bluuebunny/crossref_metadata_2025 huggingface dataset:
Platinum-based nanocages with subnanometer-thick walls and well-defined, controllable facets
Authors: Zhang Lei , Roling Luke T. , Wang Xue , Vara Madeline , Chi Miaofang , Liu Jingyue , Choi Sang-Il , Park Jinho , Herron Jeffrey A. , Xie Zhaoxiong , Mavrikakis Manos , Xia Younan
Published: July 2015

Etching platinum nanocage catalysts

Although platinum is an excellent catalyst for the oxygen reduction reaction that occurs in fuel cells, its scarcity continues to drive efforts to improve its utilization. Zhang
et al.
made nanocages of platinum by coating palladium nanocrystals with only a few layers of platinum and then etching away the palladium core (see the Perspective by Strasser). Platinum nanocages made using nanoscale octahedra and cubes of palladium displayed different catalytic activity for the oxygen reduction reaction.

Science
, this issue p.
412
; see also p.
379
Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate
Authors: Moser von Filseck Joachim , Čopič Alenka , Delfosse Vanessa , Vanni Stefano , Jackson Catherine L. , Bourguet William , Drin Guillaume
Published: July 2015

Membrane contact sites promote lipid exchange

Most membrane lipids are manufactured in the endoplasmic reticulum (ER). Different organelles and the plasma membrane (PM) have distinct phospholipid compositions. Chung
et al.
, working in mammalian cells, and Moser von Filseck
et al.
, working in yeast, both describe how a family of proteins is important in maintaining the balance of lipids within the cell. These special proteins accumulate at and tether contact sites between the ER and the PM and promote the exchange of specific phospholipids, which helps to maintain the PM's distinct identity.

Science
, this issue pp.
428
and
432
Origami of thick panels
Authors: Chen Yan , Peng Rui , You Zhong
Published: July 2015

Expanding origami to thicker materials

A vast array of origami patterns can be applied to the folding of any material of near-zero thickness, such as paper. However, the folding of thick materials requires adding material and offsets. Chen
et al.
develop a general model in which the standard spherical linkages (creases) are replaced with rigid panels connected via offset folds, so that the folding motion of both near-zero–thickness and thick objects is the same. Furthermore, the hinges are constrained to move in only one direction, which is important for applications such as unfolding solar panels in space or large engineering structures, where you want the folding to occur automatically.

Science
, this issue p.
396
Four legs too many?
Authors: Evans Susan
Published: July 2015

A long-bodied fossil snake retains fore- and hindlimbs

[Also see Report by

Martill
et al.

]
Hydro-hygrophilous vegetation diversity and distribution patterns in riverine wetlands in an agricultural landscape: a case study from the Oglio River (Po Plain, Northern Italy)
Authors: Bolpagni Rossano , Piotti Andrea
Published: July 2015

