bluuebunny/crossref_metadata_2025 huggingface dataset:
A Macro-Micro Model of Fusion Zone Microstructure Evolution in Mn-C Low-Alloy Steel Coupled With Thermal Stress Analysis
Authors: Kazemi Komeil , Artemev Andrei , Zhou Jianguo , Goldak John A.
Published: November 2015

A macro-micro-model for microstructure evolution in the fusion zone of a l.2 Mn and 0.11 C low-alloy steel is described. The macro-model is a 3D transient thermal analysis of a welded structure that resolves the weld pool with element size greater than 1 mm and time steps greater than 1 second. The micro-model has cell size of about 1 micron and time step size of about 10 micro-seconds with a grid of about 80×80×500 cells. The micro model is positioned on the liquid-solid interface of the weld pool in the macro-model. The boundary conditions for the micro-model are mapped from the macro-model. The micro-model solves the 3D transient solute diffusion equations for Mn and C. The micro-model computes the liquid-solid interface movement with local velocities determined by local temperature, compositions of solid and liquid phases and interface curvature to predict columnar or dendritic solidification structures. As the solid cools from the melting point to room temperature, the evolution of austenite, ferrite, pearlite, bainite and martensite phases are computed. The 3D transient stress due to temperature and phase changes is computed in the micro-model as it cools from the melting temperature to room temperature. At room temperature a micro-model tensile test is run to 4% strain. The macro-stress and strain is compared to the micro-stress and strain distributions. The model is intended to be used to initialize models of fracture, fatigue and creep in weld fusion zones.
Development of Laser Engineered Net Shape (LENS) Additive Manufacturing for Repair
Authors: Korinko Paul S.
Published: November 2015

Laser Engineered Net Shape processing was evaluated as a means to repair scratched gas sample bottles and mis-machined test bases. Defects to be repaired were intentionally introduced. These defects simulated scratches and holes that had been either over bored or misaligned. These parts were repaired using LENS and then tested in the baseline, as damaged, and hydrogen charged conditions. The effect of LENS repair was not detrimental to the properties of the components. Further development work and implementation of the process is recommended.
ORNL Special Form Testing of Sealed-Source Encapsulations
Authors: Martinez Oscar , Blessinger Christopher
Published: November 2015

In the United States of America all transportation of radioactive material is regulated by the Department of Transportation (DOT), along with input from the Nuclear Regulatory Commission. Beginning in 2008 a new type of sealed-source encapsulation package was developed at Oak Ridge National Laboratory (ORNL); these packages contain radioactive material and are regulated and transported in accordance with the requirements set for DOT Class 7 hazardous material. DOT provides regulations pertaining to specific package contents categorized as special form designs. The special form designation indicates that the encapsulated radioactive contents have a very low probability of dispersion even when subjected to significant structural conditions. All ORNL DOT designs have been certified by DOT as being special form materials. The special form designs have been shown to simplify the delivery, transport, acceptance, and receipt process. Simplification of the transportation process makes the sealed-source encapsulation designs very advantageous for shipment to various facilities throughout the lifetime of the special form material. To this end, DOT Certificates of Competent Authority (CoCAs) have been sought for the design suitable for containing high-alpha-activity actinide materials. This design consists of a core of porous zirconia matrix pre-encapsulated within triangular canister (ZipCan) tiles that are then enclosed by a spherical shell. This new ZipCan design and a similar rectangular ZipCube design were tested for compliance with the regulations found in Title 49, Code of Federal Regulations, Section 173.469, Tests for Special Form Class 7 (Radioactive) (49 CFR 173.469) materials. The spherical enclosure was subjected to 9 m impact, 1 m percussion, and 10-minute thermal tests. Before and after each test the designs were subjected to a helium leak check and a bubble test. The ZipCan tiles and core were subjected to the tests required for ISO 2919:1999(E), including a Class 4 impact test and heat test, and were subsequently subjected to helium leakage rate tests [49 CFR 173.469(a)(4)(i)]. The impact tile test unit contained a nonradioactive surrogate; however, the thermal test unit contained a radioactive source. All three designs are still undergoing regulatory special form testing, and all three sealed-source encapsulation designs are to be submitted to DOT for CoCAs.
Update on Stress Intensity Factor Influence Coefficients for Axial ID Surface Flaws in Cylinders for Appendix A of ASME Section XI
Authors: Xu Steven X. , Lee Darrell R. , Scarth Douglas A. , Cipolla Russell C.
Published: November 2015

Article A-3000 of Appendix A in Section XI of the ASME Boiler and Pressure Vessel Code provides linear elastic fracture mechanics based calculation procedures for the determination of stress intensity factors. The 2015 Edition of ASME Section XI implements a number of significant improvements in Article A-3000. Major improvements include the implementation of an alternate method for calculation of the stress intensity factor for a surface flaw that makes explicit use of the Universal Weight Function Method and does not require a polynomial fit to the actual stress distribution, and the inclusion of closed-form equations for stress intensity factor influence coefficients for circumferential ID surface cracks. With the inclusion of the explicit weight function approach and the closed-form relations for influence coefficients, the procedures of Appendix A for the calculation of stress intensity factors can be used more efficiently.

