A role for glycolipid biosynthesis in severe fever with thrombocytopenia syndrome virus entry

A novel bunyavirus was recently found to cause severe febrile illness with high mortality in agricultural regions of China, Japan, and South Korea. This virus, named severe fever with thrombocytopenia syndrome virus (SFTSV), represents a new group within the Phlebovirus genus of the Bunyaviridae. Little is known about the viral entry requirements beyond showing dependence on dynamin and endosomal acidification. A haploid forward genetic screen was performed to identify host cell requirements for SFTSV entry. The screen identified dependence on glucosylceramide synthase (ugcg), the enzyme responsible for initiating de novo glycosphingolipid biosynthesis. Genetic and pharmacological approaches confirmed that UGCG expression and enzymatic activity were required for efficient SFTSV entry. Furthermore, inhibition of UGCG affected a post-internalization stage of SFTSV entry, leading to the accumulation of virus particles in enlarged cytoplasmic structures, suggesting impaired trafficking and/or fusion of viral and host membranes. These findings specify a role for glucosylceramide in SFTSV entry and provide a novel target for antiviral therapies

The astrobiology primer: An outline of general knowledge - Version 1, 2006

Peer reviewe

End to End QoS Network Design: Quality of Service for Rich-Media and Cloud Networks, -2/E

Quality of Service for Rich-Media & Cloud Networks?Second Edition New best practices, technical strategies, and proven designs for maximizing QoS in complex networks This authoritative guide to deploying, managing, and optimizing QoS with Cisco technologies has been thoroughly revamped to reflect the newest applications, best practices, hardware, software, and tools for modern networks. This new edition focuses on complex traffic mixes with increased usage of mobile devices, wireless network access, advanced communications, and video. It reflects the growing heterogeneity of video traffic, including passive streaming video, interactive video, and immersive videoconferences. It also addresses shifting bandwidth constraints and congestion points; improved hardware, software, and tools; and emerging QoS applications in network security. The authors first introduce QoS technologies in high-to-mid-level technical detail, including protocols, tools, and relevant standards. They examine new QoS demands and requirements, identify reasons to reevaluate current QoS designs, and present new strategic design recommendations. Next, drawing on extensive experience, they offer deep technical detail on campus wired and wireless QoS design; next-generation wiring closets; QoS design for data centers, Internet edge, WAN edge, and branches; QoS for IPsec VPNs, and more. Tim Szigeti, CCIE No. 9794 is a Senior Technical Leader in the Cisco System Design Unit. He has specialized in QoS for the past 15 years and authored Cisco TelePresence Fundamentals. Robert Barton, CCIE No. 6660 (R&S and Security), CCDE No. 2013::6 is a Senior Systems Engineer in the Cisco Canada Public Sector Operation. A registered Professional Engineer (P. Eng), he has 15 years of IT experience and is primarily focused on wireless and security architectures. Christina Hattingh spent 13 years as Senior Member of Technical Staff in Unified Communications (UC) in Cisco’s Services Routing Technology Group (SRTG). There, she spoke at Cisco conferences, trained sales staff and partners, authored books, and advised customers. Kenneth Briley, Jr., CCIE No. 9754, is a Technical Lead in the Cisco Network Operating Systems Technology Group. With more than a decade of QoS design/implementation experience, he is currently focused on converging wired and wireless QoS. n Master a proven, step-by-step best-practice approach to successful QoS deployment n Implement Cisco-validated designs related to new and emerging applications n Apply best practices for classification, marking, policing, shaping, markdown, and congestion management/avoidance n Leverage the new Cisco Application Visibility and Control feature-set to perform deep-packet inspection to recognize more than 1000 different applications n Use Medianet architecture elements specific to QoS configuration, monitoring, and control n Optimize QoS in rich-media campus networks using the Cisco Catalyst 3750, Catalyst 4500, and Catalyst 6500 n Design wireless networks to support voice and video using a Cisco centralized or converged access WLAN n Achieve zero packet loss in GE/10GE/40GE/100GE data center networks n Implement QoS virtual access data center designs with the Cisco Nexus 1000V n Optimize QoS at the enterprise customer edge n Achieve extraordinary levels of QoS in service provider edge networks n Utilize new industry standards and QoS technologies, including IETF RFC 4594, IEEE 802.1Q-2005, HQF, and NBAR2 This book is part of the Networking Technology Series from Cisco Press®, which offers networking professionals valuable information for constructing efficient networks, understanding new technologies, and building successful careers

Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

The glycoprotein YKL-40 (CHI3L1) is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs) can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and trans-differentiated phenotypes

Immunofluorescence microscopy and quantification of incoming virus particles.

<p>(<b>A, B</b>) A549 cells prepared as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006316#ppat.1006316.g008" target="_blank">Fig 8</a> were fixed 40 minutes post-warming and co-stained for rVSV-SFTSV (red) and the early endosome marker EEA1 (green) (A) or TGN46 (B). Cells were treated with NB-DNJ (bottom panels) or left untreated (top panels). Boxes indicate zoomed-in regions. Scale bar represents 5μm. (<b>C, D</b>) Quantitative image analysis was performed to measure the volume of discrete VSV M-stained puncta within z-stack images in untreated and NB-DNJ treated cells at both 20 and 40 minutes post-warming. Puncta were counted for at least 6 independent z-stacks per sample for both rVSV-SFTSV (C) and VSV (D) infected cells. (**** p<0.0001 using Welch’s one-tailed t-test).</p

Mechanistic studies on UGCG’s role in SFTSV entry.

<p><b>(A)</b> Binding and Internalization Assay. U-2 OS cells were transfected with negative control or UGCG siRNAs, replated the following day into 24 wells dishes, and the assay was performed 72 hours post-transfection. Assay details provided in Materials and Methods. vRNA levels were normalized to GAPDH mRNA levels, and are expressed relative to bound vRNA for the negative control siRNA. Mean ± S.E.M. for 3 independent experiments. <b>(B)</b> RNA collected from (A) was also analyzed for UGCG mRNA expression. UGCG mRNA levels were measured by RT-qPCR, normalized to GAPDH mRNA levels, and expressed relative to the negative control siRNA (bound). <b>(C,D)</b> The binding and internalization assay was carried out essentially as in (A) with the exception that U-2 OS cells were instead pre-treated with NB-DNJ for 48 hours prior to binding with rVSV-SFTSV (C) or SV40 (D) and qPCR for SV40 genomes did not require reverse transcription. Mean ± S.E.M. for 3 independent experiments. ** p<0.01 using Student’s t-test.</p

Immunofluorescence microscopy of rVSV-SFTSV particles following UGCG knockdown.

<p><b>(A-D)</b> U-2 OS cells were transfected with siRNAs targeting UGCG (siUGCG) or a non-targeting control (siNegCtrl) and plated onto glass coverslips. At 72 hours post-transfection cells were chilled to 4°C on ice and rVSV-SFTSV was bound by centrifugation (1200xg, 30’, 4°C). Following centrifugation, media was replaced with pre-warmed media (37°C) and the cells placed in a 37°C incubator for 20 or 40 minutes before fixation in 1% paraformaldehyde for 15 minutes. Cells were then immunostained for viral antigen (anti-VSV M, red), cellular markers (green), and nuclei stained with DAPI (blue). Images are representative from at least 3 independent experiments. (<b>A,B</b>) U-2 OS cells fixed after 20 minutes were co-stained for rVSV-SFTSV (red) and EEA1 (A) or TGN46 (B) (green). (<b>C,D</b>) U-2 OS cells fixed after 40 minutes and stained as above. Boxes indicate zoomed-in regions. Scale bar represents 5μm.</p