In the European plains, up to eighty percent of riverine wetlands have been lost due to alteration of hydrological regime and catchment exploitation. This condition is expected to be further negatively exacerbated by climate change. To better understand the observed change in distribution
patterns of hydro-hygrophilous vegetation in temperate and Mediterranean floodplains, a vegetation survey was conducted within the lower Oglio River reach in Northern Italy. This river is a mid-size, altered and nutrient-rich left tributary of the Po River. During the 2008 growing season,
a total of 60 marginal aquatic habitats were investigated. Overall, 37 vegetation communities were detected, showing a clear predominance of hygrophilous herbaceous plant communities both in terms of representativeness (55.1%) and diversity (54.1%) with respect to woody (22.9% and 10.8%, respectively),
and obligate aquatic vegetation (22.0% and 35.1%, respectively). Our main findings were (1) the widespread presence of highly opportunistic, non-native and invasive hygrophilous plant communities (largely dominated by Amorpha fruticosa, Phragmites australis s.l., Amaranthus
spp., Bidens spp., and Cyperus spp.), and (2) the limited distribution of hydrophyte vegetation usually dominated by pleustophytes (e.g., Spirodela polyrhiza and Lemna spp.). The present study confirms the dominance of secondary plant communities characterized by
the widespread presence of alien species in lowland over-exploited riverscapes, coupled with a low local representativeness of native willow (Salix alba, S. cinerea) communities and anchored macrophyte (batrachid, ceratophyllid, elodeid, myriophyllid) meadows. Total vegetation
diversity is consistent with previous studies in similar ecological contexts; on the other hand, at the site scale, the diversity values were extremely low. This is especially true for the aquatic vegetation, and can be related to the high water turbidity and chlorophyll-a concentrations that
prevent the establishment and colonization of submerged and rooted hydrophytes. Consequently, we stress the need for metabolic and biogeochemical indicators to assess the actual trophic status of lowland wetlands in order to clarify their potential to be restored.
WetVegEurope: a database of aquatic and wetland vegetation of Europe
Authors: Landucci Flavia , Řezníčková Marcela , Šumberová Kateřina , Chytrý Milan , Aunina Liene , Biţă-Nicolae Claudia , Bobrov Alexander , Borsukevych Lyubov , Brisse Henry , Čarni Andraž , Csiky János , Cvijanović Dušanka , De Bie Els , De Ruffray Patrice , Dubyna Dmytro , Dimopoulos Panayotis , Dziuba Tetiana , FitzPatrick Úna , Font Xavier , Gigante Daniela , Golub Valentin , Hennekens Stephan M. , Hrivnák Richard , Iemelianova Svitlana , Jandt Ute , Jenačković Dragana , Jansen Florian , Kącki Zygmunt , Lájer Konrád , Matulevičiutė Dalytė , Mesterházy Attila , Michalcová Dana , Paal Jaanus , Papastergiadou Eva , Properzi Alessandro , Radulović Snežana , Rodwell John S. , Schaminée Joop H.J. , Šilc Urban , Sinkevičienė Zofija , Stančić Zvjezdana , Stepanovich Jazep , Teteryuk Boris , Tzonev Rossen , Venanzoni Roberto , Weekes Lynda , Willner Wolfgang
Published: July 2015

WetVegEurope is a research project (http://www.sci.muni.cz/botany/vegsci/wetveg) whose goal is to provide a synthesized formalized classification of the aquatic and marsh vegetation across Europe at the level of phytosociological associations. In order to achieve the project objective,
a WetVegEurope database has been created (GIVD ID: EU-00-020, http://www.givd.info/ID/EU-00-020), which currently contains 375,212 vegetation plots of aquatic, marsh and wet vegetation types from 33 European countries. The WetVegEurope database includes datasets from pre-existing national
and thematic databases and also 10,616 plots previously not digitalized or even unpublished. This database offers an extensive source of data for future studies on aquatic and marsh plant species and vegetation types at the European scale.
Guidelines for proposals to conserve or reject names of syntaxa
Authors: Willner Wolfgang , Theurillat Jean-Paul , Čarni Andraž , Pallas Jens , Weber Heinrich E.
Published: July 2015

This paper explains how to apply for conservation or rejection of names of syntaxa according to the International Code of Phytosociological Nomenclature. First, authors have to submit a written proposal with all relevant details to be published in the Section “Phytosociological
Nomenclature” of Phytocoenologia after formal editing. Then, from time to time, the Committee for Changes and Conservation of Names (CCCN) of the Working Group for Phytosociological Nomenclature (GPN) will evaluate the published proposals and make recommendations
to accept or reject them. Finally, the GPN Assembly will vote on these recommendations and publish the decision.
Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis
Authors: Coppola Mariateresa , van den Eeden Susan J. F. , Wilson Louis , Franken Kees L. M. C. , Ottenhoff Tom H. M. , Geluk Annemieke
Published: July 2015

Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions.Mycobacterium bovisBCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity againstMycobacterium tuberculosislatency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a majorM. tuberculosislatency antigen which is highly expressed by “dormant”M. tuberculosisand well recognized by T cells from latentlyM. tuberculosis-infected individuals. In order to assess itsin vivoimmunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ+/TNF+) and IFN-γ+CD4+T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs ofM. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosischallenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential ofM. tuberculosislatency antigens to improve BCG efficacy. These data suggest a promising role forM. tuberculosislatency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.
Characterization of Hepatitis C Virus Recombination in Cameroon by Use of Nonspecific Next-Generation Sequencing
Authors: Iles James C. , Njouom Richard , Foupouapouognigni Yacouba , Bonsall David , Bowden Rory , Trebes Amy , Piazza Paolo , Barnes Ellie , Pépin Jacques , Klenerman Paul , Pybus Oliver G.
Published: July 2015