Closed-form equations for stress intensity factor influence coefficients for axial ID surface cracks have been under development. Tabular data of influence coefficients for the cylinder geometry provided in API 579-1/ASME FFS-1 2007 are used as data source. A set of closed-formed equations for an axial semi-elliptical ID surface crack with depth a and length 2c in a cylinder were previously reported in a 2014 PVP paper. The smallest value for a/c is 0.03125 in the tabular data that were used to fit the equations. For practical applications, it is desirable to use axial flaw equations that allow a/c to approach zero without extrapolation. This issue is addressed in the current PVP paper.
Experimental observation of acoustic emissions generated by a pulsed proton beam from a hospital‐based clinical cyclotron
Authors: Jones Kevin C. , Vander Stappen François , Bawiec Christopher R. , Janssens Guillaume , Lewin Peter A. , Prieels Damien , Solberg Timothy D. , Sehgal Chandra M. , Avery Stephen
Published: November 2015

Purpose:

To measure the acoustic signal generated by a pulsed proton spill from a hospital‐based clinical cyclotron.

Methods:

An electronic function generator modulated the IBA C230 isochronous cyclotron to create a pulsed proton beam. The acoustic emissions generated by the proton beam were measured in water using a hydrophone. The acoustic measurements were repeated with increasing proton current and increasing distance between detector and beam.

Results:

The cyclotron generated proton spills with rise times of 18 μs and a maximum measured instantaneous proton current of 790 nA. Acoustic emissions generated by the proton energy deposition were measured to be on the order of mPa. The origin of the acoustic wave was identified as the proton beam based on the correlation between acoustic emission arrival time and distance between the hydrophone and proton beam. The acoustic frequency spectrum peaked at 10 kHz, and the acoustic pressure amplitude increased monotonically with increasing proton current.

Conclusions:

The authors report the first observation of acoustic emissions generated by a proton beam from a hospital‐based clinical cyclotron. When modulated by an electronic function generator, the cyclotron is capable of creating proton spills with fast rise times (18 μs) and high instantaneous currents (790 nA). Measurements of the proton‐generated acoustic emissions in a clinical setting may provide a method for in vivo proton range verification and patient monitoring.
Enhancing the Ability of Students to Engage With Theoretical Concepts Through the Creation of Learner-Generated Video Assessment
Authors: Walters Simon R. , Hallas Julia , Phelps Sean , Ikeda Erika
Published: November 2015

Even though technology has become a key driver in preparing sports management students for an increasingly globalized industry, it is unclear whether the affordances of these technologies contribute to the transformation of the learning environment. The purpose of this study was to investigate how a learner-generated video assessment develops students’ critical thinking and engagement with the theoretical concepts taught in an undergraduate second-year Sociology of Sport course. Data were gathered using a qualitative case study approach. Students found the video assessment enjoyable; it promoted critical thinking and engagement with theory. However, students were less interested in technology-based assessment than the need for courses to align learning strategies and assessment methods to the graduate capabilities required to transform their discipline in the workplace. We argue that it is this alignment that will lead to a transformation in the learning environment and quality student engagement, rather than the video technology itself.
Flipping a Class: Active Learning and More of It
Authors: Lumpkin Angela , Achen Rebecca M.
Published: November 2015

Despite what many claim, just because there is teaching does not mean there is learning. Clear and convincing evidence supports changing the instructional paradigm to a learner-centered classroom. Flipping a class shifts the delivery, often through technologically presented lectures, to free class time for student participation in a plethora of learning activities, such as think-pair-share and discussions, leading to student perceptions of greater learning and more enjoyment. In an action research approach with one class, 72% of juniors and seniors in an undergraduate sport finance and economics class reported out-of-class lectures often positively impacted their learning, and the remaining 28% responded these lectures did sometimes. End-of-course evaluations and surveys were overwhelmingly positive about class engagement, interaction, and enjoyment.
NIH program fails to launch blacks in biotech
Authors: Mervis Jeffrey
Published: November 2015

Small Business Innovation Research program makes almost no grants to African-Americans.
What does a disease deserve?
Authors: Kaiser Jocelyn
Published: November 2015

A move to end NIH's AIDS earmark fuels debate over whether funding should reflect disease burden.
Bidirectional Notch signaling regulates
Drosophila
intestinal stem cell multipotency
Authors: Guo Zheng , Ohlstein Benjamin
Published: November 2015

Notch role in multipotency or cell fate

Multipotent
Drosophila
intestinal stem cells (ISCs) generate either nutrient-absorbing enterocytes (ECs) or secretory enteroendocrine cells (EECs). Guo and Ohlstein investigated the role of Notch signaling in this process. They tracked ISC asymmetric divisions and found that EEC daughter cells, which have a low level of Notch, signal back to the ISC in order to keep it multipotent. However, during EC production, ISCs activate strong Notch signaling in daughters. Thus, Notch signaling functions in two directions to achieve stem cell multipotency.

