bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling the Semantic Structure of Textually Derived Learning Content and its Impact on Recipients' Response States
Authors: Munoz David , Tucker Conrad S.
Published: December 2015

In the United States, the greatest decline in the number of students in the STEM education pipeline occurs at the university level, where students, who were initially interested in STEM fields, drop-out or move on to other interests. It has been reported that “of the 23 most commonly cited reasons for switching out of STEM, all but 7 had something to do with the pedagogical experience.” Thus, understanding the characteristics of the pedagogical experience that impact students' interest in STEM is of great importance to the academic community. This work tests the hypothesis that there exists a correlation between the semantic structure of lecture content and students' affective states. Knowledge gained from testing this hypothesis will inform educators of the specific semantic structure of lecture content that enhance students' affective states and interest in course content, toward the goal of increasing STEM retention rates and overall positive experiences in STEM majors. A case study involving a series of science and engineering based digital content is used to create a semantic network and demonstrate the implications of the methodology. The results reveal that affective states such as engagement and boredom are consistently strongly correlated to the semantic network metrics outlined in the paper, while the affective state of confusion is weakly correlated with the same semantic network metrics. The results reveal semantic network relationships that are generalizable across the different textually derived information sources explored. These semantic network relationships can be explored by researchers trying to optimize their message structure in order to have its intended effect.
A Comparative Study on Motion Characteristics of Three Two-Degree-of-Freedom Pointing Mechanisms
Authors: Yu Jingjun , Wu Kang , Zong Guanghua , Kong Xianwen
Published: December 2015

Two-degree-of-freedom (2DOF) pointing mechanisms have been widely used in areas such as stabilized platforms, tracking devices, etc. Besides the commonly used serial gimbal structures, another two types of parallel pointing mechanisms, i.e., spherical parallel manipulators (SPMs) and equal-diameter spherical pure rolling (ESPR) parallel manipulators, are increasingly concerned. Although all these pointing mechanisms have two rotational DOFs, they exhibit very different motion characteristics. A typical difference existing in these three pointing mechanisms can be found from their characteristics of self-motion, also called spinning motion by the authors. In this paper, the spinning motions of three pointing mechanisms are modeled and compared via the graphical approach combined with the vector composition theorem. According to our study, the spinning motion is essentially one component of the moving platform's real rotation. Furthermore, image distortions caused by three spinning motions are identified and distinguished when the pointing mechanisms are used as tracking devices. Conclusions would facilitate the design and control of the pointing devices and potentially improve the measuring accuracy for targets pointing and tracking.
Metatarsal Loading During Gait—A Musculoskeletal Analysis
Authors: Al-Munajjed Amir A. , Bischoff Jeffrey E. , Dharia Mehul A. , Telfer Scott , Woodburn James , Carbes Sylvain
Published: December 2015

Detailed knowledge of the loading conditions within the human body is essential for the development and optimization of treatments for disorders and injuries of the musculoskeletal system. While loads in the major joints of the lower limb have been the subject of extensive study, relatively little is known about the forces applied to the individual bones of the foot. The objective of this study was to use a detailed musculoskeletal model to compute the loads applied to the metatarsal bones during gait across several healthy subjects. Motion-captured gait trials and computed tomography (CT) foot scans from four healthy subjects were used as the inputs to inverse dynamic simulations that allowed the computation of loads at the metatarsal joints. Low loads in the metatarsophalangeal (MTP) joint were predicted before terminal stance, however, increased to an average peak of 1.9 times body weight (BW) before toe-off in the first metatarsal. At the first tarsometatarsal (TMT) joint, loads of up to 1.0 times BW were seen during the early part of stance, reflecting tension in the ligaments and muscles. These loads subsequently increased to an average peak of 3.0 times BW. Loads in the first ray were higher compared to rays 2–5. The joints were primarily loaded in the longitudinal direction of the bone.
Intra-Articular Knee Contact Force Estimation During Walking Using Force-Reaction Elements and Subject-Specific Joint Model2
Authors: Jung Yihwan , Phan Cong-Bo , Koo Seungbum
Published: December 2015

Joint contact forces measured with instrumented knee implants have not only revealed general patterns of joint loading but also showed individual variations that could be due to differences in anatomy and joint kinematics. Musculoskeletal human models for dynamic simulation have been utilized to understand body kinetics including joint moments, muscle tension, and knee contact forces. The objectives of this study were to develop a knee contact model which can predict knee contact forces using an inverse dynamics-based optimization solver and to investigate the effect of joint constraints on knee contact force prediction. A knee contact model was developed to include 32 reaction force elements on the surface of a tibial insert of a total knee replacement (TKR), which was embedded in a full-body musculoskeletal model. Various external measurements including motion data and external force data during walking trials of a subject with an instrumented knee implant were provided from the Sixth Grand Challenge Competition to Predict in vivo Knee Loads. Knee contact forces in the medial and lateral portions of the instrumented knee implant were also provided for the same walking trials. A knee contact model with a hinge joint and normal alignment could predict knee contact forces with root mean square errors (RMSEs) of 165 N and 288 N for the medial and lateral portions of the knee, respectively, and coefficients of determination (R2) of 0.70 and −0.63. When the degrees-of-freedom (DOF) of the knee and locations of leg markers were adjusted to account for the valgus lower-limb alignment of the subject, RMSE values improved to 144 N and 179 N, and R2 values improved to 0.77 and 0.37, respectively. The proposed knee contact model with subject-specific joint model could predict in vivo knee contact forces with reasonable accuracy. This model may contribute to the development and improvement of knee arthroplasty.
Investigation of Swirl Ratio Impact on In-Cylinder Flow in an SIDI Optical Engine
Authors: Zhuang Hanyang , Hung David L. S. , Yang Jie , Tian Shaoxiong
Published: December 2015

