bluuebunny/crossref_metadata_2025 huggingface dataset:
Injection of a Novel Biomimetic Aggrecan for the Restoration of Intervertebral Disc Tissue Mechanics
Authors: Prudnikova Katsiaryna , Jamison David , Marcolongo Michele
Published: January 2014

Intervertebral disc degeneration and associated lower back pain is one of the leading musculoskeletal disorders confronting our health system with 15%–20% of the population experiencing lower back pain annually [1–4]. It has been shown that early in disc degeneration, the extracellular matrix of the nucleus pulposus is depleted of the proteoglycan aggrecan, resulting in loss of disc hydration, osmotic pressure and mechanical stability which leads to lower back pain [2, 5]. Early-stage restoration of the proteoglycan content within normal levels with natural aggrecan may help to restore disc functionality but it is cost prohibitive. We propose a new strategy to restore the extracellular matrix of the degenerated disc and mitigate lower back pain by molecularly engineering the disc matrix with an injection of a biomimetic aggrecan (BA) novel class of molecules that mimics the 3D bottle brush structure and physical properties of natural aggrecan.
Computational Modeling of the Canine Middle Cerebral Artery During Localized Hypothermia Stroke Treatment
Authors: Sikorski Ryan A. , Merrill Denise R. , Merrill Thomas L.
Published: January 2014

Stroke is caused by an interruption of brain blood supply and is one of the leading causes of death and disability in the United States. Each year 795,000 people experience a new stroke, of which 87% are ischemic [1]. The primary goal in stroke treatment is restoring blood flow in the effected regions of the brain, typically by using mechanical thrombectomy devices. However, after blood flow is restored, additional damage can occur in the form of reperfusion injury. Studies have shown that up to 50% of cell death from an ischemic event occurs as a result of reperfusion injury in cardiac patients [2].
Dissection of Porcine Ascending Aortic Media: Mechanical Characterization and Multiscale Model
Authors: Shah Sachin B. , Witzenburg Colleen , Hadi Mohammad F. , Wagner Hallie P. , Goodrich Janna , Barocas Victor H.
Published: January 2014

Ascending thoracic aortic aneurysm (aTAA) is a pathological condition with a high risk of dissection and rupture. Clinically, management of aTAA balances the risk of rupture with that of surgery-related complications. The risk of aneurysm rupture is known to correlate with aneurysm diameter. 1,2 Aneurysms greater than 6 cm in diameter have a significantly higher risk of rupture. 1 Current guidelines for intervention suggest surgical intervention for aTAA diameters greater than 5.5cm for patients without connective tissue disorders. 1
Effects of Bisphosphonate on Long-Term Culture of Cartilage Allografts
Authors: Zhou Yilu , Resutek Lauren , Wang Liyun , Lu X. Lucas
Published: January 2014

Zoledronic acid (ZA), an FDA approved bisphosphonate (BP) medicine, is widely used for the treatment of osteoclast-related bone loss diseases [1]. Our previous study has found that systemic administration of ZA could dramatically suppress the development of post-traumatic osteoarthritis (PTOA) in the DMM (destabilization of the medial meniscus) mouse model, a model recapitulating the altered joint loading associated with PTOA [2]. This finding is consistent with a few similar studies using different animal models [3]. However, little is known about the cellular and biochemical mechanisms of BP mediated chondro-protection in PTOA pathogenesis. Studies have shown that PTOA often initiates from the apoptosis and altered metabolism of cartilage chondrocytes. In this study, we will investigate the direct effects of ZA on the metabolisms of chondrocytes using long-term in vitro culture of cartilage allografts. As one of the earliest responses of chondrocytes to mechanical stimulation, intracellular calcium ([Ca 2+] i) signaling is the upstream of numerous mechanotransduction pathways [4]. We hypothesize that the chondro-protective mechanisms of ZA could be represented by the characteristics of [Ca 2+] i signaling of in situ chondrocytes. Our specific aims were to: (i) compare the in situ spontaneous [Ca 2+] i responses of chondrocytes cultured in non-ZA and ZA supplemented environments, and (ii) compare the biomechanical properties of cartilage allografts under the two culture conditions.
Assessment of Induced Pluripotent Stem Cell-Derived Cardiomyocyte Contractility Using Micropost Arrays
Authors: Rodriguez Marita L. , Murry Charles E. , Sniadecki Nathan J.
Published: January 2014

Cardiovascular stem cell therapies have shown increasing promise as a potential therapeutic means for reversing the effects of a myocardial infarction [1]. Out of the currently available sources of human stem cells, human induced pluripotent stem cells (hiPSCs) are very promising in that: the number of cell lines that can be induced to the pluripotent state is extremely vast, they serve as a potential source for patient-specific cardiomyocytes, and their use is non-controversial. However, before they can be used feasibly in a clinical setting, the functional engraftment of these cells into the host tissue must be improved [2]. It is hypothesized that the structural and functional maturity of the stem-cell derived cardiomyocytes prior to implantation, may significantly affect the ability of these cells to engraft with resident heart tissue [3]. One of the most important functional characteristics of a cardiomyocyte is its ability to produce contractile forces. However, assessing the contractile properties of single iPS-CMs is a difficult task. iPS-CMs generally have relatively unorganized cytoskeletons, with stress fibers in multiple directions. This trait renders one or two-point force assays ineffectual in determining total cell forces. Furthermore, iPS-CMs don’t spread well on tissue culture surfaces, which make two-dimensional force measurements almost impossible.
Focal Adhesion Mechanotransduction Regulates Stiffness-Directed Differentiation
Authors: Holle Andrew W. , Del Alamo Juan Carlos , Engler Adam J.
Published: January 2014