The importance of recombination in the evolution and genetic diversity of the hepatitis C virus (HCV) is currently uncertain. Only a small number of intergenotypic recombinants have been identified so far, and each has core and envelope genes classified as belonging to genotype 2. Here, we investigated two putative genotype 4/1 recombinants from southern Cameroon using a number of approaches, including standard Sanger sequencing, genotype-specific PCR amplification, and non-HCV-specific Illumina RNA sequencing (RNA-seq). Recombination between genotypes 1 and 4 was confirmed in both samples, and the parental lineages of each recombinant belong to HCV subtypes that are cocirculating at a high prevalence in Cameroon. Using the RNA-seq approach, we obtained a complete genome for one sample, which contained a recombination breakpoint at the E2/P7 gene junction. We developed and applied a new method, called Deep SimPlot, which can be used to visualize and identify viral recombination directly from the short sequence reads created by next-generation sequencing in conjunction with a consensus sequence.
Comparing Etest and Broth Microdilution for Antifungal Susceptibility Testing of the Most-Relevant Pathogenic Molds
Authors: Lamoth Frédéric , Alexander Barbara D.
Published: July 2015

Invasive mold infections are life-threatening diseases for which appropriate antifungal therapy is crucial. Their epidemiology is evolving, with the emergence of triazole-resistant
Aspergillus
spp. and multidrug-resistant non-
Aspergillus
molds. Despite the lack of interpretive criteria, antifungal susceptibility testing of molds may be useful in guiding antifungal therapy. The standard broth microdilution method (BMD) is demanding and requires expertise. We assessed the performance of a commercialized gradient diffusion method (Etest method) as an alternative to BMD. The MICs or minimal effective concentrations (MECs) of amphotericin B, voriconazole, posaconazole, caspofungin, and micafungin were assessed for 290 clinical isolates of the most representative pathogenic molds (154
Aspergillus
and 136 non-
Aspergillus
isolates) with the BMD and Etest methods. Essential agreements (EAs) within ±2 dilutions of ≥90% between the two methods were considered acceptable. EAs for amphotericin B and voriconazole were >90% for most potentially susceptible species. For posaconazole, the correlation was acceptable for
Mucoromycotina
but Etest MIC values were consistently lower for
Aspergillus
spp. (EAs of <90%). Excellent EAs were found for echinocandins with highly susceptible (MECs of <0.015 μg/ml) or intrinsically resistant (MECs of >16 μg/ml) strains. However, MEC determinations lacked consistency between methods for strains exhibiting mid-range MECs for echinocandins. We concluded that the Etest method is an appropriate alternative to BMD for antifungal susceptibility testing of molds under specific circumstances, including testing with amphotericin B or triazoles for non-
Aspergillus
molds (
Mucoromycotina
and
Fusarium
spp.). Additional study of molecularly characterized triazole-resistant
Aspergillus
isolates is required to confirm the ability of the Etest method to detect voriconazole and posaconazole resistance among
Aspergillus
spp.
Human Wound Infection with Mannheimia glucosida following Lamb Bite
Authors: Lau Jillian S. Y. , Omaleki Lida , Turni Conny , Barber Stuart Richard , Browning Glenn Francis , Francis Michelle J. , Graham Maryza , Korman Tony M.
Published: July 2015

Mannheimia
spp. are veterinary pathogens that can cause mastitis and pneumonia in domestic cattle and sheep. While
Mannheimia glucosida
can be found as normal flora in oral and respiratory mucosa in sheep, there have been no reported cases of human infection with this organism.
Asymptomatic Leprosy Infection among Blood Donors May Predict Disease Development and Suggests a Potential Mode of Transmission
Authors: Goulart Isabela Maria Bernardes , Araujo Sergio , Filho Adilson Botelho , de Paiva Paulo Henrique Ribeiro , Goulart Luiz Ricardo
Published: July 2015

