Porcine Sapelovirus Uses α2,3-Linked Sialic Acid on GD1a Ganglioside as a Receptor.

UNLABELLED: The receptor(s) for porcine sapelovirus (PSV), which causes diarrhea, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs, remains largely unknown. Given the precedent for other picornaviruses which use terminal sialic acids (SAs) as receptors, we examined the role of SAs in PSV binding and infection. Using a variety of approaches, including treating cells with a carbohydrate-destroying chemical (NaIO4), mono- or oligosaccharides (N-acetylneuraminic acid, galactose, and 6'-sialyllactose), linkage-specific sialidases (neuraminidase and sialidase S), lectins (Maakia amurensislectin andSambucus nigralectin), proteases (trypsin and chymotrypsin), and glucosylceramide synthase inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol and phospholipase C), we demonstrated that PSV could recognize α2,3-linked SA on glycolipids as a receptor. On the other hand, PSVs had no binding affinity for synthetic histo-blood group antigens (HBGAs), suggesting that PSVs could not use HBGAs as receptors. Depletion of cell surface glycolipids followed by reconstitution studies indicated that GD1a ganglioside, but not other gangliosides, could restore PSV binding and infection, further confirming α2,3-linked SA on GD1a as a PSV receptor. Our results could provide significant information on the understanding of the life cycle of sapelovirus and other picornaviruses. For the broader community in the area of pathogens and pathogenesis, these findings and insights could contribute to the development of affordable, useful, and efficient drugs for anti-sapelovirus therapy. IMPORTANCE: The porcine sapelovirus (PSV) is known to cause enteritis, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs. However, the receptor(s) that the PSV utilizes to enter host cells remains largely unknown. Using a variety of approaches, we showed that α2,3-linked terminal sialic acid (SA) on the cell surface GD1a ganglioside could be used for PSV binding and infection as a receptor. On the other hand, histo-blood group antigens also present in the cell surface carbohydrates could not be utilized as PSV receptors for binding and infection. These findings should contribute to the understanding of the sapelovirus life cycle and to the development of affordable, useful and efficient drugs for anti-sapelovirus therapy.This study was supported by Wellcome Trust (097997/Z/11/Z) and a grant from Basic Science Research Program through the National Research Foundation of Korea (NRF). This study was also supported by Bio-industry Technology Development Program through the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET) funded by the Ministry of Agriculture, Food and Rural Affairs, and Chonnam National University (2013). IG is a Wellcome Senior Fellow supported by the Wellcome Trust (097997/Z/11/Z).This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/JVI.02449-1

Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II.

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules

Pathogenesis of Korean SapelovirusA in piglets and chicks.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.This study was supported by a grant (2014R1A2A2A01004292) of the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning, Bio-industry Technology Development Program (315021-04) through the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET) funded by the Ministry of Agriculture, Food and Rural Affairs, and Korea Basic Science Institute grant (C33730), Republic of Korea. IG is a Wellcome Senior Fellow supported by the Wellcome Trust (097997/Z/11/Z). Chonnam National University provided funding to Mun-Il Kang (2012). The Mab against SV-A capsid protein was received as a generous gift from Dr. M. Dauber (Friedrich-Loeffler Institute, Germany).This is the accepted version of the article. The final version is available from the Microbiology Society via http://dx.doi.org/10.1099/jgv.0.00057

Long-term efficacy, safety and immunogenicity in patients with rheumatoid arthritis continuing on an etanercept biosimilar (LBEC0101) or switching from reference etanercept to LBEC0101: an open-label extension of a phase III multicentre, randomised, double-blind, parallel-group study

Background To evaluate the long-term efficacy, safety and immunogenicity of continuing LBEC0101; the etanercept (ETN) biosimilar; or switching from the ETN reference product (RP) to LBEC0101 in patients with rheumatoid arthritis (RA). Methods This multicentre, single-arm, open-label extension study enrolled patients who had completed a 52-week randomised, double-blind, parallel phase III trial of LBEC0101 vs ETN-RP. Patients treated with ETN-RP during the randomised controlled trial switched to LBEC0101; those treated with LBEC0101 continued to receive LBEC0101 in this study. LBEC0101 (50 mg) was administered subcutaneously once per week for 48 weeks with a stable dose of methotrexate. Efficacy, safety and immunogenicity of LBEC0101 were assessed up to week 100. Results A total of 148 patients entered this extension study (70 in the maintenance group and 78 in the switch group). The 28-joint disease activity scores (DAS28)-erythrocyte sedimentation rate (ESR) were maintained in both groups from week 52 to week 100 (from 3.068 to 3.103 in the maintenance group vs. from 3.161 to 3.079 in the switch group). ACR response rates at week 100 for the maintenance vs. switch groups were 79.7% vs. 83.3% for ACR20, 65.2% vs. 66.7% for ACR50 and 44.9% vs. 42.3% for ACR70. The incidence of adverse events and the proportion of patients with newly developed antidrug antibodies were similar in the maintenance and switch groups (70.0% and 70.5%, 1.4% and 1.3%, respectively). Conclusions Administration of LBEC0101 showed sustained efficacy and acceptable safety in patients with RA after continued therapy or after switching from ETN-RP to LBEC0101. Trial registration ClinicalTrials.gov, NCT02715908. Registered 22 March 2016.This extension study was funded by LG Chem, Ltd. (formerly, LG Life Sciences, Ltd), Mochida Pharmaceutical Co., Ltd. and Korea Health Industry Development Institute

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Porcine sapovirus-induced RIPK1-dependent necroptosis is proviral in LLC-PK cells