Human mesenchymal stem cells (hMSCs) are capable of differentiating into mesodermal lineages, with their fate mirroring the tissue lineage possessing a matching in vivo stiffness. The precise mechanisms responsible for this mechanotransduction-induced change in fate are unknown beyond the requirement for force transmission from the extracellular niche through to the nucleus. As a result of cellular contraction, linker proteins connecting the cytoskeleton to the extracellular matrix (ECM) are exposed to differing levels of force and deform to different extents based on the adjacent ECM’s stiffness. Therefore, some of these linker proteins could act as ‘molecular strain gauges,’ as they have been shown to unfold in response to this force. The unfolding process could result in exposure of cryptic binding sites and induction of new signaling pathways. For example, talin exposes multiple vinculin binding sites under physiological force [1]. Vinculin binds at either end to talin and actin and is thought to change its conformation in conjunction with this force [2] similar to how a strain gauge works. Here we show that force-dependent changes in vinculin recruit MAPK1, inducing a signaling cascade that results in the expression of myogenic markers. Together these data suggest that specific proteins may act as ‘molecular strain gauges’ and play a role in mechanosensitive stem cell differentiation.
Evidence for Interfibrillar Shear Load Transfer Between Sliding Fibrils in Tendon
Authors: Szczesny Spencer E. , Elliott Dawn M.
Published: January 2014

While collagen fibrils are understood to be the primary tensile load bearing components in tendon, how loads applied at the tissue level are transmitted across each element within the tissue hierarchical structure is unclear. A central unresolved question is whether collagen fibrils bear load independently or if the applied load is transferred across the fibrils through interfibrillar shear forces. Relative sliding between fibrils is suggested by findings that fibril strains within rat tail tendon fascicles do not agree with the applied tissue tensile strains [1]. Other studies using confocal microscopy have directly measured sliding behavior [2,3]; however, the impact that interfibrillar sliding has on tendon macroscale mechanics and whether sliding is associated with interfibrillar shear stresses are unknown. Therefore, the objective of this work is to quantify the contribution of interfibrillar sliding on tendon macroscale mechanics by simultaneously measuring the tissue behavior at both length-scales and interpreting the results with a micro-structural shear lag model directly incorporating interfibrillar shear stresses. We hypothesize that the reduced stiffness and increased viscosity observed in the tissue macroscale properties at higher strains are due to increases in interfibrillar sliding and that this behavior is consistent with a shear lag model involving interfibrillar shear stress.
The Rule of Mixtures: Approximating Chorioamnion Elastic Modulus in Human Placental Membranes
Authors: Barnard Carly A. , Briggs Brandi N. , Ferguson Virginia L.
Published: January 2014

The placental membranes, which surround the fetus in utero, are made of chorioamnion (CA) — a composite bilayered structure made of amnion and chorion. The CA is the only tissue in the body designed to fail, yet early CA failure causes preterm birth and is associated with elevated infant morbidity and mortality 1. While the elastic moduli, E, of the chorion and the amnion have been independently investigated 1,2,3, how these two layers work together in the composite CA bilayer has yet to be thoroughly investigated.
Relationship Between Blast Overpressure and Shell Thickness on the Fluid Pressure on a Cylinder Under Blast Loading
Authors: Selvan Veera , Chandra Namas
Published: January 2014

The mechanics of blast wave-head interaction determines the magnitude of mechanical insult to the human head during a field explosion and subsequent brain injury. In this work, blast overpressure and shell thickness are related to fluid pressure based on experimental and computational methods. A fluid-filled cylinder is idealized as a two-dimensional analog of a skull-brain complex and is subjected to a Friedlander blast wave. Strain and pressure on the surface of the cylinder and pressure in the fluid (analogue of Intracranial pressure) are experimentally measured and compared with numerical simulation results. The validated numerical model shows that fluid pressure increases linearly with increase in reflected overpressures (ROP) for a given shell thickness. When the ROP is kept constant, fluid pressure increases linearly with the decrease in shell thickness. An equation is developed for predicting the fluid pressure for a given ROP and shell thickness.
Multiscale Modeling of In Vitro Acupuncture Needling
Authors: Wagner Hallie , Shreiber David , Barocas Victor
Published: January 2014

Traditional acupuncture therapy involves inserting needles at acupuncture points across the body and twisting them, which winds soft tissues around the acupuncture needle. In order to better understand this phenomenon Julias et al [1, 2] conducted in vitro acupuncture needling on collagen gels as a soft tissue analog. Using polarized light microscopy (PLM) (Fig. 1) they observed changes in collagen alignment and measured the torque developed on the needle during twisting. While these results are insightful, what remains unknown are the forces transmitted to the tissue surrounding the acupuncture point via the needle. Computational modeling allows us to calculate these forces, which could never be measured experimentally.
Mechanics of Interaction of Blast Waves on Surrogate Head: Effect of Head Orientation
Authors: Ganpule S. , Chandra N.
Published: January 2014