Science
, this issue p.
10.1126/science.aab0988
Helminth infection, fecundity, and age of first pregnancy in women
Authors: Blackwell Aaron D. , Tamayo Marilyne A. , Beheim Bret , Trumble Benjamin C. , Stieglitz Jonathan , Hooper Paul L. , Martin Melanie , Kaplan Hillard , Gurven Michael
Published: November 2015

Parasitic worms influence human fecundity

Parasitic worms infect 2 billion people globally. Mostly, such infections are symptomless and individual worm burdens are low. Blackwell
et al.
monitored the fecundity of Tsimane women in Bolivia. These women have on average of 10 children in their lifetimes. However, if they had successive hookworm infections, lifetime births dropped to 7. Surprisingly, if the women were chronically infested with roundworm, they had as many as 12 children. These effects may relate to the balance of immune responses that the different worms induce, rather than to the physiological costs of parasitism.

Science
, this issue p.
970
Stable recombination hotspots in birds
Authors: Singhal Sonal , Leffler Ellen M. , Sannareddy Keerthi , Turner Isaac , Venn Oliver , Hooper Daniel M. , Strand Alva I. , Li Qiye , Raney Brian , Balakrishnan Christopher N. , Griffith Simon C. , McVean Gil , Przeworski Molly
Published: November 2015

Recombination: The birds and the yeast

Apes and mice have a specific gene,
PRDM9
, that is associated with genomic regions with high rates of recombination, called hotspots. In species with
PRDM9
, hotspots move rapidly within the genome, varying among populations and closely related species (see the Perspective by Lichten). To investigate recombination hotspots in species lacking
PRDM9
, Singhal
et al.
examined bird genomes, which lack a
PRDM9
gene. They looked closely at the genomes of finch species and found that recombination was localized to the promoter regions of genes and highly conserved over millions of years. Similarly, Lam and Keeney examined recombination localization within yeast, which also lacks
PRDM9
. They found a similar more-or-less fixed pattern of hotspots. Thus, recombination in species lacking a
PRDM9
gene shows similar patterns of hotspot localization and evolution.

Science
, this issue p.
913
, p.
928
; see also p.
932
Multifunctionality of chiton biomineralized armor with an integrated visual system
Authors: Li Ling , Connors Matthew J. , Kolle Mathias , England Grant T. , Speiser Daniel I. , Xiao Xianghui , Aizenberg Joanna , Ortiz Christine
Published: November 2015

A set of strong eyes

Although many biological tissues serve more than one purpose, rarely are they optimized to do multiple tasks well. When you try to optimize for one functionality, it comes at the expense of another. Li
et al.
investigated the biomineralized armor of the small mollusc chiton
Acanthopleura granulata
. The armor appears to be optimized for both mechanical strength and for image capture by hundreds of integral aragonite-based lenses.

Science
, this issue p.
952
The picture of health
Things to Do in a Retirement Home Trailer Park … When You're 29 and Unemployed
Aneurin Wright
Pennsylvania State University Press, 2015. 320 pp.
My Degeneration: A Journey Through Parkinson's
Peter Dunlap-Shohl
Pennsylvania State University Press, 2015. 106 pp.
Authors: Frank Arthur W.
Published: November 2015

A new series of graphic novels features intimate portraits of illness
100 years of continental drift
Authors: Romano Marco , Cifelli Richard L.
Published: November 2015

One hundred years ago, Alfred Wegener laid the foundations for the theory of plate tectonics
Characterization of Mucosal Immune Responses to Enterotoxigenic Escherichia coli Vaccine Antigens in a Human Challenge Model: Response Profiles after Primary Infection and Homologous Rechallenge with Strain H10407
Authors: Chakraborty Subhra , Harro Clayton , DeNearing Barbara , Ram Malathi , Feller Andrea , Cage Alicia , Bauers Nicole , Bourgeois A. Louis , Walker Richard , Sack David A.
Published: November 2015