Blood donor samples (1,007) were assessed for anti-phenolic glycolipid 1 (PGL-1) IgM antibodies and
Mycobacterium leprae
DNA presence, which had 3.8% and 0.3% positivity, respectively. After a 5-year follow-up period, six individuals with positive markers developed leprosy, raising the hypothesis that asymptomatic infection among blood donors may be an undisclosed mode of leprosy transmission via transfusion.
Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool
Authors: Song Linan , Zhao Mingwei , Duffy David C. , Hansen Joshua , Shields Kelsey , Wungjiranirun Manida , Chen Xinhua , Xu Hua , Leffler Daniel A. , Sambol Susan P. , Gerding Dale N. , Kelly Ciarán P. , Pollock Nira R.
Published: July 2015

The currently available diagnostics for
Clostridium difficile
infection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenic
C. difficile
. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinical
C. difficile
isolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.
Whole-Genome Mapping as a Novel High-Resolution Typing Tool for Legionella pneumophila
Authors: Bosch Thijs , Euser Sjoerd M. , Landman Fabian , Bruin Jacob P. , IJzerman Ed P. , den Boer Jeroen W. , Schouls Leo M.
Published: July 2015

Legionella
is the causative agent for Legionnaires' disease (LD) and is responsible for several large outbreaks in the world. More than 90% of LD cases are caused by
Legionella pneumophila
, and studies on the origin and transmission routes of this pathogen rely on adequate molecular characterization of isolates. Current typing of
L. pneumophila
mainly depends on sequence-based typing (SBT). However, studies have shown that in some outbreak situations, SBT does not have sufficient discriminatory power to distinguish between related and nonrelated
L. pneumophila
isolates. In this study, we used a novel high-resolution typing technique, called whole-genome mapping (WGM), to differentiate between epidemiologically related and nonrelated
L. pneumophila
isolates. Assessment of the method by various validation experiments showed highly reproducible results, and WGM was able to confirm two well-documented Dutch
L. pneumophila
outbreaks. Comparison of whole-genome maps of the two outbreaks together with WGMs of epidemiologically nonrelated
L. pneumophila
isolates showed major differences between the maps, and WGM yielded a higher discriminatory power than SBT. In conclusion, WGM can be a valuable alternative to perform outbreak investigations of
L. pneumophila
in real time since the turnaround time from culture to comparison of the
L. pneumophila
maps is less than 24 h.
Superbugs on Duodenoscopes: the Challenge of Cleaning and Disinfection of Reusable Devices
Authors: Humphries Romney M. , McDonnell Gerald
Published: July 2015

Inadequate flexible endoscope reprocessing has been associated with infection outbreaks, most recently caused by carbapenem-resistant
Enterobacteriaceae
. Lapses in essential device reprocessing steps such as cleaning, disinfection/sterilization, and storage have been reported, but some outbreaks have occurred despite claimed adherence to established guidelines. Recommended changes in these guidelines include the use of sterilization instead of high-level disinfection or the use of routine microbial culturing to monitor efficacy of reprocessing. This review describes the current standards for endoscope reprocessing, associated outbreaks, and the complexities associated with both microbiological culture and sterilization approaches to mitigating the risk of infection associated with endoscopy.
Rapid Identification of Bacteria Directly from Positive Blood Cultures by Use of a Serum Separator Tube, Smudge Plate Preparation, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry
Authors: Chen Yan , Porter Vanessa , Mubareka Samira , Kotowich Leona , Simor Andrew E.
Published: July 2015

We analyzed the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) of smudge plate growth for bacterial identification from 400 blood cultures. Ninety-seven percent of Gram-negative bacilli and 85% of Gram-positive organisms were correctly identified within 4 h; only eight isolates (2.0%) were misidentified. This method provided rapid and accurate microbial identification from positive blood cultures.
Development of a Multilocus Sequence Typing System for Medically Relevant Bipolaris Species
Authors: Pham Cau D. , Purfield Anne E. , Fader Robert , Pascoe Neil , Lockhart Shawn R.
Published: July 2015

