Sofosbuvir Suppresses the Genome Replication of DENV1 in Human Hepatic Huh7 Cells.

Dengue virus (DENV) causes dengue fever and dengue hemorrhagic fever, and DENV infection kills 20,000 people annually worldwide. Therefore, the development of anti-DENV drugs is urgently needed. Sofosbuvir (SOF) is an effective drug for HCV-related diseases, and its triphosphorylated metabolite inhibits viral RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of HCV. (2'R)-2'-Deoxy-2'-fluoro-2'-methyluridine (FMeU) is the dephosphorylated metabolite produced from SOF. The effects of SOF and FMeU on DENV1 replication were analyzed using two DENV1 replicon-based methods that we previously established. First, a replicon-harboring cell assay showed that DENV1 replicon replication in human hepatic Huh7 cells was decreased by SOF but not by FMeU. Second, a transient replicon assay showed that DENV1 replicon replication in Huh7 cells was decreased by SOF; however, in hamster kidney BHK-21 cells, it was not suppressed by SOF. Additionally, the replicon replication in Huh7 and BHK-21 cells was not affected by FMeU. Moreover, we assessed the effects of SOF on infectious DENV1 production. SOF suppressed infectious DENV1 production in Huh7 cells but not in monkey kidney Vero cells. To examine the substrate recognition of the HCV and DENV1 RdRps, the complex conformation of SOF-containing DENV1 RdRp or HCV RdRp was predicted using AlphaFold 2. These results indicate that SOF may be used as a treatment for DENV1 infection.

Relationship among trunk control, activities of daily living, and upper extremity function during the first week after stroke in patients with acute cerebral infarction.

[Purpose] We aimed to identify the relationship among trunk control, activities of daily living, and upper extremity function during the first week after stroke in patients with acute cerebral infarction. [Participants and Methods] Ninety-five patients with first cerebral infarction were included. Trunk control was assessed using the Postural Assessment Scale for Stroke. Additionally, activities of daily living were evaluated using the Functional Independence Measure, and upper extremity function was assessed using the upper extremity component of the Fugl-Meyer Assessment. Correlation analysis was performed to examine the relationships among these three measures. Furthermore, stepwise multiple regression analysis was performed to investigate the factors affecting activities of daily living. [Results] The total score and two subcategories of the Postural Assessment Scale for Stroke were significantly correlated with the Functional Independence Measure motor values. Stepwise multiple regression analysis revealed age and the Postural Assessment Scale for Stroke as factors influencing the Functional Independence Measure. Moreover, the Postural Assessment Scale for Stroke and upper extremity component of Fugl-Meyer Assessment showed a high correlation. [Conclusion] The trunk control ability assessed using the Postural Assessment Scale for Stroke is strongly correlated with activities of daily living estimated using the Functional Independence Measure in the first week after stroke in patients with acute cerebral infarction. The upper extremity component of Fugl-Meyer Assessment was not identified as a factor affecting the Functional Independence Measure.

Endoplasmic Reticulum Associated Degradation of Spinocerebellar Ataxia-Related CD10 Cysteine Mutant.

Spinocerebellar ataxia (SCA) is one of the most severe neurodegenerative diseases and is often associated with misfolded protein aggregates derived from the genetic mutation of related genes. Recently, mutations in CD10 such as C143Y have been identified as SCA type 43. CD10, also known as neprilysin or neuroendopeptidase, digests functional neuropeptides, such as amyloid beta, in the extracellular region. In this study, we explored the cellular behavior of CD10 C143Y to gain an insight into the functional relationship of the mutation and SCA pathology. We found that wild-type CD10 is expressed on the plasma membrane and exhibits endopeptidase activity in a cultured cell line. CD10 C143Y, however, forms a disulfide bond-mediated oligomer that does not appear by the wild-type CD10. Furthermore, the CD10 C143Y mutant was retained in the endoplasmic reticulum (ER) by the molecular chaperone BiP and was degraded through the ER-associated degradation (ERAD) process, in which representative ERAD factors including EDEM1, SEL1L, and Hrd1 participate in the degradation. Suppression of CD10 C143Y ERAD recovers intracellular transport but not enzymatic activity. Our results indicate that the C143Y mutation in CD10 negatively affects protein maturation and results in ER retention and following ERAD. These findings provide beneficial insight into SCA type 43 pathology.