Enterotoxigenic
Escherichia coli
(ETEC) bacteria are the most common bacterial cause of diarrhea in children in resource-poor settings as well as in travelers. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. A significant challenge to successful vaccine development is our poor understanding of the immune responses that correlate best with protection against ETEC illness. In this study, ETEC-specific mucosal immune responses were characterized and compared in subjects challenged with ETEC strain H10407 and in subjects rechallenged with the homologous organism. IgA responses to lipopolysaccharide (LPS), heat-labile toxin B subunit (LTB), and colonization factor antigen I (CFA/I) in antibody in lymphocyte supernatant (ALS), feces, lavage fluid, and saliva samples were evaluated. In all assay comparisons, ALS was the most sensitive indicator of a local immune response, but serum IgA was also a useful indirect marker of immune response to oral antigens. Volunteers challenged and then rechallenged with strain H10407 were protected from illness following rechallenge. Comparing mucosal antibody responses after primary and homologous rechallenge, protection against disease was reflected in reduced antibody responses to key ETEC antigens and in reduced fecal shedding of the H10407 challenge strain. Subjects challenged with strain H10407 mounted stronger antibody responses to LPS and LTB than subjects in the rechallenge group, while responses to CFA/I in the rechallenge group were higher than in the challenge group. We anticipate that this study will help provide an immunological benchmark for the evaluation of ETEC vaccines and immunization regimens in the future.
Changes in the Population of Methicillin-Resistant Staphylococcus pseudintermedius and Dissemination of Antimicrobial-Resistant Phenotypes in the Netherlands
Authors: Duim Birgitta , Verstappen Koen M. , Broens Els M. , Laarhoven Laura M. , van Duijkeren Engeline , Hordijk Joost , de Heus Phebe , Spaninks Mirlin , Timmerman Arjen J. , Wagenaar Jaap A.
Published: November 2015

Methicillin-resistant
Staphylococcus pseudintermedius
(MRSP), which is often multidrug resistant (MDR), has recently emerged as a threat to canine health worldwide. Knowledge of the temporal distribution of specific MRSP lineages, their antimicrobial resistance phenotypes, and their association with clinical conditions may help us to understand the emergence and spread of MRSP in dogs. The aim of this study was to determine the yearly proportions of MRSP lineages and their antimicrobial-resistant phenotypes in the Netherlands and to examine possible associations with clinical conditions. MRSP was first isolated from a canine specimen submitted for diagnostics to the Faculty of Veterinary Medicine of Utrecht University in 2004. The annual cumulative incidence of MRSP among
S. pseudintermedius
increased from 0.9% in 2004 to 7% in 2013. MRSP was significantly associated with pyoderma and, to a lesser extent, with wound infections and otitis externa. Multilocus sequence typing (MLST) of 478 MRSP isolates yielded 39 sequence types (ST) belonging to 4 clonal complexes (CC) and 15 singletons. CC71 was the dominant lineage that emerged since 2004, and CC258, CC45, and several unlinked isolates became more frequent during the following years. All but two strains conferred an MDR phenotype, but strains belonging to CC258 or singletons were less resistant. In conclusion, our study showed that MDR CC71 emerged as the dominant lineage from 2004 and onward and that less-resistant lineages were partly replacing CC71.
Usefulness of High-Quality Core Genome Single-Nucleotide Variant Analysis for Subtyping the Highly Clonal and the Most Prevalent Salmonella enterica Serovar Heidelberg Clone in the Context of Outbreak Investigations
Authors: Bekal S. , Berry C. , Reimer A. R. , Van Domselaar G. , Beaudry G. , Fournier E. , Doualla-Bell F. , Levac E. , Gaulin C. , Ramsay D. , Huot C. , Walker M. , Sieffert C. , Tremblay C.
Published: November 2015

Salmonella enterica
serovar Heidelberg is the second most frequently occurring serovar in Quebec and the third-most prevalent in Canada. Given that conventional pulsed-field gel electrophoresis (PFGE) subtyping for common
Salmonella
serovars, such as
S
. Heidelberg, yields identical subtypes for the majority of isolates recovered, public health laboratories are desperate for new subtyping tools to resolve highly clonal
S
. Heidelberg strains involved in outbreak events. As PFGE was unable to discriminate isolates from three epidemiologically distinct outbreaks in Quebec, this study was conducted to evaluate whole-genome sequencing (WGS) and phylogenetic analysis as an alternative to conventional subtyping tools. Genomes of 46 isolates from 3 Quebec outbreaks (2012, 2013, and 2014) supported by strong epidemiological evidence were sequenced and analyzed using a high-quality core genome single-nucleotide variant (hqSNV) bioinformatics approach (SNV phylogenomics [SNVphyl] pipeline). Outbreaks were indistinguishable by conventional PFGE subtyping, exhibiting the same PFGE pattern (SHEXAI.0001/SHEBNI.0001). Phylogenetic analysis based on hqSNVs extracted from WGS separated the outbreak isolates into three distinct groups, 100% concordant with the epidemiological data. The minimum and maximum number of hqSNVs between isolates from the same outbreak was 0 and 4, respectively, while >59 hqSNVs were measured between 2 previously indistinguishable outbreaks having the same PFGE and phage type, thus corroborating their distinction as separate unrelated outbreaks. This study demonstrates that despite the previously reported high clonality of this serovar, the WGS-based hqSNV approach is a superior typing method, capable of resolving events that were previously indistinguishable using classic subtyping tools.
Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452Antigen for Detecting Antibodies against Aleutian Mink Disease Virus
Authors: Chen Xiaowei , Song Cailing , Liu Yun , Qu Liandong , Liu Dafei , Zhang Yun , Liu Ming
Published: November 2015