Advanced powertrain technologies have improved engine performance with higher power output, lower exhaust emission, and better controllability. Chief among them is the development of spark-ignition direct-injection (SIDI) engines in which the in-cylinder processes control the air flow motion, fuel–air mixture formation, combustion, and soot formation. Specifically, intake air with strong swirl motion is usually introduced to form a directional in-cylinder flowfield. This approach improves the mixing process of air and fuel as well as the propagation of flame. In this study, the effect of intake air swirl on in-cylinder flow characteristics was experimentally investigated. High-speed particle image velocimetry (PIV) was conducted in an optical SIDI engine to record the flowfield on a swirl plane. The intake air swirl motion was achieved by adjusting the opening of a swirl ratio (SR) control valve which was installed in one of the two intake ports in the optical engine. Ten opening angles of the SR control valve were adjusted to produce an intake SR from 0.55 to 5.68. The flow structures at the same crank angle degree (CAD), but under different SR, were compared and analyzed using proper orthogonal decomposition (POD). The flow dominant structures and variation structures were interpreted by different POD modes. The first POD mode captured the most dominant flowfield structure characteristics; the corresponding mode coefficients showed good linearity with the measured SR at the compression stroke when the flow was swirling and steady. During the intake stroke, strong intake air motion took place, and the structures and coefficients of the first modes varied along different SR. These modes captured the flow properties affected by the intake swirl motion. Meanwhile, the second and higher modes captured the variation feature of the flow at various CADs. In summary, this paper demonstrated a promising approach of using POD to interpret the effectiveness of swirl control valve on in-cylinder swirl flow characteristics, providing better understanding for engine intake system design and optimization.
Improved performance of InGaN/GaN flip-chip light-emitting diodes through the use of robust Ni/Ag/TiW mirror contacts
Authors: Hasanov Namig , Zhu Binbin , Sharma Vijay Kumar , Lu Shunpeng , Zhang Yiping , Liu Wei , Tan Swee Tiam , Sun Xiao Wei , Demir Hilmi Volkan
Published: December 2015

In this work, the authors report the incorporation of TiW alloy in InGaN/GaN-based flip-chip light-emitting diodes (LEDs). The advantages provided by the use of TiW are analyzed in detail. InGaN/GaN multiple quantum well LEDs with a Ni/Ag/TiW metal stack are found to tolerate high-temperature annealing better than those with a Ni/Ag metal stack. Highly improved current–voltage characteristics and enhanced optical output power are achieved for the devices with a TiW thin layer. These changes are ascribed to the higher reflectivity, smoother surface, and better ohmic properties of the device containing TiW after annealing. Better heat management of the device with TiW is demonstrated by comparing electroluminescence spectra of the two device structures. Overall, these factors resulted in devices with TiW exhibiting a higher external quantum efficiency than devices without TiW. Detailed x-ray photoelectron spectroscopy analyses of the reflector metal stacks reveal little intermixing of the layers after annealing in the devices with TiW. The results show that incorporation of TiW is a promising approach for the fabrication of high-performance InGaN/GaN flip-chip LEDs.
As Ebola epidemic draws to a close, a thin scientific harvest
Authors: Cohen Jon , Enserink Martin
Published: December 2015

Thirteen clinical trials in West Africa have yielded only one unequivocally positive result.
Unfilled Vials
Authors: Cohen Jon
Published: December 2015

Scientifically feasible vaccines against major diseases are stalled for lack of funds.
Science
names 10 top candidates that need a boost.
A shot at migraine
Authors: Underwood Emily
Published: January 2016

Drug companies are racing to prove antibody treatments can prevent the debilitating headaches.
Structural basis for leucine sensing by the Sestrin2-mTORC1 pathway
Authors: Saxton Robert A. , Knockenhauer Kevin E. , Wolfson Rachel L. , Chantranupong Lynne , Pacold Michael E. , Wang Tim , Schwartz Thomas U. , Sabatini David M.
Published: December 2015

From sensing leucine to metabolic control

The mTORC1 protein kinase complex plays central roles in regulating cell growth and metabolism and is implicated in common human diseases such as diabetes and cancer. The level of the amino acid leucine tells an organism a lot about its physiological state, including how much food is available, how much insulin is going to be needed, and whether new muscle mass can be made (see the Perspective by Buel and Blenis). Wolfson
et al.
identified a biochemical sensor of leucine, Sestrin2, which connects the concentration of leucine to the control of organismal metabolism and growth. When leucine bound to Sestrin2, it was released from a complex with the mTORC1 regulatory factor GATOR2, activating the mTORC1 complex. Saxton
et al.
describe the crystal structure of Sestrin2 and show how it specifically detects leucine. Aylett
et al.
determined the structure of human mTORC1 by cryoelectron microscopy and the crystal structure of a regulatory subunit, Raptor. The results reveal the structural basis for the function and intricate regulation of this important enzyme, which is also a strategic drug target.