A case of Basaloid Follicular Hamartoma.

We here describe a case of solitary basaloid follicular hamartoma (BFH) : the case developing in company with senile lentigo on the nose. BFH is a relatively rare benign follicular neoplasm of undetermined etiology. Histologically, the specimen consisted of small-sized squamoid or basaloid cells and follicular germ-like cells in the periphery of the tumor nests. There were no infundibular cysts. BFH should be differentiated from infundibulocystic basal cell carcinoma (BCC), which consists of squamoid or basaloid cells in company with infundibular cysts, tumor of follicular infundibulum or trichoepithelioma. We analyzed the immunohistochemical findings of the case in comparison with those of BCC and trichoepithelioma. An immunohistochemical examination revealed 1) that Bcl-2 and CD10 was preferentially expressed in the outermost cells in the tumor nests consisting of follicular germ-like cells, 2) that most of the tumor cells, especially germ-like cells, were strongly positive for Ber-EP4, and 3) that peritumoral stroma was positive for CD34. The immunohistochemical findings of our cases supported that BFH should be differentiated from BCC, a common malignant neoplasm.

Prophylactic and therapeutic vaccine against Pseudomonas aeruginosa keratitis using bacterial membrane vesicles.

PURPOSE: Pseudomonas aeruginosa (P. aeruginosa) infection is one of the major causes of keratitis. However, effective prophylactic and therapeutic vaccines against P. aeruginosa keratitis have yet to be developed. In this study, we explored the use of P. aeruginosa membrane vesicles (MVs) as a prophylactic vaccine as well as the use of immune sera derived from P. aeruginosa MV-immunized animals as a treatment for P. aeruginosa corneal infections in C57BL/6 mice. METHODS: C57BL/6 mice were intramuscularly immunized with P. aeruginosa MVs; the mouse corneas were then scarified and topically infected with several P. aeruginosa strains, followed by determination of corneal clinical score and corneal bacterial load. Next, immune sera derived from P. aeruginosa MV-immunized ICR mice were administered intraperitoneally to naïve C57BL/6 mice, followed by topical P. aeruginosa challenge. Finally, the immune sera were also used as a topical treatment in the mice with established P. aeruginosa corneal infections. RESULTS: P. aeruginosa-specific IgG and IgA antibodies induced by intramuscular immunization were detected not only in the sera but also in the eye-wash solution. Both active and passive immunization significantly inhibited P. aeruginosa corneal infection. Finally, topical treatment with immune sera in the mice with established P. aeruginosa corneal infections notably decreased the corneal clinical score and corneal bacterial load. CONCLUSIONS: P. aeruginosa keratitis can be attenuated by vaccination of P. aeruginosa MVs and topical application of P. aeruginosa MV-specific immune sera.

Vaccination inhibits the human adenoviral transduction in a mouse keratoconjunctivitis model.

Adenovirus infections are a major cause of epidemic keratoconjunctivitis (EKC), which can lead to corneal subepithelial infiltrates and multifocal corneal opacity. In the current study, we investigated the use of an E1/E3-deleted adenovirus serotype 5 (Ad5) vector as a vaccine administered intramuscularly (IM) or intranasally (IN) against subsequent challenges with a luciferase-expressing Ad5 (Ad5-Luci) vector via eyedrop. We evaluated the adaptive immune response to Ad5 vector vaccination and confirmed a robust polyfunctional CD8 T cell response in splenic cells. Neutralizing Ad5 antibodies were also measured in the sera of vaccinated mice as well as Ad5 antibody in the eye wash solutions. Upon challenge with Ad5-Luci vector 8 weeks post the primary immunization, transduction was significantly reduced by > 70% in the vaccinated mice, which was slightly better in IM- vs. that in IN-vaccinated animals. Resistance to subsequent challenge was observed 10 months post primary IM vaccination, with sustained reduction up to 60% in the Ad5-Luci vector transduction. Passive immunization of naive mice with antisera from IM to vaccinated mice subsequently challenged with the Ad5-Luci vector resulted in approximately 40% loss in transduction efficiency. Furthermore, the mice that received IM immunization with or without CD8 T cell depletion showed > 40% and 70% reductions, respectively, in Ad8 genomic copies after Ad8 topical challenge. We conclude that Ad-vector vaccination successfully induced an adaptive immune response that prevented subsequent Ad transduction in the cornea and conjunctiva-associated tissues in a mouse model of adenovirus keratoconjunctivitis, and that both cellular and humoral immunity play an important role in preventing Ad transduction.