Blast induced Traumatic Brain Injury (bTBI) has emerged as the most significant injury to warfighters in recent conflicts. How blast waves interact with the head and head protection device (helmet), and induce biomechanical loading on the head are not fully understood. This work focuses on the mechanics of blast wave head interactions on a surrogate head for various orientations (front, back, side and 45°) using experiments and numerical models. The role of the orientation of the head on the mechanical load experienced by the head is studied by monitoring pressures on the surface of a surrogate head subjected to planar blast waves. Validated numerical models are further employed to understand the experimental results and to interpret various aspects of flow mechanics (e.g. flow separation, flow reunion) around the head. Our results indicate that geometry of the head and its orientation with respect to the direction of the blast, govern the flow dynamics around the head and this alters the surface pressures experienced by the head. Orientation dependent responses predicted by the experiments and numerical models suggest that direction-specific tolerances are needed in the helmet design, in order to offer multi-directional protection under blast loading conditions.
Initial Steps Toward Development of a Lumped-Parameter Model of the Lymphatic Network
Authors: Jamalian Samira , Bertram Christopher D. , Moore James E.
Published: January 2014

One of the primary functions of the lymphatic system is maintaining fluid and protein balance in the body. The system holds this balance by collecting about four liters of fluid every day from the interstitial space and returning it back to the subclavian vein. In contrast to the blood circulation system that relies on the heart for pumping, there is no central pump in the lymphatic system. Thus, the transport of viscous fluid against gravity and pressure difference occurs by recruiting extrinsic and intrinsic pumping mechanisms. Extrinsic pumping is the transport of lymph due to the movements outside the lymphatic vessel such as the pulse in blood vessels, whereas the intrinsic pumping is transport of lymph by contraction of lymphatic muscle cells embedded in the walls of lymphatic vessels. Similar to the veins, the bi-leaflet valves throughout the lymphatic network prevent backflow. Lymphatic valves are biased open and allow for small amounts of back flow before they completely shut.
Deposition and characterization of Cd1−xMgxTe thin films grown by a novel cosublimation method
Authors: Kobyakov Pavel S. , Moore Andrew , Raguse John M. , Swanson Drew E. , Sampath Walajabad S.
Published: January 2014

Photovoltaic cells utilizing the CdS/CdTe structure have improved substantially in the past few years. Despite the recent advances, the efficiency of CdS/CdTe cells is still significantly below their Shockley–Queisser limit. CdTe based ternary alloy thin films, such as Cd1−xMgxTe (CMT), could be used to improve efficiency of CdS/CdTe photovoltaic cells. Higher band gap Cd1−xMgxTe films can be the absorber in top cells of a tandem structure or an electron reflector layer in CdS/CdTe cells. A novel cosublimation method to deposit CMT thin films has been developed. This method can deposit CMT films of band gaps ranging from 1.5 to 2.3 eV. The cosublimation method is fast, repeatable, and scalable for large areas, making it suitable for implementing into large-scale manufacturing. Characterization of as-deposited CMT films, with x varying from 0 to 0.35, reveals a linear relationship between Mg content measured by energy dispersive x-ray spectroscopy and the optical band gap. Glancing angle x-ray diffraction (GAXRD) measurements of Cd1−xMgxTe films show a zinc-blende structure similar to CdTe. Furthermore, increasing Mg content decreases the lattice parameter and the grain size. GAXRD shows the films are under mild tension after deposition.
Drug Policy as Conservation Policy: Narco-Deforestation
Authors: McSweeney Kendra , Nielsen Erik A. , Taylor Matthew J. , Wrathall David J. , Pearson Zoe , Wang Ophelia , Plumb Spencer T.
Published: January 2014

Drug trafficking is taking a toll on Central America's biodiverse forests.
Life in a Fluid Landscape
Dancing with the River
People and Life on the Chars of South Asia

by Kuntala Lahiri-Dutt and Gopa Samanta

Yale University Press, New Haven, CT, 2013. 296 pp. $65, £40. ISBN 9780300188301. Yale Agrarian Studies.
Authors: Nuckolls Charles W.
Published: January 2014

Using their ethnographic work in deltaic Bengal, Lahiri-Dutt and Samanta consider the residents' resilience, vulnerability, and adaptation to change.
In the Playing Ground of Consciousness
Consciousness and the Brain
Deciphering How the Brain Codes Our Thoughts

by Stanislas Dehaene

Viking, New York, 2014. 350 pp. $27.95, C$32.95. ISBN 9780670025435.
Authors: Koch Christof
Published: January 2014

Dehaene summarizes two decades of experimentation and modeling in attempts to understand consciousness.
When Electrons Leave Holes in Organic Solar Cells
Authors: Bredas Jean-Luc
Published: January 2014

Ultrafast spectroscopy reveals how the charge carriers in organic solar cells separate at interfaces and avoid substantial energy loss.

[Also see Report by

Gélinas
et al.

]
Delayed Gratification—Waiting to Terminate Stem Cell Identity
Authors: Zhang Xiaoyu
Published: January 2014

A cell-cycle timing mechanism in
Arabidopsis
is critical for flower development through the staggered expression of two transcription factors.

[Also see Research Article by

Sun
et al.