Multilocus sequence typing (MLST) is the gold standard genotyping technique for many microorganisms. This classification approach satisfies the requirements for a high-resolution, standardized, and archivable taxonomic system. Here, we describe the development of a novel MLST system to assist with the investigation of an unusual cluster of surgical site infections caused by
Bipolaris
spp. in postoperative cardiothoracic surgery (POCS) patients during January 2008 to December 2013 in the southeastern United States. We also used the same MLST system to perform a retrospective analysis on isolates from a 2012
Bipolaris
endophthalmitis outbreak caused by a contaminated product. This MLST system showed high intraspecies discriminatory power for
Bipolaris spicifera
,
B. hawaiiensis
, and
B. australiensis
. Based on the relatedness of the isolates, the MLST data supported the hypothesis that infections in the POCS cluster were from different environmental sources while confirming that the endophthalmitis outbreak resulted from a point source, which was a contaminated medication.
Apolipoprotein E, but Not Apolipoprotein B, Is Essential for Efficient Cell-to-Cell Transmission of Hepatitis C Virus
Authors: Gondar Virgínia , Molina-Jiménez Francisca , Hishiki Takayuki , García-Buey Luisa , Koutsoudakis George , Shimotohno Kunitada , Benedicto Ignacio , Majano Pedro L.
Published: July 2015

Hepatitis C virus (HCV) infects hepatocytes through two different routes: (i) cell-free particle diffusion followed by engagement with specific cellular receptors and (ii) cell-to-cell direct transmission mediated by mechanisms not well defined yet. HCV exits host cells in association with very-low-density lipoprotein (VLDL) components. VLDL particles contain apolipoproteins B (ApoB) and E (ApoE), which are required for viral assembly and/or infectivity. Based on these precedents, we decided to study whether these VLDL components participate in HCV cell-to-cell transmission
in vitro
. We observed that cell-to-cell viral spread was compromised after ApoE interference in donor but not in acceptor cells. In contrast, ApoB knockdown in either donor or acceptor cells did not impair cell-to-cell viral transmission. Interestingly, ApoB participated in the assembly of cell-free infective virions, suggesting a differential regulation of cell-to-cell and cell-free HCV infection. This study identifies host-specific factors involved in these distinct routes of infection that may unveil new therapeutic targets and advance our understanding of HCV pathogenesis.

IMPORTANCE
This work demonstrates that cell-to-cell transmission of HCV depends on ApoE but not ApoB. The data also indicate that ApoB is required for the assembly of cell-free infective particles, strongly suggesting the existence of mechanisms involving VLDL components that differentially regulate cell-free and cell-to-cell HCV transmission. These data clarify some of the questions regarding the role of VLDL in HCV pathogenesis and the transmission of the virus cell to cell as a possible mechanism of immune evasion and open the door to therapeutic intervention.
Experimental Evolution Identifies Vaccinia Virus Mutations in A24R and A35R That Antagonize the Protein Kinase R Pathway and Accompany Collapse of an Extragenic Gene Amplification
Authors: Brennan Greg , Kitzman Jacob O. , Shendure Jay , Geballe Adam P.
Published: July 2015

Most new human infectious diseases emerge from cross-species pathogen transmissions; however, it is not clear how viruses adapt to productively infect new hosts. Host restriction factors represent one species-specific barrier that viruses may initially have little ability to inhibit in new hosts. For example, viral antagonists of protein kinase R (PKR) vary in their ability to block PKR-mediated inhibition of viral replication, in part due to PKR allelic variation between species. We previously reported that amplification of a weak PKR antagonist encoded by rhesus cytomegalovirus,
rhtrs1
, improved replication of a recombinant poxvirus (VVΔEΔK+RhTRS1) in several resistant primate cells. To test whether amplification increases the opportunity for mutations that improve virus replication with only a single copy of
rhtrs1
to evolve, we passaged
rhtrs1
-amplified viruses in semipermissive primate cells. After passage, we isolated two viruses that contained only a single copy of
rhtrs1
yet replicated as well as the amplified virus. Surprisingly,
rhtrs1
was not mutated in these viruses; instead, we identified mutations in two vaccinia virus (VACV) genes, A24R and A35R, either of which was sufficient to improve VVΔEΔK+RhTRS1 replication. Neither of these genes has previously been implicated in PKR antagonism. Furthermore, the mutation in A24R, but not A35R, increased resistance to the antipoxviral drug isatin-β-thiosemicarbazone, suggesting that these mutations employ different mechanisms to evade PKR. This study supports our hypothesis that gene amplification may provide a “molecular foothold,” broadly improving replication to facilitate rapid adaptation, while subsequent mutations maintain this efficient replication in the new host without requiring gene amplification.