For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452ELISA may be a preferable method for detecting antibodies against AMDV.
Ancestral Mutations Acquired in Refrex-1, a Restriction Factor against Feline Retroviruses, during its Cooption and Domestication
Authors: Ito Jumpei , Baba Takuya , Kawasaki Junna , Nishigaki Kazuo
Published: November 2015

Endogenous retroviruses (ERVs) are remnants of ancestral retroviral infections of germ cells. Retroviral endogenization is an adaptation process for the host genome, and ERVs are gradually attenuated or inactivated by mutation. However, some ERVs that have been “domesticated” by their hosts eventually gain physiological functions, such as placentation or viral resistance. We previously reported the discovery of Refrex-1, a soluble antiretroviral factor in domestic cats that specifically inhibits infection by feline leukemia virus subgroup D (FeLV-D), a chimeric virus of FeLV, and a feline ERV, ERV-DC. Refrex-1 is a truncated envelope protein (Env) encoded by both ERV-DC7 and ERV-DC16 proviral loci. Here, we reconstituted ancestral and functional Env from ERV-DC7 and ERV-DC16 envelope genes (
env
) by inducing reverse mutations. Unexpectedly, ERV-DC7 and ERV-DC16 full-length Env (ERV-DC7 fl and ERV-DC16 fl), reconstructed by removing stop codons, did not produce infectious viral particles. ERV-DC7 fl and ERV-DC16 fl were highly expressed in cells but were not cleaved into surface subunits (SU) and transmembrane subunits, nor were they incorporated into virions. G407R/N427I-A429T and Y431D substitutions within the SU C-terminal domain of ERV-DC7 fl and ERV-DC16 fl, respectively, caused these dysfunctions. The residues glycine 407 and tyrosine 431 are relatively conserved among infectious gammaretroviruses, and their substitution causes the same dysfunctions as the tested retroviruses. Our results reveal that specific mutations within the SU C-terminal domain suppressed Env cleavage and incorporation into virions and indicate that these mutations contributed to the domestication of Refrex-1 through multistep events that occurred in the postintegration period.

IMPORTANCE
Domestic cats are colonized with various exogenous retroviruses (exRVs), such as feline leukemia virus (FeLV), and their genomes contain numerous ERVs, some of which are replication-competent proviruses. The feline hosts, exRVs, and ERVs have complicated genetic interactions and provide an interesting field model for triangular relationships: recombination between FeLV and ERV-DC, which is a feline ERV, generated FeLV-D, a chimeric virus, and FeLV-D is restricted by Refrex-1, an antiretroviral factor corresponding to truncated Env of ERV-DC7 and ERV-DC16. Here, we reconstructed ancestral, functional Env from ERV-DC7 and ERV-DC16
env
by inducing reverse mutations to elucidate how Refrex-1 was generated from its ancestor. Our results reveal that they were repeatedly inactivated by mutations preventing Env maturation. Our results provide insights into how ERVs were “domesticated” by their hosts and identify the mutations that mediated these evolutions. Notably, experiments that restore inactivated ERVs might uncover previously unrecognized features or properties of retroviruses.
A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection
Authors: Riblett Amber M. , Blomen Vincent A. , Jae Lucas T. , Altamura Louis A. , Doms Robert W. , Brummelkamp Thijn R. , Wojcechowskyj Jason A.
Published: November 2015

Rift Valley fever virus (RVFV) causes recurrent insect-borne epizootics throughout the African continent, and infection of humans can lead to a lethal hemorrhagic fever syndrome. Deep mutagenesis of haploid human cells was used to identify host factors required for RVFV infection. This screen identified a suite of enzymes involved in glycosaminoglycan (GAG) biogenesis and transport, including several components of the
cis
-oligomeric Golgi (COG) complex, one of the central components of Golgi complex trafficking. In addition, disruption of
PTAR1
led to RVFV resistance as well as reduced heparan sulfate surface levels, consistent with recent observations that PTAR1-deficient cells exhibit altered Golgi complex morphology and glycosylation defects. A variety of biochemical and genetic approaches were utilized to show that both pathogenic and attenuated RVFV strains require GAGs for efficient infection on some, but not all, cell types, with the block to infection being at the level of virion attachment. Examination of other members of the
Bunyaviridae
family for GAG-dependent infection suggested that the interaction with GAGs is not universal among bunyaviruses, indicating that these viruses, as well as RVFV on certain cell types, employ additional unidentified virion attachment factors and/or receptors.

