HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication.

Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.

Metachronous Esophageal Ulcers after Immune-mediated Colitis Due to Immune Checkpoint Inhibitor Therapy: A Case Report and Literature Review.

Although cases of gastrointestinal toxicity of pembrolizumab have been reported, cases of acute immune-mediated colitis accompanied with metachronous esophageal disorders (esophagitis and ulcer) are rare. We herein report a case of acute colitis and metachronous esophageal ulcers due to an immune-related adverse event following concomitant pembrolizumab chemotherapy for lung adenocarcinoma. To our knowledge, there have so far been no reports of cases in which both acute immune-mediated colitis and metachronous esophageal ulcers developed. We therefore report the details of this case along with a review of the pertinent literature.

Comparative effectiveness and safety of warfarin and dabigatran in patients with non-valvular atrial fibrillation in Japan: A claims database analysis.

BACKGROUND: Antithrombotic therapy, including direct oral anticoagulants, is recommended in patients with non-valvular atrial fibrillation (NVAF) who are at intermediate-to-high risk of stroke. The aims of this study were to assess the patterns of oral anticoagulant (OAC) prescription in Japanese patients with NVAF and compare the effectiveness and safety of dabigatran and warfarin. METHODS: This was a retrospective observational study of adults with NVAF who initiated dabigatran or warfarin between March 14, 2011 and June 30, 2016, using electronic claims data of approximately 12.94 million patients from 230 hospitals. Propensity score matching was used to derive equal patient cohorts. Outcomes included the combined incidence of stroke, systemic embolism, and intracranial bleeding (primary endpoint) and the incidence of major bleeding (secondary endpoint). RESULTS: Overall, 400,884 patients were included. Among those prescribed an OAC, warfarin was the most common (34.3%). For the comparison of dabigatran and warfarin, 4606 patients were propensity-score matched in each cohort. Dabigatran recipients had lower incidences of stroke, systemic embolism, and intracranial bleeding [29.0 vs. 35.6 per 1000 patient-years; hazard ratio (HR), 0.72; 95% confidence interval (CI): 0.53-0.97; p=0.031] and major bleeding (6.4 vs. 11.3 per 1000 patient-years; HR, 0.55; 95% CI: 0.30-0.99; p=0.048). The most common type of bleeding in both groups was gastrointestinal and the incidence was lower in dabigatran recipients (1.6 vs. 6.4 per 1000 patient-years; HR, 0.24; 95% CI: 0.08-0.69; p=0.009). CONCLUSIONS: In Japan, dabigatran was associated with a lower risk of stroke, systemic embolism, and intracranial bleeding and major bleeding compared with warfarin in patients with NVAF.

Large-cell neuroendocrine carcinoma arising from a gastritis cystica polyposa.

Gastritis cystica polyposa is a polypoid lesion that arises from the gastric mucosa at the gastrojejunal anastomotic site and is characterized by cystic dilation of the gastric glands. A 78-year-old man who underwent distal gastrectomy for a gastric ulcer with Billroth II reconstruction approximately 40 years previously, exhibited a gastritis cystica polyposa at the anastomotic site. Ulceration was observed on an annual endoscopic examination. Endoscopic ultrasonography revealed a submucosal hypoechoic mass with multiple cystic lesions. Gastrectomy was performed and histological examination revealed a large-cell neuroendocrine carcinoma with cystic dilation of the gastric glands. Here, we report the first case of a large-cell neuroendocrine carcinoma arising from a gastritis cystica polyposa. Endoscopic ultrasonography was effective at diagnosing a submucosal hypoechoic mass with cystic dilation of the gastric glands.

The efficacy and long-term safety of a triple combination of 80 mg telmisartan, 5 mg amlodipine and 12.5 mg hydrochlorothiazide in Japanese patients with essential hypertension: a randomized, double-blind study with open-label extension.