Influence of local industrial changes on reef coral calcification.

Coral reefs are currently facing multiple disturbances caused by natural/anthropogenic factors. Recent industrial development might influence reef environments and ecosystems; however, few direct comparisons of coral calcification with the histories of local industries exist. We show the coral Ba/Ca record and growth histories for 46 years collected from Sumiyo Bay, Amami-Oshima Island, Japan. Coral Ba/Ca was mainly controlled by the sediment loads in seawater, which are introduced through the two local rivers. Coral Ba/Ca records have been characterized by two distinct historical periods: the decadal fluctuation corresponding to the traditional silk fabric industry (1960s ~ 1995) and the increasing trend corresponding to the development of quarries and the construction industry (1996 ~). Coral Ba/Ca records and local industrial histories were also linked to coral calcification. A long-term quantitative assessment of reef environments and local industrial changes could provide an evaluation of the survival strategies of reef-building corals in the future.

Design of a novel HIV-1 fusion inhibitor that displays a minimal interface for binding affinity.

Reported herein are the design, biological activities, and biophysical properties of a novel HIV-1 membrane fusion inhibitor. alpha-Helix-inducible X-EE-XX-KK motifs were applied to design an enfuvirtide analogue 2 that exhibited highly potent anti-HIV activity against wild-type HIV-1, enfuvirtide-resistant HIV-1 strains, and an HIV-2 strain in vitro. Indispensable residues for bioactivity of enfuvirtide, including the residues interacting with the N-terminal heptad repeat and the C-terminal hydrophobic residues, were identified.

Efficacy and safety of secukinumab in patients with generalized pustular psoriasis: A 52-week analysis from phase III open-label multicenter Japanese study.

Generalized pustular psoriasis (GPP) is a severe inflammatory skin disease characterized by the presence of sterile pustules covering almost the entire body and systemic symptoms such as fever. Secukinumab, a fully human-recombinant anti-interleukin-17A monoclonal antibody was indicated for psoriasis vulgaris and psoriatic arthritis in Japan but is not yet investigated for GPP. In this phase III, open-label multicenter single arm study, the efficacy and safety of secukinumab as monotherapy or with co-medication was evaluated in 12 Japanese patients with GPP. All the patients received secukinumab 150 mg s.c. at baseline, week 1, 2, 3 and 4, and then every 4 weeks. Two non-responders were up-titrated to 300 mg. Change in GPP severity from baseline was evaluated by clinical global impression (CGI) categorized as "worsened", "no change", "minimally improved", "much improved" or "very much improved". Treatment success was achieved by 83.3% (n = 10) of patients at week 16 (primary end-point) with CGI evaluated as "very much improved" (n = 9) and "much improved" (n = 1). Moreover, the area of erythema with pustules improved as early as week 1 and resolved by week 16 in most of the patients. The improvements were sustained throughout 52 weeks. Over the 52-week treatment period, secukinumab was well tolerated with no unexpected safety signals. Nasopharyngitis, urticaria, diabetes mellitus and arthralgia were the frequent adverse events reported. The data from this study shows that secukinumab can become one of the potent treatment options for GPP.

Molecular design and biological potential of galacto-type trehalose as a nonnatural ligand of shiga toxins.

Galacto-type trehalose, a "C-4 epimer of trehalose", possesses a stereochemical structure around the alpha(1-1)-linkage analogous to that of the globobiosyl alpha(1-4)-linkage in Gb(2) and Gb(3) ceramides, which are known as the ligands of Shiga toxins produced by pathogenic E. coli. This paper presents evidence supporting the new idea of using a trehalosyl alpha(1-1)-linkage as a substitute for the galactobiosyl alpha(1-4)-linkage.

Four-dimensional measurement of the displacement of internal fiducial markers during 320-multislice computed tomography scanning of thoracic esophageal cancer.