IMPORTANCE
Rift Valley fever virus (RVFV) is an emerging pathogen that can cause severe disease in humans and animals. Epizootics among livestock populations lead to high mortality rates and can be economically devastating. Human epidemics of Rift Valley fever, often initiated by contact with infected animals, are characterized by a febrile disease that sometimes leads to encephalitis or hemorrhagic fever. The global burden of the pathogen is increasing because it has recently disseminated beyond Africa, which is of particular concern because the virus can be transmitted by widely distributed mosquito species. There are no FDA-licensed vaccines or antiviral agents with activity against RVFV, and details of its life cycle and interaction with host cells are not well characterized. We used the power of genetic screening in human cells and found that RVFV utilizes glycosaminoglycans to attach to host cells. This furthers our understanding of the virus and informs the development of antiviral therapeutics.
Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein
Authors: Tang Wen-Fang , Huang Ru-Ting , Chien Kun-Yi , Huang Jo-Yun , Lau Kean-Seng , Jheng Jia-Rong , Chiu Cheng-Hsun , Wu Tzong-Yuan , Chen Chung-Yung , Horng Jim-Tong
Published: November 2015

Enterovirus 71 (EV71), a member of
Picornaviridae
, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3′ untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication.

IMPORTANCE
Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.
Machupo Virus Expressing GPC of the Candid#1 Vaccine Strain of Junin Virus Is Highly Attenuated and Immunogenic
Authors: Koma Takaaki , Patterson Michael , Huang Cheng , Seregin Alexey V. , Maharaj Payal D. , Miller Milagros , Smith Jeanon N. , Walker Aida G. , Hallam Steven , Paessler Slobodan
Published: November 2015

Machupo virus (MACV) is the causative agent of Bolivian hemorrhagic fever. Our previous study demonstrated that a MACV strain with a single amino acid substitution (F438I) in the transmembrane domain of glycoprotein is attenuated but genetically unstable in mice. MACV is closely related to Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever. Others and our group have identified the glycoprotein to be the major viral factor determining JUNV attenuation. In this study, we tested the compatibility of the glycoprotein of the Candid#1 live-attenuated vaccine strain of JUNV in MACV replication and its ability to attenuate MACV
in vivo
. Recombinant MACV with the Candid#1 glycoprotein (rMACV/Cd#1-GPC) exhibited growth properties similar to those of Candid#1 and was genetically stable
in vitro
. In a mouse model of lethal infection, rMACV/Cd#1-GPC was fully attenuated, more immunogenic than Candid#1, and fully protective against MACV infection. Therefore, the MACV strain expressing the glycoprotein of Candid#1 is safe, genetically stable, and highly protective against MACV infection in a mouse model.

IMPORTANCE
Currently, there are no FDA-approved vaccines and/or treatments for Bolivian hemorrhagic fever, which is a fatal human disease caused by MACV. The development of antiviral strategies to combat viral hemorrhagic fevers, including Bolivian hemorrhagic fever, is one of the top priorities of the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. Here, we demonstrate for the first time that MACV expressing glycoprotein of Candid#1 is a safe, genetically stable, highly immunogenic, and protective vaccine candidate against Bolivian hemorrhagic fever.
A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner
Authors: Koehler Alexander , Kolesnikova Larissa , Welzel Ulla , Schudt Gordian , Herwig Astrid , Becker Stephan
Published: November 2015

Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the viral matrix protein VP40 at position 184 (VP40
D184N
). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARV
VP40(D184N)
] grew to higher titers than wild-type recombinant MARV (rMARV
WT
) in guinea pig cells. Moreover, rMARV
VP40(D184N)
displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other viral proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40
D184N
-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved viral fitness of rMARV
VP40(D184N)
in guinea pig cells was due to the better viral assembly function of VP40
D184N
and its lower inhibitory effect on viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling.