Science
, this issue p.
43
, p.
48
, p.
53
; see also p.
25
Multidrug evolutionary strategies to reverse antibiotic resistance
Authors: Baym Michael , Stone Laura K. , Kishony Roy
Published: December 2015

Evolving antibiotic rescue stratagems

Antibiotic resistance threatens to put modern medicine into reverse. But we are not at the end of our options for currently available drugs. Baym
et al.
review what can be done by using combinations of antibiotics to circumvent bacteria's evolutionary strategies. For instance, resistance to one drug may cause sensitivity to another, the effectiveness of two drugs can be synergized by a resistance mutation, and some negative drug interactions may even be beneficial in selecting against resistance. Although not simple to assess, drug combinations still have something to offer for the development of sorely needed anti-infectives.

Science
, this issue p.
10.1126/science.aad3292
Winning the war
The Death of Cancer
Vincent T. DeVita Jr. and Elizabeth DeVita-Raeburn
Sarah Crichton Books, 2015. 349 pp.
Authors: Woolfson Adrian
Published: December 2015

An insider's guide to the politics and personalities of America's war on cancer
The screams of a star being ripped apart
Authors: Bower Geoffrey C.
Published: December 2015

A star being devoured by a black hole provides a route to study accretion and jet formation

[Also see Report by

van Velzen
et al.

]
Catalytic conjunctive cross-coupling enabled by metal-induced metallate rearrangement
Authors: Zhang Liang , Lovinger Gabriel J. , Edelstein Emma K. , Szymaniak Adam A. , Chierchia Matteo P. , Morken James P.
Published: December 2015

A two-for-one twist on Suzuki coupling

The Suzuki-Miyaura coupling reaction is one of the most widely used ways of making carbon-carbon bonds. Essentially a palladium catalyst activates one carbon fragment and then links it to a second fragment pulled from boron. Zhang
et al.
now demonstrate a twist on the conventional pathway (see the Perspective by Fyfe and Watson). In their system, the palladium initially coaxes together two carbon fragments on one boron center. Then the catalyst stitches a second C-C bond to a third, external fragment. A chiral ligand renders the reaction highly enantio-selective.

Science
, this issue p.
70
; see also p.
26
Illuminating anhedonia
Authors: Robbins Trevor W.
Published: December 2015

Optogenetics and fMRI reveal the brain circuitry of anhedonia

[Also see Research Article by

Ferenczi
et al.

]
Concentration of Sonication Fluid through Centrifugation Is Superior to Membrane Filtration for Microbial Diagnosis of Orthopedic Implant-Associated Infection
Authors: Zitron Renato , Wajsfeld Tali , Klautau Giselle Burlamaqui , da Silva Cely Barreto , Nigro Stanley , Mercadante Marcelo Tomanik , Polesello Giancarlo Cavalli , Cury Ricardo de Paula Leite , Salles Mauro Jose Costa
Published: December 2015

Microbial identification of orthopedic implant-associated infections using sonication fluid (SF) submitted to a concentration step by membrane filtration (SMF) was compared with the standard centrifugation (SC) method. Among 33 retrieved infected implants, sonication identified microorganisms in 26 (78.8%). The sensitivity of SC was higher than that of SMF (78.8% versus 30.3%;
P
< 0.001).
Coproduction of KPC-18 and VIM-1 Carbapenemases by Enterobacter cloacae: Implications for Newer β-Lactam–β-Lactamase Inhibitor Combinations
Authors: Thomson Gina K. , Snyder James W. , McElheny Christi L. , Thomson Kenneth S. , Doi Yohei
Published: December 2015

Enterobacter cloacae
strain G6809 with reduced susceptibility to carbapenems was identified from a patient in a long-term acute care hospital in Kentucky. G6809 belonged to sequence type (ST) 88 and carried two carbapenemase genes,
bla
KPC-18
and
bla
VIM-1
. Whole-genome sequencing localized
bla
KPC-18
to the chromosome and
bla
VIM-1
to a 58-kb plasmid. The strain was highly resistant to ceftazidime-avibactam. Insidious coproduction of metallo-β-lactamase with KPC-type carbapenemase has implications for the use of next-generation β-lactam–β-lactamase inhibitor combinations.
Comparison of Vitek MS and MALDI Biotyper for Identification of Actinomycetaceae of Clinical Importance
Authors: Ferrand Janina , Hochard Hélène , Girard Victoria , Aissa Nejla , Bogard Baptiste , Alauzet Corentine , Lozniewski Alain
Published: December 2015