]
Selecting the Direction of Sound Transmission
Authors: Cummer Steven A.
Published: January 2014

A device containing a circulating fluid breaks the symmetry of acoustic waves and allows one-way transmission of sound.

[Also see Report by

Fleury
et al.

]
Pathogen Specialization
Authors: Coaker Gitta
Published: January 2014

The ability to infect new hosts can drive the evolution and specialization of secreted pathogen proteins.

[Also see Report by

Dong
et al.

]
A Unified Process for Neurological Disease
Authors: Singleton Andrew B.
Published: January 2014

The discovery of mutations associated with disease pathogenesis does not have to rest solely on genetic evidence.

[Also see Research Article by

Novarino
et al.

]
Visa Labyrinth
Authors: Poliakoff Martyn
Published: January 2014

The date of an exciting overseas academic conference is approaching, and you discover that you need a visa. You have to fill out an endless form (do you really have to list every country you've visited over the past 5, or even worse 10, years?). You may need to travel to a distant town for an interview. The visa may cost as much as your airline ticket. You begin to question whether you really need to go to this conference at all. And you wonder what things are like for someone trying to get a visa to attend a conference in your own country or work in your institution.
Neandertals and Moderns Made Imperfect Mates
Authors: Gibbons Ann
Published: January 2014

Our extinct cousins left us some key genes, according to two studies of Neandertal DNA in living people, but much of their genetic legacy has been wiped out of modern genomes.
Ecosystems Say 'Pass the Salt!'
Authors: Pennisi Elizabeth
Published: January 2014

In sodium-poor soil, a University of Oklahoma ecologist has found, small amounts of added salt boost invertebrate biomass and increase decomposition—so much so, his latest work suggests, that a lack of salt could have a major impact on the global carbon cycle.
Online Video Game Plugs Players Into Remote-Controlled Biochemistry Lab
Authors: Bohannon John
Published: January 2014

In a new twist to online science, researchers have crowdsourced their experiments by connecting players of a video game to an actual remote-controlled biochemistry lab.
Comparative Analysis of Bacillus subtilis Spores and Monophosphoryl Lipid A as Adjuvants of Protein-Based Mycobacterium tuberculosis-Based Vaccines: Partial Requirement for Interleukin-17A for Induction of Protective Immunity
Authors: Esparza-Gonzalez Sandra C. , Troy Amber R. , Izzo Angelo A.
Published: January 2014

The development of adjuvants for vaccines has become an important area of research as the number of protein-based vaccines against infectious pathogens increases. Currently, there are a number of adjuvant-basedMycobacterium tuberculosisvaccines in clinical trials that have shown efficacy in animal models. Despite these novel adjuvants, there is still a need to design new and more versatile adjuvants that have minimal adverse side effects but produce robust long-lasting adaptive immune responses. To this end, we hypothesized thatBacillus subtilisspores may provide the appropriate innate signals that are required to generate such vaccine-mediated responses, which would be sufficient to reduce the mycobacterial burden after infection withM. tuberculosis. In addition, we compared the response generated byB. subtilisspores to that generated by monophosphoryl lipid A (MPL), which has been used extensively to test tuberculosis vaccines. The well-characterized, 6-kDa early secretory antigenic target ofM. tuberculosis(ESAT-6; Rv3875) was used as a test antigen to determine the T cell activation potential of each adjuvant. Inoculated into mice,B. subtilisspores induced a strong proinflammatory response and Th1 immunity, similar to MPL; however, unlike MPL formulated with dimethyldioctadecylammonium (DDA) bromide, it failed to induce significant levels of interleukin-17A (IL-17A) and was unable to significantly reduce the mycobacterial burden after pulmonary infection withM. tuberculosis. Further analysis of the activity of MPL-DDA suggested that IL-17A was required for protective immunity. Taken together, the data emphasize the requirement for a network of cytokines that are essential for protective immunity.
Fatal Fulminant Hepatitis E Associated with Autoimmune Hepatitis and Excessive Paracetamol Intake in Southeastern France
Authors: Doudier Barbara , Vencatassin Hugues , Aherfi Sarah , Colson Philippe
Published: January 2014

We present, to our knowledge, the first case of fatal fulminant liver failure associated with hepatitis E virus infection, autoimmune hepatitis, and excessive paracetamol intake, which occurred in a 77-year-old woman. Hepatitis E testing should be performed in severe acute liver failure cases, even when another cause has been identified.
Evaluation of a Short, On-Plate Formic Acid Extraction Method for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Identification of Clinically Relevant Yeast Isolates
Authors: Gorton Rebecca L. , Seaton Shila , Ramnarain Purnima , McHugh Timothy D. , Kibbler Christopher C.
Published: January 2014

This report describes a short, on-plate formic acid (FA) extraction method for the identification of clinical yeast isolates using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS). A total of 41.1% (78/190) and 63.7% (121/190) of yeasts were identified using species log score thresholds of >2.0 and >1.9, respectively. Overall, 97.4% (185/190) of yeasts were identified in combination with conventional FA extraction.
Abdominal Abscess Caused by Mycobacterium llatzerense
Authors: Cárdenas Ana María , Gomila Margarita , Lalucat Jorge , Edelstein Paul H.
Published: January 2014