IMPORTANCE
The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the viral genome. Analyzing how these mutations affect the functions of viral proteins and the ability of the virus to grow within new host cells helps in the understanding of the molecular mechanisms increasing virulence. Using a reverse genetics approach, we demonstrated that a single mutation in MARV VP40 detected in a guinea pig-adapted MARV provided a replicative advantage of rMARV
VP40(D184N)
in guinea pig cells. Our studies show that this replicative advantage of rMARV VP40
D184N
was based on the improved functions of VP40 in iVLP assembly and in the regulation of transcription and replication rather than on the ability of VP40 to combat the host innate immunity.
Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting
BAD
-Mediated
caspase-3
-Dependent Apoptosis
Authors: Kim Hyoji , Choi Hoyun , Lee Suk Kyeong
Published: November 2015

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the
in vivo
triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes
BRLF1
and
BZLF1
as well as
Bcl-2
-associated death promoter (
BAD
) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of
BRLF1
and
BZLF1
were suppressed in cells following
BAD
knockdown and increased after
BAD
overexpression. Progeny virus production was also downregulated by specific knockdown of
BAD
. Our results demonstrated that
caspase-3
-dependent apoptosis is a prerequisite for
BAD
-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting
BAD
-mediated
caspase-3
-dependent apoptosis, which would trigger immediate early gene expression.

IMPORTANCE
EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing
BAD
-induced
caspase-3
-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis.
Oral Norovirus Infection Is Blocked in Mice Lacking Peyer's Patches and Mature M Cells
Authors: Kolawole Abimbola O. , Gonzalez-Hernandez Mariam B. , Turula Holly , Yu Chenchen , Elftman Michael D. , Wobus Christiane E.
Published: November 2015

A critical early step in murine norovirus (MNV) pathogenesis is crossing the intestinal epithelial barrier to reach the target cells for replication, i.e., macrophages, dendritic cells, and B cells. Our previous work showed that MNV replication decreases in the intestines of mice conditionally depleted of microfold (M) cells. To define the importance of Peyer's patch (PP) M cells during MNV pathogenesis, we used a model of BALB/c mice deficient in recombination-activating gene 2 (Rag2) and the common gamma chain (γc) (Rag-γc
−/−
), which lack gut-associated lymphoid tissues (GALT), such as Peyer's patches, and mature GP2
+
M cells. Rag-γc
−/−
mice were infected intraperitoneally or perorally with MNV-1 or CR3 for 24 or 72 h. Although the intestinal laminae propriae of Rag-γc
−/−
mice have a higher frequency of certain MNV target cells (dendritic cells and macrophages) than those of wild-type mice and lack others (B cells), Rag-γc
−/−
and wild-type BALB/c mice showed relatively similar viral loads in the intestine following infection by the intraperitoneal route, which provides direct access to target cells. However, Rag-γc
−/−
mice were not productively infected with MNV by the oral route, in which virions must cross the intestinal epithelial barrier. These data are consistent with a model whereby PP M cells are the primary route by which MNV crosses the intestinal epithelia of BALB/c mice.

IMPORTANCE
Noroviruses (NoVs) are prevalent pathogens that infect their hosts via the intestine. Identifying key factors during the initial stages of virus infection in the host may provide novel points of intervention. Microfold (M) cells, antigen-sampling cells in the intestine, were previously shown to provide a gateway for murine NoV (MNV) into the host, but the relative importance of this uptake pathway remained unknown. Here we show that the absence of gut-associated lymphoid tissues (GALT), such as Peyer's patches, which contain high numbers of mature M cells, renders BALB/c mice refractory to oral infection with MNV. These findings are consistent with the model that M cells represent the primary route by which MNV crosses the intestinal epithelial barrier and infects underlying immune cells during a productive infection.
Protocol for extended antibiotic therapy after laparoscopic cholecystectomy for acute calculous cholecystitis (Cholecystectomy Antibiotic Randomised Trial, CHART)
Authors: Pellegrini Pablo , Campana Juan Pablo , Dietrich Agustín , Goransky Jeremías , Glinka Juan , Giunta Diego , Barcan Laura , Alvarez Fernando , Mazza Oscar , Sánchez Claria Rodrigo , Palavecino Martin , Arbues Guillermo , Ardiles Victoria , de Santibañes Eduardo , Pekolj Juan , de Santibañes Martin
Published: November 2015

Introduction

Acute calculous cholecystitis represents one of the most common complications of cholelithiasis. While laparoscopic cholecystectomy is the standard treatment in mild and moderate forms, the need for antibiotic therapy after surgery remains undefined. The aim of the randomised controlled Cholecystectomy Antibiotic Randomised Trial (CHART) is therefore to assess if there are benefits in the use of postoperative antibiotics in patients with mild or moderate acute cholecystitis in whom a laparoscopic cholecystectomy is performed.