The Vitek MS
in vitro
diagnostic (IVD) and MALDI Biotyper IVD systems were evaluated for the identification of 158 strains of
Actinomycetaceae
. Correct species-level identification rates of 60.7% and 58.2% were obtained with the Vitek MS system after direct deposit and with the MALDI Biotyper system after on-plate formic acid treatment, respectively.
Hemagglutinin Stalk Immunity Reduces Influenza Virus Replication and Transmission in Ferrets
Authors: Nachbagauer Raffael , Miller Matthew S. , Hai Rong , Ryder Alex B. , Rose John K. , Palese Peter , García-Sastre Adolfo , Krammer Florian , Albrecht Randy A.
Published: December 2015

We assessed whether influenza virus hemagglutinin stalk-based immunity protects ferrets against aerosol-transmitted H1N1 influenza virus infection. Immunization of ferrets by a universal influenza virus vaccine strategy based on viral vectors expressing chimeric hemagglutinin constructs induced stalk-specific antibody responses. Stalk-immunized ferrets were cohoused with H1N1-infected ferrets under conditions that permitted virus transmission. Hemagglutinin stalk-immunized ferrets had lower viral titers and delayed or no virus replication at all following natural exposure to influenza virus.
Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation
Authors: Tran Giao V. Q. , Luong Trang T. D. , Park Eun-Mee , Kim Jong-Wook , Choi Jae-Woong , Park Chorong , Lim Yun-Sook , Hwang Soon B.
Published: December 2015

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for virus propagation. To identify the cellular factors involved in HCV propagation, we recently performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of 90 cellular protein candidates, we selected the soluble resistance-related calcium-binding protein (sorcin) for further characterization. Sorcin is a calcium-binding protein and is highly expressed in certain cancer cells. We verified that NS5A interacted with sorcin through domain I of NS5A, and phosphorylation of the threonine residue 155 of sorcin played a crucial role in protein interaction. Small interfering RNA (siRNA)-mediated knockdown of sorcin impaired HCV propagation. Silencing of sorcin expression resulted in a decrease of HCV assembly without affecting HCV RNA and protein levels. We further demonstrated that polo-like kinase 1 (PLK1)-mediated phosphorylation of sorcin was increased by NS5A. We showed that both phosphorylation and calcium-binding activity of sorcin were required for HCV propagation. These data indicate that HCV modulates sorcin activity via NS5A protein for its own propagation.

IMPORTANCESorcin is a calcium-binding protein and regulates intracellular calcium homeostasis. HCV NS5A interacts with sorcin, and phosphorylation of sorcin is required for protein interaction. Gene silencing of sorcin impaired HCV propagation at the assembly step of the HCV life cycle. Sorcin is phosphorylated by PLK1 via protein interaction. We showed that sorcin interacted with both NS5A and PLK1, and PLK1-mediated phosphorylation of sorcin was increased by NS5A. Moreover, calcium-binding activity of sorcin played a crucial role in HCV propagation. These data provide evidence that HCV regulates host calcium metabolism for virus propagation, and thus manipulation of sorcin activity may represent a novel therapeutic target for HCV.
Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation
Authors: Eberle Kirsten C. , McGill Jodi L. , Reinhardt Timothy A. , Sacco Randy E.
Published: December 2015

Parainfluenza viruses are known to inhibit type I interferon (IFN) production; however, there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, IFN-λR1/interleukin-10R2 (IL-10R2), which is primarily expressed by epithelial cells. Parainfluenza virus 3 (PIV-3) infection is highly restricted to the airway epithelium. We therefore sought to examine type III IFN signaling pathways during PIV-3 infection of epithelial cells. We used three strains of PIV-3: human PIV-3 (HPIV-3), bovine PIV-3 (BPIV-3), and dolphin PIV-1 (
Tursiops truncatus
PIV-1, or TtPIV-1). Here, we show that message levels of IL-29 are significantly increased during PIV-3 infection, yet downstream antiviral signaling molecules are not upregulated to levels similar to those of the positive control. Furthermore, in Vero cells infected with PIV-3, stimulation with recombinant IL-29/-28A/-28B does not cause upregulation of downstream antiviral molecules, suggesting that PIV-3 interferes with the JAK/STAT pathway downstream of the IFN-λR1/IL-10R2 receptor. We used Western blotting to examine the phosphorylation of Stat1 and Stat2 in Vero cells and the bronchial epithelial cell line BEAS-2B. In Vero cells, we observed reduced phosphorylation of the serine 727 (S727) site on Stat1, while in BEAS-2B cells Stat1 phosphorylation was decreased at the tyrosine 701 (Y701) site during PIV-3 infection. PIV-3 therefore interferes with the phosphorylation of Stat1 downstream of the type III IFN receptor. These data provide new evidence regarding strategies employed by parainfluenza viruses to effectively circumvent respiratory epithelial cell-specific antiviral immunity.