Mycobacterium llatzerense
was cultured from a subdiaphragmatic abscess. To our knowledge, this is the first report of isolation of this rapidly growing mycobacterium from a human. Growth characteristics and antimicrobial susceptibilities different from those previously reported for environmental isolates were observed.
Activation of the Interferon Induction Cascade by Influenza A Viruses Requires Viral RNA Synthesis and Nuclear Export
Authors: Killip Marian J. , Smith Matt , Jackson David , Randall Richard E.
Published: January 2014

We have examined the requirements for virus transcription and replication and thus the roles of input and progeny genomes in the generation of interferon (IFN)-inducing pathogen-associated molecular patterns (PAMPs) by influenza A viruses using inhibitors of these processes. Using IFN regulatory factor 3 (IRF3) phosphorylation as a marker of activation of the IFN induction cascade that occurs upstream of the IFN-β promoter, we demonstrate strong activation of the IFN induction cascade in A549 cells infected with a variety of influenza A viruses in the presence of cycloheximide or nucleoprotein (NP) small interfering RNA (siRNA), which inhibits viral protein synthesis and thus complementary ribonucleoprotein (cRNP) and progeny viral RNP (vRNP) synthesis. In contrast, activation of the IFN induction cascade by influenza viruses was very effectively abrogated by treatment with actinomycin D and other transcription inhibitors, which correlated with the inhibition of the synthesis of all viral RNA species. Furthermore, 5,6-dichloro-1-β-
d
-ribofuranosyl-benzimidazole, an inhibitor that prevents viral RNA export from the nucleus, was also a potent inhibitor of IRF3 activation; thus, both viral RNA synthesis and nuclear export are required for IFN induction by influenza A viruses. While the exact nature of the viral PAMPs remains to be determined, our data suggest that in this experimental system the major influenza A virus PAMPs are distinct from those of incoming genomes or progeny vRNPs.

IMPORTANCE
The host interferon system exerts an extremely potent antiviral response that efficiently restricts virus replication and spread; the interferon response can thus dictate the outcome of a virus infection, and it is therefore important to understand how viruses induce interferon. Both input and progeny genomes have been linked to interferon induction by influenza viruses. However, our experiments in tissue culture cells show that viral RNA synthesis and nuclear export are required to activate this response. Furthermore, the interferon induction cascade is activated under conditions in which the synthesis of progeny genomes is inhibited. Therefore, in tissue culture cells, input and progeny genomes are not the predominant inducers of interferon generated by influenza A viruses; the major viral interferon inducer(s) still remains to be identified.
Inhibition of Dengue and Chikungunya Virus Infections by RIG-I-Mediated Type I Interferon-Independent Stimulation of the Innate Antiviral Response
Authors: Olagnier David , Scholte Florine E. M. , Chiang Cindy , Albulescu Irina C. , Nichols Carmen , He Zhong , Lin Rongtuan , Snijder Eric J. , van Hemert Martijn J. , Hiscott John
Published: January 2014

RIG-I is a cytosolic sensor critically involved in the activation of the innate immune response to RNA virus infection. In the present study, we evaluated the inhibitory effect of a RIG-I agonist on the replication of two emerging arthropod-borne viral pathogens, dengue virus (DENV) and chikungunya virus (CHIKV), for which no therapeutic options currently exist. We demonstrate that when a low, noncytotoxic dose of an optimized 5′triphosphorylated RNA (5′pppRNA) molecule was administered, RIG-I stimulation generated a robust antiviral response against these two viruses. Strikingly, 5′pppRNA treatment before or after challenge with DENV or CHIKV provided protection against infection. In primary human monocytes and monocyte-derived dendritic cells, the RIG-I agonist blocked both primary infection and antibody-dependent enhancement of DENV infection. The protective response against DENV and CHIKV induced by 5′pppRNA was dependent on an intact RIG-I/MAVS/TBK1/IRF3 axis and was largely independent of the type I IFN response. Altogether, this
in vitro
analysis of the antiviral efficacy of 5′pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.

IMPORTANCE
DENV and CHIKV are two reemerging mosquito-borne viruses for which no therapeutic options currently exist. Both viruses overlap geographically in tropical regions of the world, produce similar fever-like symptoms, and are difficult to diagnose. This study investigated the inhibitory effect of a RIG-I agonist on the replication of these two viruses. RIG-I stimulation using 5′pppRNA before or after DENV or CHIKV infection generated a protective antiviral response against both pathogens in immune and nonimmune cells; interestingly, the protective response against the viruses was largely independent of the classical type I interferon response. The antiviral efficacy of 5′pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.
Large-Scale Nucleotide Optimization of Simian Immunodeficiency Virus Reduces Its Capacity To Stimulate Type I Interferon
In Vitro
Authors: Vabret Nicolas , Bailly-Bechet Marc , Lepelley Alice , Najburg Valérie , Schwartz Olivier , Verrier Bernard , Tangy Frédéric
Published: January 2014

Lentiviral RNA genomes present a strong bias in their nucleotide composition with extremely high frequencies of A nucleotide in human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). Based on the observation that human optimization of RNA virus gene fragments may abolish their ability to stimulate the type I interferon (IFN-I) response, we identified the most biased sequences along the SIV genome and showed that they are the most potent IFN-I stimulators. With the aim of designing an attenuated SIV genome based on a reduced capacity to activate the IFN-I response, we synthesized artificial SIV genomes whose biased sequences were optimized toward macaque average nucleotide composition without altering their regulatory elements or amino acid sequences. A synthetic SIV optimized with 169 synonymous mutations in
gag
and
pol
genes showed a 100-fold decrease in replicative capacity. Interestingly, a synthetic SIV optimized with 70 synonymous mutations in
pol
had a normal replicative capacity. Its ability to stimulate IFN-I was reduced when infected cells were cocultured with reporter cells. IFN regulatory factor 3 (IRF3) transcription factor was required for IFN-I stimulation, implicating cytosolic sensors in the detection of SIV-biased RNA in infected cells. No reversion of introduced mutations was observed for either of the optimized viruses after 10 serial passages. In conclusion, we have designed large-scale nucleotide-modified SIVs that may display attenuated pathogenic potential.