IMPORTANCE
Understanding how viruses adapt to a new host may help identify viruses poised to cross species barriers before an outbreak occurs. Amplification of
rhtrs1
, a weak viral antagonist of the host antiviral protein PKR, enabled a recombinant vaccinia virus to replicate in resistant cells from humans and other primates. After serial passage of
rhtrs1
-amplified viruses, there arose in two vaccinia virus genes mutations that improved viral replication without requiring
rhtrs1
amplification. Neither of these genes has previously been associated with inhibition of the PKR pathway. These data suggest that gene amplification can improve viral replication in a resistant host species and facilitate the emergence of novel adaptations that maintain the foothold needed for continued replication and spread in the new host.
Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate
Authors: Maestrale Caterina , Cancedda Maria G. , Pintus Davide , Masia Mariangela , Nonno Romolo , Ru Giuseppe , Carta Antonello , Demontis Francesca , Santucciu Cinzia , Ligios Ciriaco
Published: July 2015

Thirty-seven goats carrying different prion protein genotypes (
PRNP
) were orally infected with a classical scrapie brain homogenate from wild-type (ARQ/ARQ) sheep and then mated to obtain 2 additional generations of offspring, which were kept in the same environment and allowed to be naturally exposed to scrapie. Occurrence of clinical or subclinical scrapie was observed in the experimentally infected goats (F
0
) and in only one (F
1b
) of the naturally exposed offspring groups. In both groups (F
0
and F
1b
), goats carrying the R154H, H154H, R211Q, and P168Q-P240P dimorphisms died of scrapie after a longer incubation period than wild-type, G37V, Q168Q-P240P, and S240P goats. In contrast, D145D and Q222K goats were resistant to infection. The immunobiochemical signature of the scrapie isolate and its pathological aspects observed in the sheep donors were substantially maintained over 2 goat generations, i.e., after experimental and natural transmission. This demonstrates that the prion protein gene sequence, which is shared by sheep and goats, is more powerful than any possible but unknown species-related factors in determining scrapie phenotypes. With regard to genetics, our study confirms that the K222 mutation protects goats even against ovine scrapie isolates, and for the first time, a possible association of D145 mutation with scrapie resistance is shown. In addition, it is possible that the sole diverse frequencies of these genetic variants might, at least in part, shape the prevalence of scrapie among naturally exposed progenies in affected herds.

IMPORTANCE
This study was aimed at investigating the genetic and pathological features characterizing sheep-to-goat transmission of scrapie. We show that in goats with different prion protein gene mutations, the K222 genetic variant is associated with scrapie resistance after natural and experimental exposure to ovine prion infectivity. In addition, we observed for the first time a protective effect of the D145 goat variant against scrapie. Importantly, our results demonstrate that the phenotypic characteristic of the wild-type sheep scrapie isolate is substantially preserved in goats carrying different susceptible
PRNP
gene variants, thus indicating that the prion protein gene sequence, which is shared by sheep and goats, plays a fundamental role in determining scrapie phenotypes.
Modulation of Glycosaminoglycans Affects PrPScMetabolism but Does Not Block PrPScUptake
Authors: Wolf Hanna , Graßmann Andrea , Bester Romina , Hossinger André , Möhl Christoph , Paulsen Lydia , Groschup Martin H. , Schätzl Hermann , Vorberg Ina
Published: July 2015

Mammalian prions are unconventional infectious agents composed primarily of the misfolded aggregated host prion protein PrP, termed PrPSc. Prions propagate by the recruitment and conformational conversion of cellular prion protein into abnormal prion aggregates on the cell surface or along the endocytic pathway. Cellular glycosaminoglycans have been implicated as the first attachment sites for prions and cofactors for cellular prion replication. Glycosaminoglycan mimetics and obstruction of glycosaminoglycan sulfation affect prion replication, but the inhibitory effects on different strains and different stages of the cell infection have not been thoroughly addressed. We examined the effects of a glycosaminoglycan mimetic and undersulfation on cellular prion protein metabolism, prion uptake, and the establishment of productive infections in L929 cells by two mouse-adapted prion strains. Surprisingly, both treatments reduced endogenous sulfated glycosaminoglycans but had divergent effects on cellular PrP levels. Chemical or genetic manipulation of glycosaminoglycans did not prevent PrPScuptake, arguing against their roles as essential prion attachment sites. However, both treatments effectively antagonizedde novoprion infection independently of the prion strain and reduced PrPScformation in chronically infected cells. Our results demonstrate that sulfated glycosaminoglycans are dispensable for prion internalization but play a pivotal role in persistently maintained PrPScformation independent of the prion strain.