IMPORTANCE
Parainfluenza virus (PIV) in humans is associated with bronchiolitis and pneumonia and can be especially problematic in infants and the elderly. Also seen in cattle, bovine PIV-3 causes respiratory infections in young calves. In addition, PIV-3 is one of a number of pathogens that contribute to the bovine respiratory disease complex (BRDC). As their name suggests, interferons (IFNs) are produced by cells to interfere with viral replication. Paramyxoviruses have previously been shown to block production and downstream signaling of type I IFNs. For the first time, it is shown here that PIV-3 can induce protective type III IFNs in epithelial cells, the primary site of PIV-3 infection. However, we found that PIV-3 modulates signaling pathways downstream of the type III IFN receptor to block production of several specific molecules that aid in a productive antiviral response. Importantly, this work expands our understanding of how PIV-3 effectively evades host innate immunity.
Human Cytomegalovirus US28 Is Important for Latent Infection of Hematopoietic Progenitor Cells
Authors: Humby Monica S. , O'Connor Christine M.
Published: December 2015

Human cytomegalovirus (HCMV) resides latently in hematopoietic progenitor cells (HPCs). During latency, only a subset of HCMV genes is transcribed, including one of the four virus-encoded G protein-coupled receptors (GPCRs), US28. Although US28 is a multifunctional lytic protein, its function during latency has remained undefined. We generated a panel of US28 recombinant viruses in the bacterial artificial chromosome (BAC)-derived clinical HCMV strain TB40/E-mCherry. We deleted the entire US28 open reading frame (ORF), deleted all four of the viral GPCR ORFs, or deleted three of the HCMV GPCRs but not the US28 wild-type protein. Using these recombinant viruses, we assessed the requirement for US28 during latency in the Kasumi-3in vitrolatency model system and in primaryex vivo-cultured CD34+HPCs. Our data suggest that US28 is required for latency as infection with viruses lacking the US28 ORF alone or in combination with the remaining HCMV-encoded GPCR results in transcription from the major immediate early promoter, the production of extracellular virions, and the production of infectious virus capable of infecting naive fibroblasts. The other HCMV GPCRs are not required for this phenotype as a virus expressing only US28 but not the remaining virus-encoded GPCRs is phenotypically similar to that of wild-type latent infection. Finally, we found that US28 copurifies with mature virions and is expressed in HPCs upon virus entry although its expression at the time of infection does not complement the US28 deletion latency phenotype. This work suggests that US28 protein functions to promote a latent state within hematopoietic progenitor cells.

IMPORTANCEHuman cytomegalovirus (HCMV) is a widespread pathogen that, once acquired, remains with its host for life. HCMV remains latent, or quiescent, in cells of the hematopoietic compartment and upon immune challenge can reactivate to cause disease. HCMV-encoded US28 is one of several genes expressed during latency although its biological function during this phase of infection has remained undefined. Here, we show that US28 aids in promoting experimental latency in tissue culture.
Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins
Authors: Valdivieso-Torres Leonardo , Sarangi Anindita , Whidby Jillian , Marcotrigiano Joseph , Roth Monica J.
Published: December 2015

Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways.

IMPORTANCE
Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines within the randomized region have the highest transduction efficiencies. This emphasizes the importance of considering cysteine pairs in the design of new libraries. Furthermore, our data clearly indicate that these cysteines are essential for viral infectivity by presenting essential residues to the host cell receptor. These studies facilitate the screening of Env libraries for functional entry into target cells, allowing the identification of novel gammaretroviral Envs targeting alternative host cell receptors for gene and protein delivery.
Isolation and Characterization of a Novel Bat Coronavirus Closely Related to the Direct Progenitor of Severe Acute Respiratory Syndrome Coronavirus
Authors: Yang Xing-Lou , Hu Ben , Wang Bo , Wang Mei-Niang , Zhang Qian , Zhang Wei , Wu Li-Jun , Ge Xing-Yi , Zhang Yun-Zhi , Daszak Peter , Wang Lin-Fa , Shi Zheng-Li
Published: December 2015

We report the isolation and characterization of a novel bat coronavirus which is much closer to the severe acute respiratory syndrome coronavirus (SARS-CoV) in genomic sequence than others previously reported, particularly in its S gene. Cell entry and susceptibility studies indicated that this virus can use ACE2 as a receptor and infect animal and human cell lines. Our results provide further evidence of the bat origin of the SARS-CoV and highlight the likelihood of future bat coronavirus emergence in humans.
Defining the Microglia Response during the Time Course of Chronic Neurodegeneration
Authors: Vincenti James E. , Murphy Lita , Grabert Kathleen , McColl Barry W. , Cancellotti Enrico , Freeman Tom C. , Manson Jean C.
Published: December 2015

Inflammation has been proposed as a major component of neurodegenerative diseases, although the precise role it plays has yet to be defined. We examined the role of key contributors to this inflammatory process, microglia, the major resident immune cell population of the brain, in a prion disease model of chronic neurodegeneration. Initially, we performed an extensive reanalysis of a large study of prion disease, where the transcriptome of mouse brains had been monitored throughout the time course of disease. Our analysis has provided a detailed classification of the disease-associated genes based on cell type of origin and gene function. This revealed that the genes upregulated during disease, regardless of the strain of mouse or prion protein, are expressed predominantly by activated microglia. In order to study the microglia contribution more specifically, we established a mouse model of prion disease in which the 79A murine prion strain was introduced by an intraperitoneal route into BALB/cJFms-EGFP/−mice, which express enhanced green fluorescent protein under the control of thec-fmsoperon. Samples were taken at time points during disease progression, and histological analysis of the brain and transcriptional analysis of isolated microglia was carried out. The analysis of isolated microglia revealed a disease-specific, highly proinflammatory signature in addition to an upregulation of genes associated with metabolism and respiratory stress. This study strongly supports the growing recognition of the importance of microglia within the prion disease process and identifies the nature of the response through gene expression analysis of isolated microglia.