IMPORTANCE
In this study, we synthesized artificial SIV genomes in which the most hyperbiased sequences were optimized to bring them closer to the nucleotide composition of the macaque SIV host. Interestingly, we generated a stable synthetic SIV optimized with 70 synonymous mutations in
pol
gene, which had a normal replicative capacity but a reduced ability to stimulate type I IFN. This demonstrates the possibility to rationally change viral nucleotide composition to design replicative and genetically stable lentiviruses with attenuated pathogenic potentials.
Surfactant-Modified Nanoclay Exhibits an Antiviral Activity with High Potency and Broad Spectrum
Authors: Liang Jian-Jong , Wei Jiun-Chiou , Lee Yi-Ling , Hsu Shan-hui , Lin Jiang-Jen , Lin Yi-Ling
Published: January 2014

Nanomaterials have the characteristics associated with high surface-to-volume ratios and have been explored for their antiviral activity. Despite some success, cytotoxicity has been an issue in nanomaterial-based antiviral strategies. We previously developed a novel method to fully exfoliate montmorillonite clay to generate the most fundamental units of nanoscale silicate platelet (NSP). We further modified NSP by capping with various surfactants and found that the surfactant-modified NSP (NSQ) was less cytotoxic. In this study, we tested the antiviral potentials of a series of natural-clay-derived nanomaterials. Among the derivatives, NSP modified with anionic sodium dodecyl sulfate (NSQc), but not the pristine clay, unmodified NSP, a silver nanoparticle-NSP hybrid, NSP modified with cationicn-octadecanylamine hydrochloride salt, or NSP modified with nonionic Triton X-100, significantly suppressed the plaque-forming ability of Japanese encephalitis virus (JEV) at noncytotoxic concentrations. NSQc also blocked infection with dengue virus (DEN) and influenza A virus. Regarding the antiviral mechanism, NSQc interfered with viral binding through electrostatic interaction, since its antiviral activity can be neutralized by Polybrene, a cationic polymer. Furthermore, NSQc reduced the lethality of JEV and DEN infection in mouse challenge models. Thus, the surfactant-modified exfoliated nanoclay NSQc may be a novel nanomaterial with broad and potent antiviral activity.

IMPORTANCENanomaterials have being investigated as antimicrobial agents, yet their antiviral potential is overshadowed by their cytotoxicity. By using a novel method, we fully exfoliated montmorillonite clay to generate the most fundamental units of nanoscale silicate platelet (NSP). Here, we show that the surfactant-modified NSP (NSQ) is less cytotoxic and that NSQc (NSP modified with sodium dodecyl sulfate) could potently block infection by dengue virus (DEN), Japanese encephalitis virus (JEV), and influenza A virus at noncytotoxic concentrations. For the antiviral mechanism, we find that the electrostatic interaction between the negatively charged NSQc and the positively charged virus particles blocks viral binding. Furthermore, we used mouse challenge models of JEV and DEN to demonstrate thein vivoantiviral potential of NSQc. Thus, NSQc may function as a potent and safe antiviral nanohybrid against several viruses, and our success in synthesizing surfactant-modified NSP with antiviral activity may shed some light on future antiviral development.
Forkhead Box Transcription Factor Regulation and Lipid Accumulation by Hepatitis C Virus
Authors: Bose Sandip K. , Kim Hangeun , Meyer Keith , Wolins Nathan , Davidson Nicholas O. , Ray Ranjit
Published: January 2014

We have previously shown that hepatitis C virus (HCV) infection modulates the expression of forkhead box transcription factors, including FoxO1 and FoxA2, which play key roles in gluconeogenesis and β-oxidation of fatty acid, respectively. The aim of the present study was to determine the role of forkhead box transcription factors in modulating lipid metabolism. HCV infection or core protein expression alone in transfected Huh7.5 cells increased expression of sterol regulatory element binding protein 1c (SREBP-1c) and its downstream target, fatty acid synthase (FASN), which are key proteins involved in lipid synthesis. Knockdown of FoxO1 by small interfering RNA in HCV-infected cells significantly decreased SREBP-1c and FASN expression. Further, HCV infection or core protein expression in Huh7.5 cells significantly decreased the expression of medium-chain acyl coenzyme A dehydrogenase (MCAD) and short-chain acyl coenzyme A dehydrogenase (SCAD), involved in the regulation of β-oxidation of fatty acids. Ectopic expression of FoxA2 in HCV-infected cells rescued the expression of MCAD and SCAD. Oil red O and neutral lipid staining indicated that HCV infection significantly increases lipid accumulation compared to that in the mock-infected control. This was further verified by the increased expression of perilipin-2 and decreased activity of hormone-sensitive lipase (HSL) in HCV-infected hepatocytes, implying increased accumulation of neutral lipids. Knockdown of FoxO1 and ectopic expression of FoxA2 significantly decreased HCV replication. Taken together, these results suggest that HCV modulates forkhead box transcription factors which together increase lipid accumulation and promote viral replication.