Methods and analysis

A single-centre, double-blind, randomised trial. After screening for eligibility and informed consent, 300 patients admitted for acute calculus cholecystitis will be randomised into two groups of treatment, either receiving amoxicillin/clavulanic acid or placebo for 5 consecutive days. Postoperative evaluation will take place during the first 30 days. Postoperative infectious complications are the primary end point. Secondary end points are length of hospital stay, readmissions, need of reintervention (percutaneous or surgical reinterventions) and overall mortality. The results of this trial will provide strong evidence to either support or abandon the use of antibiotics after surgery, impacting directly in the incidence of adverse events associated with the use of antibiotics, the emergence of bacterial resistance and treatment costs.

Ethics and dissemination

This study and informed consent sheets have been approved by the Research Projects Evaluating Committee (CEPI) of Hospital Italiano de Buenos Aires (protocol N° 2111).

Results

The results of the trial will be reported in a peer-reviewed publication.

Trial registration number

NCT02057679.
A quality improvement project to improve early sepsis care in the emergency department
Authors: Gatewood Medley O'Keefe , Wemple Matthew , Greco Sheryl , Kritek Patricia A , Durvasula Raghu
Published: November 2015

Background

Sepsis causes substantial morbidity and mortality in hospitalised patients. Although many studies describe the use of protocols in the management of patients with severe sepsis and septic shock, few have addressed emergency department (ED) screening and management for patients initially presenting with uncomplicated sepsis (ie, patients without organ failure or hypotension).

Objective

A quality improvement task force at a large, quaternary care referral hospital sought to develop a protocol focusing on early identification of patients with uncomplicated sepsis, in addition to severe sepsis and septic shock.

Intervention

The three-tiered intervention consisted of (1) a nurse-driven screening tool and management protocol to identify and initiate early treatment of patients with sepsis, (2) a computer-assisted screening algorithm that generated a ‘Sepsis Alert’ pop-up screen in the electronic medical record for treating clinical healthcare providers and (3) automated suggested sepsis-specific order sets for initial workup and resuscitation, antibiotic selection and goal-directed therapy.

Design

A before and after retrospective cohort study was undertaken to determine the intervention's impact on compliance with recommended sepsis management, including serum lactate measured in the ED, 2 L of intravenous fluid administered within 2 h of triage, antibiotics administered within 3 h of triage and blood cultures drawn before antibiotic administration. Mortality rates for patients in the ED with a sepsis-designated ICD-9 code present on admission were also analysed.

Results

Overall bundle compliance increased by 154%, from 28% at baseline to 71% in the last quarter of the study (p<0.001). Bundle, antibiotic and intravenous fluid compliance all increased significantly after launch of the sepsis initiative (eg, bundle and intravenous fluid compliance increased by 74% and 54%, respectively; p<0.001). Bundle and antibiotic compliance both showed further significant increases after implementation of suggested order sets (31% and 25% increases, respectively; p<0.001). The mortality rate for patients in the ED admitted with sepsis was 13.3% before implementation and fell to 11.1% after (p=0.230); mortality in the last two quarters of the study was 9.3% (p=0.107).

Conclusions

The new protocol demonstrates that early screening interventions can lead to expedited delivery of care to patients with sepsis in the ED and could serve as a model for other facilities. Mortality was not significantly improved by our intervention, which included patients with uncomplicated sepsis.
Accuracy of prognosis prediction by PPI in hospice inpatients with cancer: a multi-centre prospective study
Authors: Subramaniam Sivakumar , Thorns Andrew , Ridout Martin , Thirukkumaran Thiru , Osborne Thomas Richard
Published: November 2015

The Palliative Prognostic Index (PPI) is a prognostication tool for palliative care patients based on clinical indices developed in Japan and further validated by one study in the UK. The aim of this study was to test its prediction accuracy in a large inpatient hospice sample. The admitting doctor in three inpatient hospices calculated the PPI score on admission. Two hundred and sixty-two patients were included in this study. Based on the PPI score, three subgroups were identified. Group 1 corresponded to patients with PPI ≤4 and the median survival of 53 days (95% CI 40 to 80 days). Group 2 corresponded to those with PPI >4 and ≤6 and the median survival 15 days (95% CI 12 to 26 days) and Group 3 corresponded to patients with PPI >6 and the median survival of 5 days (95% CI 3 to 7 days). In this study, PPI was able to identify patients’ likelihood of dying within 3 weeks with a sensitivity of 64% and specificity of 83%. It was able to identify a 6-week survival chance with a sensitivity of 62% and specificity of 86%. A one-unit increase in PPI score was estimated to increase the hazard for death by a factor of 1.33 (95% CI 1.26 to 1.40), based on fitting a stratified Cox proportional hazards model. The authors conclude that PPI can be used to predict prognosis for patients with advanced cancer.