. Positive results were obtained for several antiganglioside antibodies. Corticosteroid treatment proved effective. The neuropathy was considered to represent a mixture of polyneuropathy as Guillain-Barré syndrome and multiple mononeuropathy via Sjögren's syndrome. We speculate that Guillain-Barré syndrome occurred in the patient and Guillain-Barré syndrome itself activated multiple mononeuropathy via Sjögren's syndrome.

Next-generation sequencing of 28 ALS-related genes in a Japanese ALS cohort.

We investigated the frequency and contribution of variants of the 28 known amyotrophic lateral sclerosis (ALS)-related genes in Japanese ALS patients. We designed a multiplex, polymerase chain reaction-based primer panel to amplify the coding regions of the 28 ALS-related genes and sequenced DNA samples from 257 Japanese ALS patients using an Ion Torrent PGM sequencer. We also performed exome sequencing and identified variants of the 28 genes in an additional 251 ALS patients using an Illumina HiSeq 2000 platform. We identified the known ALS pathogenic variants and predicted the functional properties of novel nonsynonymous variants in silico. These variants were confirmed by Sanger sequencing. Known pathogenic variants were identified in 19 (48.7%) of the 39 familial ALS patients and 14 (3.0%) of the 469 sporadic ALS patients. Thirty-two sporadic ALS patients (6.8%) harbored 1 or 2 novel nonsynonymous variants of ALS-related genes that might be deleterious. This study reports the first extensive genetic screening of Japanese ALS patients. These findings are useful for developing genetic screening and counseling strategies for such patients.

Structural basis of Cu, Zn-superoxide dismutase amyloid fibril formation involves interaction of multiple peptide core regions.

Cu, Zn-superoxide dismutase (SOD1), an enzyme implicated in the progression of familial amyotrophic lateral sclerosis (fALS), forms amyloid fibrils under certain experimental conditions. As part of our efforts to understand ALS pathogenesis, in this study we found that reduction of the intramolecular disulfide bond destabilized the tertiary structure of metal free wild-type SOD1 and greatly enhanced fibril formation in vitro. We also identified fibril core peptides that are resistant to protease digestion by using mass spectroscopy and Edman degradation analyses. Three regions dispersed throughout the sequence were detected as fibril core sequences of SOD1. Interestingly, by using three synthetic peptides that correspond to these identified regions, we determined that each region was capable of fibril formation, either alone or in a mixture containing multiple peptides. It was also revealed that by reducing the disulfide bond and causing a decrease in the structural stability, the amyloid fibril formation of a familial mutant SOD1 G93A was accelerated even under physiological conditions. These results demonstrate that by destabilizing the structure of SOD1 by removing metal ions and breaking the intramolecular disulfide bridge, multiple fibril-forming core regions are exposed, which then interact with each another and form amyloid fibrils under physiological conditions.