IMPORTANCE
Hepatic steatosis is a frequent complication associated with chronic HCV infection. Its presence is a key prognostic indicator associated with the progression to hepatic fibrosis and hepatocellular carcinoma. Several mechanisms have been proposed to account for the development of steatosis and fatty liver during HCV infection. We observed that HCV infection increases expression of both SREBP-1c and FASN. Further investigation suggested that the expression of SREBP-1c and FASN is controlled by the transcription factor FoxO1 during HCV infection. In addition, HCV infection significantly decreased both MCAD and SCAD expression, which is controlled by FoxA2. HCV infection also increased lipid droplet accumulation, increased perilipin-2 expression, and decreased HSL activity. Thus, knockdown of FoxO1 (decreased lipogenesis) and overexpression of FoxA2 (increased β-oxidation) resulted in a significant disruption of the platform and, hence, a decrease in HCV genome replication. Thus, targeting of FoxO1 and FoxA2 might be useful in developing a therapeutic approach against HCV infection.
Reversible and Efficient Activation of HIV-1 Cell Entry by a Tyrosine-Sulfated Peptide Dissects Endocytic Entry and Inhibitor Mechanisms
Authors: Platt Emily J. , Gomes Michelle M. , Kabat David
Published: January 2014

HIV-1 membranes contain gp120-gp41 trimers. Binding of gp120 to CD4 and a coreceptor (CCR5 or CXCR4) reduces the constraint on metastable gp41, enabling a series of conformational changes that cause membrane fusion. An analytic difficulty occurs because these steps occur slowly and asynchronously within cohorts of adsorbed virions. We previously isolated HIV-1
JRCSF
variants that efficiently use CCR5 mutants severely damaged in the tyrosine-sulfated amino terminus or extracellular loop 2. Surprisingly, both independent adaptations included gp120 mutations S298N, F313L, and N403S, supporting other evidence that they function by weakening gp120's grip on gp41 rather than by altering gp120 binding to specific CCR5 sites. Although several natural HIV-1 isolates reportedly use CCR5(Δ18) (CCR5 with a deletion of 18 N-terminal amino acids, including the tyrosine-sulfated region) when the soluble tyrosine-sulfated peptide is present, we show that HIV-1
JRCSF
with the adaptive mutations [HIV-1
JRCSF
(Ad)] functions approximately 100 times more efficiently and that coreceptor activation is reversible, enabling synchronous efficient entry control under physiological conditions. This system revealed that three-stranded gp41 folding intermediates susceptible to the inhibitor enfuvirtide form slowly and asynchronously on cell surface virions but resolve rapidly, with virions generally forming only one target. Adsorbed virions asynchronously and transiently become competent for entry at 37°C but are inactivated if the CCR5 peptide is absent during their window of opportunity. This competency is conferred by endocytosis, which results in inactivation if the peptide is absent. For both wild-type and adapted HIV-1 isolates, early gp41 refolding steps obligatorily occur on cell surfaces, whereas the final step(s) is endosomal. This system powerfully dissects HIV-1 entry and inhibitor mechanisms.

IMPORTANCE
We present a powerful means to reversibly and efficiently activate or terminate HIV-1 entry by adding or removing a tyrosine-sulfated CCR5 peptide from the culture medium. This system uses stable cell clones and a variant of HIV-1
JRCSF
with three adaptive mutations. It enabled us to show that CCR5 coreceptor activation is rapidly reversible and to dissect aspects of entry that had previously been relatively intractable. Our analyses elucidate enfuvirtide (T-20) function and suggest that HIV-1 virions form only one nonredundant membrane fusion complex on cell surfaces. Additionally, we obtained novel and conclusive evidence that HIV-1 entry occurs in an assembly line manner, with some steps obligatorily occurring on cell surfaces and with final membrane fusion occurring in endosomes. Our results were confirmed for wild-type HIV-1. Thus, our paper provides major methodological and mechanistic insights about HIV-1 infection.
Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis
Authors: Wei Yongwei , Zhang Yu , Cai Hui , Mirza Anne M. , Iorio Ronald M. , Peeples Mark E. , Niewiesk Stefan , Li Jianrong
Published: January 2014

Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif (
329
RGD
331
). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5β1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the
329
RGD
331
motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5β1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines.