Sapoviruses belonging to the genus Sapovirus within the family Caliciviridae are commonly responsible for severe acute gastroenteritis in both humans and animals. Caliciviruses are known to induce intrinsic apoptosis in vitro and in vivo, however, calicivirus-induced necroptosis remains to be fully elucidated. Here, we demonstrate that infection of porcine kidney LLC-PK cells with porcine sapovirus (PSaV) Cowden strain as a representative of caliciviruses induces receptor-interacting protein kinase 1 (RIPK1)-dependent necroptosis and acts as proviral compared to the antiviral function of PSaV-induced apoptosis. Infection of LLC-PK cells with PSaV Cowden strain showed that the interaction of phosphorylated RIPK1 (pRIPK1) with RIPK3 (pRIPK3), mixed lineage kinase domain-like protein (pMLKL) increased in a time-dependent manner, indicating induction of PSaV-induced RIPK1-dependent necroptosis. Interfering of PSaV-infected cells with each necroptotic molecule (RIPK1, RIPK3, or MLKL) by treatment with each specific chemical inhibitor or knockdown with each specific siRNA significantly reduced replication of PSaV but increased apoptosis and cell viability, implying proviral action of PSaV-induced necroptosis. In contrast, treatment of PSaV-infected cells with pan-caspase inhibitor Z-VAD-FMK increased PSaV replication and necroptosis, indicating an antiviral action of PSaV-induced apoptosis. These results suggest that PSaV-induced RIPK1-dependent necroptosis and apoptosis‒which have proviral and antiviral effects, respectively‒counterbalanced each other in virus-infected cells. Our study contributes to understanding the nature of PSaV-induced necroptosis and apoptosis and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections

Selective Trimerization of α-Olefins with Immobilized Chromium Catalyst for Lubricant Base Oils

The demand for poly(α-olefin)s (PAOs), which are high-performance group IV lubricant base oils, is increasingly high. PAOs are generally produced via the cationic oligomerization of 1-decene, wherein skeleton rearrangement inevitably occurs in the products. Hence, a transition-metal-based catalytic process that avoids rearrangement would be a valuable alternative for cationic oligomerization. In particular, transition-metal-catalyzed selective trimerization of α-olefins has the potential for success. In this study, (N,N′,N″-tridodecyltriazacyclohexane)CrCl3 complex was reacted with MAO-silica (MAO, methylaluminoxane) for the preparation of a supported catalyst, which exhibited superior performance in selective α-olefin trimerization compared to that of the corresponding homogeneous catalyst, enabling the preparation of α-olefin trimers at ~200 g scale. Following hydrogenation, the prepared 1-decene trimer (C30H62) exhibited better lubricant properties than those of commercial-grade PAO-4 (kinematic viscosity at 40 °C, 15.1 vs. 17.4 cSt; kinematic viscosity at 100 °C, 3.9 vs. 3.9 cSt; viscosity index, 161 vs. 123). Moreover, it was shown that 1-octene/1-dodecene mixed co-trimers (i.e., a mixture of C24H50, C28H58, C32H66, and C36H74), generated by the selective supported Cr catalyst, exhibited outstanding lubricant properties analogous to those observed for the 1-decene trimer (C30H62)

Feline calicivirus- and murine norovirus-induced COX-2/PGE2 signaling pathway has proviral effects.

Cyclooxygenases (COXs)/prostaglandin E2 (PGE2) signaling pathways are known to modulate a variety of homeostatic processes and are involved in various pathophysiological conditions. COXs/PGE2 signaling pathways have also been demonstrated to have proviral or antiviral effects, which appeared different even in the same virus family. A porcine sapovirus Cowden strain, a member of genus Sapovirus within the Caliciviridae family, induces strong COX-2/PGE2 but transient COX-1/PGE2 signaling to enhance virus replication. However, whether infections of other viruses in the different genera activate COXs/PGE2 signaling, and thus affect the replication of viruses, remains unknown. In the present study, infections of cells with the feline calicivirus (FCV) F9 strain in the genus Vesivirus and murine norovirus (MNV) CW-1 strain in the genus Norovirus only activated the COX-2/PGE2 signaling in a time-dependent manner. Treatment with pharmacological inhibitors or transfection of small interfering RNAs (siRNAs) against COX-2 enzyme significantly reduced the production of PGE2 as well as FCV and MNV replications. The inhibitory effects of these pharmacological inhibitors against COX-2 enzyme on the replication of both viruses were restored by the addition of PGE2. Silencing of COX-1 via siRNAs and inhibition of COX-1 via an inhibitor also decrease the production of PGE2 and replication of both viruses, which can be attributed to the inhibition COX-1/PGE2 signaling pathway. These data indicate that the COX-2/PGE2 signaling pathway has proviral effects for the replication of FCV and MNV, and pharmacological inhibitors against these enzymes serve as potential therapeutic candidates for treating FCV and MNV infections

Investigation of impact resistance performance of carbon fiber reinforced polypropylene composites with different lamination to applicate fender parts

Thermoplastic composites are rapidly emerging as alternative materials for auto parts owing to recyclability as well as excellent stiffness- and strength-to weight ratios. In this study, an optimal stacking sequence using twillweave carbon fiber-reinforced polypropylene (CF/PP) prepregs and random fiber-reinforced polypropylene (RFP) sheets were investigated for the fabrication of an automobile fender. The lamination consisting of CF/PP only incurred imperfect resin impregnation. RFP insertion between CF/PP layers improved the resin impregnation. The hybrid stacking sequence not only reduced internal void contents but also increased the flexural and impact strength of the composite laminate. Using the optimal stacking sequence, a real-scale composite fender was fabricated and impact tests were performed. The test results were compared with the ones from an existing steel fender. In case of the steel fender, permanent deformation was observed even in one attempt, while no visible damage was found from the composite fender even after five consecutive impact tests