The aim of this study was to compare 80 mg telmisartan/5 mg amlodipine/12.5 mg hydrochlorothiazide (T80/A5/H12.5) with 80 mg telmisartan/12.5 mg hydrochlorothiazide (T80/H12.5) to determine their relative blood pressure (BP) lowering effects in essential hypertensive patients with inadequate control and to evaluate the long-term safety of T80/A5/H12.5 in a 52-week extension period. Patients (n=132) were randomly assigned to receive double-blind treatment with T80/A5/H12.5 or T80/H12.5 for 8 weeks after a 6-week run-in-period of T80/H12.5. All 126 patients who completed the double-blind period entered the 52-week open-label extension and received T80/A5/H12.5. The adjusted mean changes from the reference baseline of the trough-seated systolic and diastolic BP (SBP/DBP) at week 8 were significantly larger in the T80/A5/H12.5 group (-10.6/-8.8 mm Hg) than in the T80/H12.5 group (-2.3/-1.3 mm Hg) (P<0.0001). The BP-lowering effect of T80/A5/H12.5 was maintained over the 52-week extension period. The adverse events (AEs) during both treatment periods were generally mild. Drug-related AEs were reported in one patient in each group in the double-blind period and in five patients exposed to T80/A5/H12.5 in the double-blind and/or open-label extension period. T80/A5/H12.5 therapy was clinically and statistically superior to T80/H12.5 therapy for the reduction of BP in patients with essential hypertension uncontrolled with T80/H12.5, and its BP-lowering effect was maintained in the long term. T80/A5/H12.5 was generally well-tolerated.

Effect of hydrochlorothiazide in addition to telmisartan/amlodipine combination for treating hypertensive patients uncontrolled with telmisartan/amlodipine: a randomized, double-blind study.

The efficacy and safety of telmisartan 80 mg/amlodipine 5 mg plus hydrochlorothiazide 12.5 mg (T80/A5/H12.5) was examined for its ability to treat hypertension in Japanese patients whose hypertension is uncontrolled with telmisartan 80 mg/amlodipine 5 mg (T80/A5). Patients aged ⩾20 years who had essential hypertension despite taking two or three antihypertensive drugs entered a 6-week run-in period on T80/A5. Patients whose hypertension remained uncontrolled were randomly assigned to either the T80/A5/H12.5 group (n=149) or the T80/A5 group (n=160), once daily for 8 weeks. After 8 weeks, patients in the T80/A5/H12.5 group showed a significantly greater adjusted mean reduction in both seated diastolic blood pressure and seated systolic blood pressure than those in the T80/A5 group. Furthermore, more patients achieved a diastolic/systolic blood pressure of <90/140 mm Hg in the T80/A5/H12.5 group compared with the T80/A5 group. The most common adverse events were nasopharyngitis, elevated blood uric acid levels and hyperuricemia, and the latter two events were more frequent in the T80/A5/H12.5 group than in the T80/A5 group. Overall, T80/A5/H12.5 administered for 8 weeks significantly reduced systolic and diastolic blood pressure and was well tolerated by patients with hypertension uncontrolled with T80/A5.

Characterization of polystyrene-binding peptides (PS-tags) for site-specific immobilization of proteins.

In this study, we characterized polystyrene-binding peptides (PS-tags) that possess a specific binding affinity for hydrophilic polystyrene (phi-PS) plates. Both the FITC-labeled PS19-1 (RAFIASRRIKRP) and PS19-6 (RIIIRRIRR) peptides showed strong binding affinity for commercially available hydrophilic, but not hydrophobic, PS plates in the presence of the non-ionic surfactant Tween 20. The dissociation constants (K(d)) of the PS19-1 and PS19-6 peptides for the hydrophilic PS-A plate were 169 and 86 nM, respectively, and the K(d) of both peptides increased with the concentration of NaCl or urea. Based on adsorption yield and residual activity of glutathione S-transferase (GST) after fusion with the PS19-6 peptide or its variants, it was found that the basic amino acid in the PS-tags, i.e., Arg was essential for the strong binding affinity of PS-tags in both the peptide and peptide-fused protein forms The aliphatic amino acids in PS19-6 and PS19-6L, such as Ile or Leu, were also effective. Thus, a series of PS-tags that possess this unusual feature, especially the peptides PS19-6 (RIIIRRIRR) and PS19-6L (RLLLRRLRR), are potential candidate affinity peptide tags for site-specific immobilization of proteins onto hydrophilic PS plates, which show potential as solid supports for protein-based biochips.

Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag).

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.