IMPORTANCE
Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral fusion (F) protein mediates both attachment and fusion activity. Recently, it was suggested that hMPV F protein utilizes integrins as receptors for entry via a poorly understood mechanism. Here, we show that α5β1 and αv integrins are essential for hMPV infectivity and F protein-mediated cell-cell fusion and that the integrin-binding motif in the F protein plays a crucial role in these functions. Our results also identify the integrin-binding motif to be a new, attenuating target for the development of a live vaccine for hMPV. These findings not only will facilitate the development of antiviral drugs targeting viral entry steps but also will lead to the development new live attenuated vaccine candidates for hMPV.
Attenuation and Restoration of Severe Acute Respiratory Syndrome Coronavirus Mutant Lacking 2′-O-Methyltransferase Activity
Authors: Menachery Vineet D. , Yount Boyd L. , Josset Laurence , Gralinski Lisa E. , Scobey Trevor , Agnihothram Sudhakar , Katze Michael G. , Baric Ralph S.
Published: January 2014

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and, more recently, Middle Eastern respiratory syndrome CoV (MERS-CoV) underscores the importance of understanding critical aspects of CoV infection and pathogenesis. Despite significant insights into CoV cross-species transmission, replication, and virus-host interactions, successful therapeutic options for CoVs do not yet exist. Recent identification of SARS-CoV NSP16 as a viral 2′-O-methyltransferase (2′-O-MTase) led to the possibility of utilizing this pathway to both attenuate SARS-CoV infection and develop novel therapeutic treatment options. Mutations were introduced into SARS-CoV NSP16 within the conserved KDKE motif and effectively attenuated the resulting SARS-CoV mutant viruses both
in vitro
and
in vivo
. While viruses lacking 2′-O-MTase activity had enhanced sensitivity to type I interferon (IFN), they were not completely restored in their absence
in vivo
. However, the absence of either MDA5 or IFIT1, IFN-responsive genes that recognize unmethylated 2′-O RNA, resulted in restored replication and virulence of the dNSP16 mutant virus. Finally, using the mutant as a live-attenuated vaccine showed significant promise for possible therapeutic development against SARS-CoV. Together, the data underscore the necessity of 2′-O-MTase activity for SARS-CoV pathogenesis and identify host immune pathways that mediate this attenuation. In addition, we describe novel treatment avenues that exploit this pathway and could potentially be used against a diverse range of viral pathogens that utilize 2′-O-MTase activity to subvert the immune system.

IMPORTANCE
Preventing recognition by the host immune response represents a critical aspect necessary for successful viral infection. Several viruses, including SARS-CoV, utilize virally encoded 2′-O-MTases to camouflage and obscure their viral RNA from host cell sensing machinery, thus preventing recognition and activation of cell intrinsic defense pathways. For SARS-CoV, the absence of this 2′-O-MTase activity results in significant attenuation characterized by decreased viral replication, reduced weight loss, and limited breathing dysfunction in mice. The results indicate that both MDA5, a recognition molecule, and the IFIT family play an important role in mediating this attenuation with restored virulence observed in their absence. Understanding this virus-host interaction provided an opportunity to design a successful live-attenuated vaccine for SARS-CoV and opens avenues for treatment and prevention of emerging CoVs and other RNA virus infections.
Transcriptional Derepression of the
ERVWE1
Locus following Influenza A Virus Infection
Authors: Li Fang , Nellåker Christoffer , Sabunciyan Sarven , Yolken Robert H. , Jones-Brando Lorraine , Johansson Anne-Sofie , Owe-Larsson Björn , Karlsson Håkan
Published: January 2014

Syncytin-1, a fusogenic protein encoded by a human endogenous retrovirus of the W family (HERV-W) element (
ERVWE1
), is expressed in the syncytiotrophoblast layer of the placenta. This locus is transcriptionally repressed in adult tissues through promoter CpG methylation and suppressive histone modifications. Whereas syncytin-1 appears to be crucial for the development and functioning of the human placenta, its ectopic expression has been associated with pathological conditions, such as multiple sclerosis and schizophrenia. We previously reported on the transactivation of HERV-W elements, including
ERVWE1
, during influenza A/WSN/33 virus infection in a range of human cell lines. Here we report the results of quantitative PCR analyses of transcripts encoding syncytin-1 in both cell lines and primary fibroblast cells. We observed that spliced
ERVWE1
transcripts and those encoding the transcription factor glial cells missing 1 (GCM1), acting as an enhancer element upstream of
ERVWE1
, are prominently upregulated in response to influenza A/WSN/33 virus infection in nonplacental cells. Knockdown of GCM1 by small interfering RNA followed by infection suppressed the transactivation of
ERVWE1
. While the infection had no influence on CpG methylation in the
ERVWE1
promoter, chromatin immunoprecipitation assays detected decreased H3K9 trimethylation (H3K9me3) and histone methyltransferase SETDB1 levels along with influenza virus proteins associated with
ERVWE1
and other HERV-W loci in infected CCF-STTG1 cells. The present findings suggest that an exogenous influenza virus infection can transactivate
ERVWE1
by increasing transcription of
GCM1
and reducing H3K9me3 in this region and in other regions harboring HERV-W elements.

IMPORTANCE
Syncytin-1, a protein encoded by the
env
gene in the HERV-W locus
ERVWE1
, appears to be crucial for the development and functioning of the human placenta and is transcriptionally repressed in nonplacental tissues. Nevertheless, its ectopic expression has been associated with pathological conditions, such as multiple sclerosis and schizophrenia. In the present paper, we report findings suggesting that an exogenous influenza A virus infection can transactivate
ERVWE1
by increasing the transcription of
GCM1
and reducing the repressive histone mark H3K9me3 in this region and in other regions harboring HERV-W elements. These observations have implications of potential relevance for viral pathogenesis and for conditions associated with the aberrant transcription of HERV-W loci.