IMPORTANCEInflammation has been proposed as a major component of neurodegenerative diseases. We have examined the role of key contributors to this inflammatory process, microglia, the major resident immune cell population of the brain, in a murine prion disease model of chronic neurodegeneration. Our study demonstrates that genes upregulated throughout the disease process are expressed predominantly by microglia. A disease-specific, highly proinflammatory signature was observed in addition to an upregulation of genes associated with metabolism and respiratory stress. This study strongly supports the growing recognition of the important contribution of microglia to a chronic neurodegenerative disease process.
Membrane Perturbation-Associated Ca
2+
Signaling and Incoming Genome Sensing Are Required for the Host Response to Low-Level Enveloped Virus Particle Entry
Authors: Hare David N. , Collins Susan E. , Mukherjee Subhendu , Loo Yueh-Ming , Gale Michael , Janssen Luke J. , Mossman Karen L.
Published: December 2015

The type I interferon (IFN) response is an important aspect of innate antiviral defense, and the transcription factor IRF3 plays an important role in its induction. Membrane perturbation during fusion, a necessary step for enveloped virus particle entry, appears sufficient to induce transcription of a subset of IFN-stimulated genes (ISGs) in an IRF3-dependent, IFN-independent fashion. IRF3 is emerging as a central node in host cell stress responses, although it remains unclear how different forms of stress activate IRF3. Here, we investigated the minimum number of Sendai virus (SeV) and human cytomegalovirus (HCMV) particles required to activate IRF3 and trigger an antiviral response. We found that Ca
2+
signaling associated with membrane perturbation and recognition of incoming viral genomes by cytosolic nucleic acid receptors are required to activate IRF3 in response to fewer than 13 particles of SeV and 84 particles of HCMV per cell. Moreover, it appears that Ca
2+
signaling is important for activation of STING and IRF3 following HCMV particle entry, suggesting that Ca
2+
signaling sensitizes cells to recognize genomes within incoming virus particles. To our knowledge, this is the first evidence that cytosolic nucleic acid sensors recognize genomes within incoming virus particles prior to virus replication. These studies highlight the exquisite sensitivity of the cellular response to low-level stimuli and suggest that virus particle entry is sensed as a stress signal.

IMPORTANCE
The mechanism by which replicating viruses trigger IRF3 activation and type I IFN induction through the generation and accumulation of viral pathogen-associated molecular patterns has been well characterized. However, the mechanism by which enveloped virus particle entry mediates a stress response, leading to IRF3 activation and the IFN-independent response, remained elusive. Here, we find that Ca
2+
signaling associated with membrane perturbation appears to sensitize cells to recognize genomes within incoming virus particles. To our knowledge, this is the first study to show that cytosolic receptors recognize genomes within incoming virus particles prior to virus replication. These findings not only highlight the sensitivity of cellular responses to low-level virus particle stimulation, but provide important insights into how nonreplicating virus vectors or synthetic lipid-based carriers used as clinical delivery vehicles activate innate immune responses.
Structure-Guided Redesign Increases the Propensity of HIV Env To Generate Highly Stable Soluble Trimers
Authors: Guenaga Javier , Dubrovskaya Viktoriya , de Val Natalia , Sharma Shailendra K. , Carrette Barbara , Ward Andrew B. , Wyatt Richard T.
Published: December 2015

Due to high viral diversity, an effective HIV-1 vaccine will likely require Envs derived from multiple subtypes to generate broadly neutralizing antibodies (bNAbs). Soluble Env mimics, like the native flexibly linked (NFL) and SOSIP trimers, derived from the subtype A BG505 Env, form homogeneous, stable native-like trimers. However, other Env sequences, such as JRFL and 16055 from subtypes B and C, do so to a lesser degree. The high-resolution BG505 SOSIP crystal structures permit the identification and redesign of Env elements involved in trimer stability. Here, we identified structure trimer-derived (TD) residues that increased the propensity of the subtype B JRFL and subtype C 16055 Env sequences to form well-ordered, homogenous, and highly stable soluble trimers. The generation of these spike mimics no longer required antibody-based selection, positive or negative. Using the redesigned subtype B and C trimer representatives as respective foundations, we further stabilized the NFL TD trimers by engineering an intraprotomer disulfide linkage in the prebridging sheet, I201C-A433C (CC), that locks the gp120 in the receptor nontriggered state. We demonstrated that this disulfide pair prevented CD4 induced-conformational rearrangements in NFL trimers derived from the prototypic subtype A, B, and C representatives. Coupling the TD-based design with the engineered disulfide linkage, CC, increased the propensity of Env to form soluble highly stable spike mimics that are resistant to CD4-induced changes. These advances will allow testing of the hypothesis that such stabilized immunogens will more efficiently elicit neutralizing antibodies in small-animal models and primates.