IMPORTANCERecently, glycosaminoglycans (GAGs) became the focus of neurodegenerative disease research as general attachment sites for cell invasion by pathogenic protein aggregates. GAGs influence amyloid formationin vitro. GAGs are also found in intra- and extracellular amyloid deposits. In light of the essential role GAGs play in proteinopathies, understanding the effects of GAGs on protein aggregation and aggregate dissemination is crucial for therapeutic intervention. Here, we show that GAGs are dispensable for prion uptake but play essential roles in downstream infection processes. GAG mimetics also affect cellular GAG levels and localization and thus might affect prion propagation by depleting intracellular cofactor pools.
Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation
Authors: Cui Xiuji , Luckenbaugh Laurie , Bruss Volker , Hu Jianming
Published: July 2015

Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA.

IMPORTANCE
Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes.
A Novel PDZ Domain Interaction Mediates the Binding between Human Papillomavirus 16 L2 and Sorting Nexin 27 and Modulates Virion Trafficking
Authors: Pim David , Broniarczyk Justyna , Bergant Martina , Playford Martin P. , Banks Lawrence
Published: July 2015

Previous studies have demonstrated an interaction between sorting nexin 17 and the L2 capsid proteins from a variety of papillomavirus types. This interaction is required for late endosomal trafficking of the L2 protein and entry of the L2/DNA complex into the nucleus during infection. Here we show an interaction between papillomavirus L2 proteins and the related PX-FERM family member sorting nexin 27 (SNX27), which is mediated in part by a novel interaction between the PDZ domain of SNX27 and sequences in a central portion of L2. The interaction is direct and, unlike that with SNX17, is variable in strength depending on the papillomavirus type. We show that small interfering RNA (siRNA)-mediated knockdown of SNX27 alone leads to a marginal reduction in the efficiency of viral infection but that double knockdown of both sorting nexins results in a striking reduction in infection, greater than that observed for the knockdown of either sorting nexin alone. These results suggest that the HPV L2 proteins can interact through distinct mechanisms with multiple components of the cellular cargo-sorting machinery.

IMPORTANCE
The trafficking of papillomaviruses to the host cell nucleus during their natural infectious life cycle is an incompletely understood process. Studies have suggested that the virus minor capsid protein L2 can interact with the endosomal recycling pathway, in part by association with sorting nexin 17, to ensure that virus DNA bound to L2 is recycled through the trans-Golgi network rather than back to the plasma membrane. In this study, we characterize the interaction between L2 and a second sorting nexin, SNX27, which is also part of the retromer complex. The study furthers our understanding of papillomavirus infection dynamics and provides potential tools for the further dissection of endosomal structure and function.
Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells
Authors: Bleau Christian , Filliol Aveline , Samson Michel , Lamontagne Lucie
Published: July 2015

Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB
in vivo
and in brain microvascular endothelial cells (BMECs)
in vitro
. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-β) production. Our findings highlight the importance of IFN-β production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain.

IMPORTANCE
Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-β by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion.
A Higher Frequency of NKG2A+than of NKG2A−NK Cells Responds to Autologous HIV-Infected CD4 Cells irrespective of Whether or Not They Coexpress KIR3DL1
Authors: Lisovsky Irene , Isitman Gamze , Song Rujun , DaFonseca Sandrina , Tremblay-McLean Alexandra , Lebouché Bertrand , Routy Jean-Pierre , Bruneau Julie , Bernard Nicole F.
Published: July 2015

