Cytomegalovirus Vaccine Strain Towne-Derived Dense Bodies Induce Broad Cellular Immune Responses and Neutralizing Antibodies That Prevent Infection of Fibroblasts and Epithelial Cells

Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.     

Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.     

Lineage and Genogroup-Defining Single Nucleotide Polymorphisms of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP)-based typing panel was developed that redundantly identified 11 genogroups that span six of the eight lineages recently described for E. coli O157:H7 (J. L. Bono, T. P. Smith, J. E. Keen, G. P. Harhay, T. G. McDaneld, R. E. Mandrell, W. K. Jung, T. E. Besser, P. Gerner-Smidt, M. Bielaszewska, H. Karch, M. L. Clawson, Mol. Biol. Evol. 29:2047–2062, 2012) and additionally defined subgroups within four of those lineages. This assay was applied to 530 isolates from human and bovine sources. The SNP-based lineage groups were concordant with previously identified E. coli O157:H7 genotypes identified by other methods and were strongly associated with carriage of specific Stx genes. Two SNP lineages (Ia and Vb) were disproportionately represented among cattle isolates, and three others (IIa, Ib, and IIb) were disproportionately represented among human clinical isolates. This 48-plex SNP assay efficiently and economically identifies biologically relevant lineages within E. coli O157:H7.     

SHORT Syndrome with Partial Lipodystrophy Due to Impaired Phosphatidylinositol 3 Kinase Signaling

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.     

Apoptosis signal-regulating kinase-1 aggravates ROS-mediated striatal degeneration in 3-nitropropionic acid-infused mice

Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been suggested to participate in the pathology of neurodegenerative diseases, which may be associated with environmental factors that impact the diseases. Although it is not entirely elucidated, 3-nitropropionic acid (3-NP) provokes mitochondrial dysfunction and selectively forms striatal lesions similar to those found in Huntington’s disease. The current study investigated whether ASK1 is involved in striatal pathology following chronic systemic infusion of 3-NP. The results show that ASK1 acts as a primary mediator of there active oxygen species (ROS) cell death signal cascade in the 3-NP-damaged striatal region by disrupting the positive feedback cycle. In 3-NP-infused striatal lesions, ROS increased ASK1. Superoxide dismutase transgenic (SOD-tg) mice reduced ASK1by scavenging ROS, and reduction of ASK1leads to a reduction in cell death. However, ASK1 down-regulation in 3-NP infusion mice also decreased striatal cell death without scavenging ROS. In contrast decreasing cell death by si-ASK1 treatment along with 3-NP in both SOD tg and wild-type mice (wt), cell death rebounded when ASK1 peptide was added to SOD tg mice. The present study suggests that ROS-inducing ASK1 may be an important step in the pathogenesis of 3-NP infused striatal lesions in murine brains.     

Isolation of (-)-olivil-9′-O-β-d-glucopyranoside from Sambucus williamsii and its antifungal effects with membrane-disruptive action

In this study, we isolated (-)-olivil-9′-O-β-d-glucopyranoside (OLI9G), a phytochemical from the stem bark of Sambucus williamsii, and investigated the antifungal mechanism of OLI9G against Candida albicans. First of all, the antifungal susceptibility testing and hemolysis assay showed that OLI9G exerted a potent activity without hemolysis compared to the activity of amphotericin B. To investigate the mechanism of action of OLI9G, we first examined membrane depolarization using cyanine dye, 3,3′-dipropylthiacarbocyanine iodide (diSC₃5). The results showed that OLI9G significantly changed the fungal membrane potential. To further understand this activity on the membrane, we did the propidium iodide (PI) influx assay. From the results, OLI9G caused membrane permeabilization in the fungal membrane, and the three dimensional (3D) flow cytometric contour plot from the PI influx assay further showed that the cells had shrunk due to the membrane damage. Finally, the membrane-active mechanism of OLI9G was confirmed by synthesizing a model membrane, calcein-encapsulating large unilamellar vesicles (LUVs). The calcein leakage showed the membrane-disruptive effects caused by direct action of OLI9G. In conclusion, the current study suggests that OLI9G exerts its antifungal activity through a membrane-disruptive action.     

Inhibitory effect on NO production of triterpenes from the fruiting bodies of Ganoderma lucidum

Four new lanostane triterpenes, butyl lucidenate P (1), butyl lucidenate D₂ (2), butyl lucidenate E₂ (3) and butyl lucidenate Q (4) along with 11 known compounds (5–15) were isolated from the fruiting bodies of Ganoderma lucidum. Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against LPS-induced NO production in macrophage RAW 264.7 cells. Compounds 1, 3, 4, 9, 10 and 15 showed inhibitory potency with IC₅₀ values of 7.4, 6.4, 4.3, 9.4, 9.2 and 4.5 μM, respectively. Compounds 1, 3 and 15 dose-dependently reduced the LPS-induced iNOS expressions. Preincubation of cell with 1, 3 and 15 significantly suppressed LPS-induced expression of COX-2 protein.     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

Structure–activity relationship of human glutaminyl cyclase inhibitors having an N-(5-methyl-1H-imidazol-1-yl)propyl thiourea template

In an effort to design inhibitors of human glutaminyl cyclase (QC), we have synthesized a library of N-aryl N-(5-methyl-1H-imidazol-1-yl)propyl thioureas and investigated the contribution of the aryl region of these compounds to their structure–activity relationships as cyclase inhibitors. Our design was guided by the proposed binding mode of the preferred substrate for the cyclase. In this series, compound 52 was identified as the most potent QC inhibitor with an IC₅₀ value of 58 nM, which was two-fold more potent than the previously reported lead 2. Compound 52 is a most promising candidate for future evaluation to monitor its ability to reduce the formation of pGlu-Aβ and Aβ plaques in cells and transgenic animals.