PURPOSE: To investigate the three-dimensional movement of internal fiducial markers placed near esophageal cancers using 320-multislice CT. METHODS AND MATERIALS: This study examined 22 metal markers in the esophageal wall near the primary tumors of 12 patients treated with external-beam photon radiotherapy. Motion assessment was analyzed in 41 respiratory phases during 20 s of cine CT in the radiotherapy position. RESULTS: Motion in the cranial-caudal (CC) direction showed a strong correlation (R(2) > 0.4) with the respiratory curve in most markers (73%). The average absolute amplitude of the marker movement was 1.5 ± 1.6 mm, 1.6 ± 1.7 mm, and 3.3 ± 3.3 mm in the left-right (LR), anterior-posterior (AP), and CC directions, respectively. The average marker displacements in the CC direction between peak exhalation and inhalation for the 22 clips were 1.1 mm (maximum, 5.5 mm), 3.0 mm (14.5 mm), and 5.1 mm (16.3 mm) for the upper, middle, and lower thoracic esophagus, respectively. CONCLUSIONS: Motion in primary esophagus tumor was evaluated with 320-multislice CT. According to this study, 4.3 mm CC, 1.5 mm AP, and 2.0 mm LR in the upper, 7.4 mm CC, 3.0 mm AP, and 2.4 mm LR in the middle, and 13.8 mm CC, 6.6 mm AP, and 6.8 mm LR in the lower thoracic esophagus provided coverage of tumor motion in 95% of the cases in our study population.

Design of peptide-based inhibitors for human immunodeficiency virus type 1 strains resistant to T-20.

Enfuvirtide (T-20) is a fusion inhibitor that suppresses replication of human immunodeficiency virus (HIV) variants with multi-drug resistance to reverse transcriptase and protease inhibitors. It is a peptide derived from the C-terminal heptad repeat (C-HR) of HIV-1 gp41, and it prevents interactions between the C-HR and the N-terminal HR (N-HR) of gp41, thus interfering with conformational changes that are required for viral fusion. However, prolonged therapies with T-20 result in the emergence of T-20-resistant strains that contain primary mutations such as N43D in the N-HR of gp41 (where T-20 and C-HR bind) that help the virus escape at a fitness cost. Such variants often go on to acquire a secondary mutation, S138A, in the C-HR of gp41 region that corresponds to the sequence of T-20. We demonstrate here that the role of S138A is to compensate for the impaired fusion kinetics of HIV-1s carrying primary mutations that abrogate binding of T-20. To preempt this escape strategy, we designed a modified T-20 variant containing the S138A substitution and showed that it is a potent inhibitor of both T-20-sensitive and T-20-resistant viruses. Circular dichroism analysis revealed that the S138A provided increased stability of the 6-helix bundle. We validated our approach on another fusion inhibitor, C34. In this case, we designed a variant of C34 with the secondary escape mutation N126K and showed that it can effectively inhibit replication of C34-resistant HIV-1. These results prove that it is possible to design improved peptide-based fusion inhibitors that are efficient against a major mechanism of drug resistance.

Electrostatically constrained alpha-helical peptide inhibits replication of HIV-1 resistant to enfuvirtide.

Alpha-helical peptides, such as T-20 (enfuvirtide) and C34, derived from the gp41 carboxyl-terminal heptad repeat (C-HR) of HIV-1, inhibit membrane fusion of HIV-1 and the target cells. Although T-20 effectively suppresses the replication of multi-drug resistant HIV variants both in vitro and in vivo, prolonged therapy with T-20 induces emergence of T-20 resistant variants. In order to suppress the emergence of such resistant variants, we introduced charged and hydrophilic amino acids, glutamic acid (E) and lysine (K), at the solvent accessible site of C34. In particular, the modified peptide, SC34EK, demonstrates remarkably potent inhibition of membrane fusion by the resistant HIV-1 variants as well as wild-type viruses. The activity was specific to HIV-1 and little influenced by serum components. We found a strong correlation between the anti-HIV-1 activities of these peptides and the thermostabilities of the 6-helix bundles that are formed with these peptides. We also obtained the crystal structure of SC34EK in complex with a 36 amino acid sequence (N36) comprising the amino-terminal heptad repeat of HIV-1. The EK substitutions in the sequence of SC34EK were directed toward the solvent and generated an electrostatic potential, which may result in enhanced alpha-helicity of the peptide inhibitor. The 6-helix bundle complex of SC34EK with N36 appears to be structurally similar to that of C34 and N36. Our approach to enhancing alpha-helicity of the peptide inhibitor may enable future design of highly effective and specific HIV-1 inhibitors.