IMPORTANCE
HIV-1 displays unprecedented global diversity circulating in the human population. Since the envelope glycoprotein (Env) is the target of neutralizing antibodies, Env-based vaccine candidates that address such diversity are needed. Soluble well-ordered Env mimics, typified by NFL and SOSIP trimers, are attractive vaccine candidates. However, the current designs do not allow most Envs to form well-ordered trimers. Here, we made design modifications to increase the propensity of representatives from two of the major HIV subtypes to form highly stable trimers. This approach should be applicable to other viral Envs, permitting the generation of a repertoire of homogeneous, highly stable trimers. The availability of such an array will allow us to assess if sequential or cocktail immune strategies can overcome some of the vaccine challenges presented by HIV diversity.
Tumor Suppressor Interferon-Regulatory Factor 1 Counteracts the Germinal Center Reaction Driven by a Cancer-Associated Gammaherpesvirus
Authors: Mboko Wadzanai P. , Olteanu Horatiu , Ray Avijit , Xin Gang , Darrah Eric J. , Kumar Suresh N. , Kulinski Joseph M. , Cui Weiguo , Dittel Bonnie N. , Gauld Stephen B. , Tarakanova Vera L.
Published: December 2015

Gammaherpesviruses are ubiquitous pathogens that are associated with the development of B cell lymphomas. Gammaherpesviruses employ multiple mechanisms to transiently stimulate a broad, polyclonal germinal center reaction, an inherently mutagenic stage of B cell differentiation that is thought to be the primary target of malignant transformation in virus-driven lymphomagenesis. We found that this gammaherpesvirus-driven germinal center expansion was exaggerated and lost its transient nature in the absence of interferon-regulatory factor 1 (IRF-1), a transcription factor with antiviral and tumor suppressor functions. Uncontrolled and persistent expansion of germinal center B cells led to pathological changes in the spleens of chronically infected IRF-1-deficient animals. Additionally, we found decreased IRF-1 expression in cases of human posttransplant lymphoproliferative disorder, a malignant condition associated with gammaherpesvirus infection. The results of our study define an unappreciated role for IRF-1 in B cell biology and provide insight into the potential mechanism of gammaherpesvirus-driven lymphomagenesis.

IMPORTANCE
Gammaherpesviruses establish lifelong infection in most adults and are associated with B cell lymphomas. While the infection is asymptomatic in many hosts, it is critical to identify individuals who may be at an increased risk of virus-induced cancer. Such identification is currently impossible, as the host risk factors that predispose individuals toward viral lymphomagenesis are poorly understood. The current study identifies interferon-regulatory factor 1 (IRF-1) to be one of such candidate host factors. Specifically, we found that IRF-1 enforces long-term suppression of an inherently mutagenic stage of B cell differentiation that gammaherpesviruses are thought to target for transformation. Correspondingly, in the absence of IRF-1, chronic gammaherpesvirus infection induced pathological changes in the spleens of infected animals. Further, we found decreased IRF-1 expression in human gammaherpesvirus-induced B cell malignancies.
Cucumber Necrosis Virus Recruits Cellular Heat Shock Protein 70 Homologs at Several Stages of Infection
Authors: Alam Syed Benazir , Rochon D'Ann
Published: December 2015

RNA viruses often depend on host factors for multiplication inside cells due to the constraints of their small genome size and limited coding capacity. One such factor that has been exploited by several plant and animal viruses is heat shock protein 70 (HSP70) family homologs which have been shown to play roles for different viruses in viral RNA replication, viral assembly, disassembly, and cell-to-cell movement. Using next generation sequence analysis, we reveal that several isoforms of Hsp70 and Hsc70 transcripts are induced to very high levels during cucumber necrosis virus (CNV) infection of
Nicotiana benthamiana
and that HSP70 proteins are also induced by at least 10-fold. We show that HSP70 family protein homologs are co-opted by CNV at several stages of infection. We have found that overexpression of Hsp70 or Hsc70 leads to enhanced CNV genomic RNA, coat protein (CP), and virion accumulation, whereas downregulation leads to a corresponding decrease. Hsc70-2 was found to increase solubility of CNV CP
in vitro
and to increase accumulation of CNV CP independently of viral RNA replication during coagroinfiltration in
N. benthamiana
. In addition, virus particle assembly into virus-like particles in CP agroinfiltrated plants was increased in the presence of Hsc70-2. HSP70 was found to increase the targeting of CNV CP to chloroplasts during infection, reinforcing the role of HSP70 in chloroplast targeting of host proteins. Hence, our findings have led to the discovery of a highly induced host factor that has been co-opted to play multiple roles during several stages of the CNV infection cycle.