Epidemiological and functional studies implicate NK cells in HIV control. However, there is little information available on which NK cell populations, as defined by the inhibitory NK cell receptors (iNKRs) they express, respond to autologous HIV-infected CD4+(iCD4) T cells. NK cells acquire antiviral functions through education, which requires signals received from iNKRs, such as NKG2A and KIR3DL1 (here, 3DL1), engaging their ligands. NKG2A interacts with HLA-E, and 3DL1 interacts with HLA-A/B antigens expressing the Bw4 epitope. HIV-infected cells downregulate HLA-A/B, which should interrupt negative signaling through 3DL1, leading to NK cell activation, provided there is sufficient engagement of activating NKRs. We examined the functionality of NK cells expressing or not NKG2A and 3DL1 stimulated by HLA-null and autologous iCD4 cells. Flow cytometry was used to gate on each NKG2A+/NKG2A−3DL1+/3DL1−(NKG2A+/−3DL1+/−) population and to measure the frequency of all possible combinations of CD107a expression and gamma interferon (IFN-γ) and CCL4 secretion. The highest frequency of functional NK cells responding to HLA-null cell stimulation was the NKG2A+3DL1+NK cell population. The highest frequencies of functional NK cells responding to autologous iCD4 cells were those expressing NKG2A; coexpression of 3DL1 did not further modulate responsiveness. This was the case for the functional subsets characterized by the sum of all functions tested (total responsiveness), as well as by the trifunctional CD107a+IFN-γ+CCL4+, CD107a+IFN-γ+, total CD107a+, and total IFN-γ+functional subsets. These results indicate that the NKG2A receptor has a role in NK cell-mediated anti-HIV responses.

IMPORTANCEHIV-infected CD4 (iCD4) cells activate NK cells, which then control HIV replication. However, little is known regarding which NK cell populations iCD4 cells stimulate to develop antiviral activity. Here, we examine the frequency of NK cell populations, defined by the presence/absence of the NK cell receptors (NKRs) NKG2A and 3DL1, that respond to iCD4 cells. NKG2A and 3DL1 are involved in priming NK cells for antiviral functions upon encountering virus-infected cells. A higher frequency of NKG2A+than NKG2A−NK cells responded to iCD4 cells by developing antiviral functions such as CD107a expression, which correlates with NK cell killing, and secretion of gamma interferon and CCL4. Coexpression of 3DL1 on the NKG2A+and NKG2A−NK cells did not modulate responses to iCD4 cells. Understanding the mechanisms underlying the interaction of NK cells with iCD4 cells that lead to HIV control may contribute to developing strategies that harness NK cells for preventing or controlling HIV infection.
Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation
Authors: Zhang Hua , Song Lei , Cong Haolong , Tien Po
Published: July 2015

Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES)trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation.

IMPORTANCEThe nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection.
Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry
Authors: Park Chorong , Min Saehong , Park Eun-Mee , Lim Yun-Sook , Kang Sangmin , Suzuki Tetsuro , Shin Eui-Cheol , Hwang Soon B.
Published: July 2015

The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both
in vitro
pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry.

IMPORTANCE
Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell entry factor but also as a new anti-HCV therapeutic target.
Progressive wheeze: atrial myxoma masquerading as chronic obstructive pulmonary disease
Authors: Sinha Aish , Apps Andrew , Liong Wei Chuen , Firoozan Soroosh
Published: July 2015

Atrial myxoma, the commonest primary cardiac neoplasm, presents with symptoms of heart failure, embolic phenomena or constitutional upset. We present an atypical case, with wheeze and symptomatic exacerbations typical of chronic obstructive pulmonary disease. With no early clinical evidence of heart failure, the patient was managed with inhaled steroids and bronchodilators, with little relief. Only when the patient was in extremis requiring intubation, due to respiratory failure, did clinical evidence of left heart failure become apparent, with echocardiography demonstrating a massive left atrial myxoma obstructing the mitral valve annulus. Following successful surgical resection, the patient's symptoms fully abated. This case highlights the importance of considering cardiac wheeze in those initially managed as obstructive airway disease not responding in a typical fashion to initial bronchodilator therapy, and particularly in those with rapidly progressive symptoms. Such patients should be referred early for cardiac imaging. The excellent prognosis and quick recovery after timely surgical resection of a myxoma are also highlighted.