IMPORTANCE
Because of the small size of its RNA genome, CNV is dependent on interaction with host cellular components to successfully complete its multiplication cycle. We have found that CNV induces HSP70 family homologs to a high level during infection, possibly as a result of the host response to the high levels of CNV proteins that accumulate during infection. Moreover, we have found that CNV co-opts HSP70 family homologs to facilitate several aspects of the infection process such as viral RNA, coat protein and virus accumulation. Chloroplast targeting of the CNV CP is also facilitated, which may aid in CNV suppression of host defense responses. Several viruses have been shown to induce HSP70 during infection and others to utilize HSP70 for specific aspects of infection such as replication, assembly, and disassembly. We speculate that HSP70 may play multiple roles in the infection processes of many viruses.
Naturally Occurring Major and Minor Capsid Protein Variants of Human Papillomavirus 45 (HPV45): Differential Recognition by Cross-Neutralizing Antibodies Generated by HPV Vaccines
Authors: Godi Anna , Facchetti Alessandra , Bissett Sara L. , Cocuzza Clementina , Miller Elizabeth , Beddows Simon
Published: December 2015

We investigated naturally occurring variation within the major (L1) and minor (L2) capsid proteins of human papillomavirus genotype 45 (HPV45). Pseudoviruses (PsVs) representing HPV45 sublineages A1, A2, A3, B1, and B2 exhibited comparable particle-to-infectivity ratios and morphologies but demonstrated both increased (A2, A3, and B1) and decreased (B2) sensitivities to cross-neutralization by HPV vaccine antibodies compared to that of the A1 sublineage. Mutant PsVs identified HI loop residue 357 as being critical for conferring this differential sensitivity.
Nef Proteins from HIV-1 Elite Controllers Are Inefficient at Preventing Antibody-Dependent Cellular Cytotoxicity
Authors: Alsahafi Nirmin , Ding Shilei , Richard Jonathan , Markle Tristan , Brassard Nathalie , Walker Bruce , Lewis George K. , Kaufmann Daniel E. , Brockman Mark A. , Finzi Andrés
Published: December 2015

Impairment of Nef function, including reduced CD4 downregulation, was described in a subset of HIV-1-infected individuals that control viral replication without antiretroviral treatment (elite controllers [EC]). Elimination of HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) requires the presence of envelope glycoproteins (Env) in the CD4-bound conformation, raising the possibility that accumulating CD4 at the surface of virus-infected cells in EC could interact with Env and thereby sensitize these cells to ADCC. We observed a significant increase in the exposure of Env epitopes targeted by ADCC-mediating antibodies at the surface of cells expressing Nef isolates from EC; this correlated with enhanced susceptibility to ADCC. Altogether, our results suggest that enhanced susceptibility of HIV-1-infected cells to ADCC may contribute to the EC phenotype.

IMPORTANCENef clones derived from elite controllers (EC) have been shown to be attenuated for CD4 downregulation; how this contributes to the nonprogressor phenotype of these infected individuals remains uncertain. Increasing evidence supports a role for HIV-specific antibody-dependent cellular cytotoxicity (ADCC) in controlling viral infection and replication. Here, we show that residual CD4 left at the surface of cells expressing Nef proteins isolated from ECs are sufficient to allow Env-CD4 interaction, leading to increased exposure of Env CD4-induced epitopes and increased susceptibility of infected cells to ADCC. Our results suggest that ADCC might be an active immune mechanism in EC that helps to maintain durable suppression of viral replication and low plasma viremia level in this rare subset of infected individuals. Therefore, targeting Nef's ability to downregulate CD4 could render HIV-1-infected cells susceptible to ADCC and thus have therapeutic utility.
Network Analysis of the Chronic Hepatitis C Virome Defines Hypervariable Region 1 Evolutionary Phenotypes in the Context of Humoral Immune Responses
Authors: Palmer Brendan A. , Schmidt-Martin Daniel , Dimitrova Zoya , Skums Pavel , Crosbie Orla , Kenny-Walsh Elizabeth , Fanning Liam J.
Published: December 2015

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) comprises the first 27 N-terminal amino acid residues of E2. It is classically seen as the most heterogeneous region of the HCV genome. In this study, we assessed HVR1 evolution by using ultradeep pyrosequencing for a cohort of treatment-naive, chronically infected patients over a short, 16-week period. Organization of the sequence set into connected components that represented single nucleotide substitution events revealed a network dominated by highly connected, centrally positioned master sequences. HVR1 phenotypes were observed to be under strong purifying (stationary) and strong positive (antigenic drift) selection pressures, which were coincident with advancing patient age and cirrhosis of the liver. It followed that stationary viromes were dominated by a single HVR1 variant surrounded by minor variants comprised from conservative single amino acid substitution events. We present evidence to suggest that neutralization antibody efficacy was diminished for stationary-virome HVR1 variants. Our results identify the HVR1 network structure during chronic infection as the preferential dominance of a single variant within a narrow sequence space.

IMPORTANCE
HCV infection is often asymptomatic, and chronic infection is generally well established in advance of initial diagnosis and subsequent treatment. HVR1 can undergo rapid sequence evolution during acute infection, and the variant pool is typically seen to diverge away from ancestral sequences as infection progresses from the acute to the chronic phase. In this report, we describe HVR1 viromes in chronically infected patients that are defined by a dominant epitope located centrally within a narrow variant pool. Our findings suggest that weakened humoral immune activity, as a consequence of persistent chronic infection, allows for the acquisition and maintenance of host-specific adaptive mutations at HVR1 that reflect virus fitness.