bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessment of magnetic resonance techniques to measure muscle damage 24 h after eccentric exercise
Authors: Fulford J. , Eston R. G. , Rowlands A. V. , Davies R. C.
Published: April 2014

The study examined which of a number of different magnetic resonance (MR) methods were sensitive to detecting muscle damage induced by eccentric exercise. Seventeen healthy, physically active participants, with muscle damage confirmed by non‐MR methods were tested 24 h after performing eccentric exercise. Techniques investigated whether damage could be detected within the quadriceps muscle as a whole, and individually within the rectus femoris, vastus lateralis (VL), vastus medialis (VM), and vastus intermedius (VI). Relative to baseline values, significant changes were seen in leg and muscle cross‐sectional areas and volumes and the resting inorganic phosphate concentration. Significant time effects over all muscles were also seen in the transverse relaxation time (T2) and apparent diffusion coefficient (ADC) values, with individually significant changes seen in the VL, VM, and VI for T2 and in the VI for ADC. A significant correlation was found between muscle volume and the average T2 change (r = 0.59) but not between T2 and ADC or Pi alterations. There were no significant time effects over all muscles for magnetization transfer contrast images, for baseline pH, phosphocreatine (PCr), phosphodiester, or ATP metabolite concentrations or the time constant describing the rate of PCr recovery following exercise.
Collective goals and shared tasks: Interdependence structure and perceptions of individual sport team environments
Authors: Evans M. B. , Eys M. A.
Published: April 2014

Across two studies, we tested the proposition that interdependence structures (i.e., task interaction among teammates during competition, competition against teammates, presence of a collective outcome) influence interdependence perceptions among teammates as well as perceptions of group cohesion, competitiveness, and satisfaction. Study 1 was a paper‐and‐pencil survey completed by 210 individual sport athletes from 12 univ ersity‐ and college‐level teams. Multiple mediation analyses demonstrated that participants who had to work alongside teammates during competition reported increased interdependence perceptions that were, in turn, associated with increased cohesion and satisfaction as well as decreased competitiveness. There were no differences according to whether participants competed in the same event as all of their teammates or not. Study 2 involved a weekly e‐mail survey with 17 university‐level individual sport athletes who reported interdependence perceptions on a continual basis over the course of their competitive season. Interdependence perceptions were higher during weeks that were close in time to competitions with a collective group outcome. These studies reveal how interdependence structures shape the group environment and support applied efforts that consider ways to structure teammate interdependencies in ways to optimize group functioning and promote member satisfaction.
CladispaBaly: revision, biology and reassignment of the genus to the tribe Spilophorini (Coleoptera: Chrysomelidae: Cassidinae)
Authors: SEKERKA LUKÁŠ , WINDSOR DONALD , DURY GUILLAUME
Published: April 2014

The genus, Cladispa Baly 1858, is transferred from the tribe Imatidiini (= Cephaloleiini Chapuis, 1875) to Spilophorini Chapuis, 1875 based on the review of type material, newly collected specimens and molecular phylogenetic analysis. The type species, C. quadrimaculata Baly, 1858, is redescribed, and two new species, C. amboroensis sp.n. from Bolivia (Santa Cruz Department) and C. ecuadorica sp.n. from Ecuador (Pastaza Province), are described and figured. The morphology of C. amboroensis sp.n. immature stages is broadly consistent with other Spilophorini. Field observations document that both C. quadrimaculata and C. amboroensis sp.n. are trophic specialists on Orchideaceae. Keys to Cladispa species and Spilophorini genera are provided. Trophic associations of other Cassidinae and Orchideaceae are discussed.

This published work has been registered in ZooBank,
http://zoobank.org/urn:lsid:zoobank.org:pub:42A1ECF3‐2030‐4938‐8F3D‐FE7EC36F303A
Abscisic acid inhibits root growth in Arabidopsis through ethylene biosynthesis
Authors: Luo Xingju , Chen Zhizhong , Gao Junping , Gong Zhizhong
Published: April 2014

When first discovered in 1963, abscisic acid (ABA) was called abscisin II because it promotes abscission. Later, researchers found that ABA accelerates abscission via ethylene. In Arabidopsis, previous studies have shown that high concentrations of ABA inhibit root growth through ethylene signaling but not ethylene production. In the present study in Arabidopsis, we found that ABA inhibits root growth by promoting ethylene biosynthesis. The ethylene biosynthesis inhibitor L–α–(2‐aminoethoxyvinyl)‐glycine reduces ABA inhibition of root growth, and multiple mutants of ACS (1–aminocyclopropane‐1–carboxylate synthase) are more resistant to ABA in terms of root growth than the wild‐type is. Two ABA‐activated calcium‐dependent protein kinases, CPK4 and CPK11, phosphorylate the C–terminus of ACS6 and increase the stability of ACS6 in ethylene biosynthesis. Plants expressing an ACS6 mutant that mimics the phosphorylated form of ACS6 produce more ethylene than the wild‐type. Our results reveal an important mechanism by which ABA promotes ethylene production. This mechanism may be highly conserved among higher plants.
Iron deficiency in blood donors: a national cross‐sectional study
Authors: Salvin Hannah E. , Pasricha Sant‐Rayn , Marks Denese C. , Speedy Joanna
Published: April 2014

Background

Iron deficiency (ID) is an important consequence of blood donation. The epidemiology of this problem in the blood donor population was therefore studied to enable appropriate targeting of potential solutions to donor ID.

Study Design and Methods

A nationally representative, cluster‐based cross‐sectional study of Australian blood donors was performed. Donors were eligible for inclusion if they fulfilled criteria for blood donation or were deferred due to low or falling hemoglobin. Ferritin was measured and demographic and donation data were collected.

Results

A total of 3094 blood donors were recruited, of which samples were collected from 3049 donors; 1873 had exclusively donated whole blood (WB only), 242 had exclusively made apheresis donations, and 530 had not donated (“new” donors) in the previous 24 months. The prevalence of ID in new female donors was 12.0% compared with 1.3% in males. The prevalence of ID in female WB‐only donors was 26.4%; it increased with donation frequency and decreased with age. The prevalence in male WB‐only donors was 6.3% with no evident change with age or donation frequency. The prevalence of ID in apheresis‐only donors (females 6.3%; males 2.2%) did not significantly exceed that of new donors nor did it change with donation frequency. Importantly, the risk of ID could not be satisfactorily predicted in an individual donor by his or her anemia status or with predictive modeling incorporating demographic and donation data.

Conclusion

ID is especially prevalent in female, premenopausal, frequent WB donors. Strategies to combat ID should be implemented, specifically targeting this group.
Particulate Matter Matters
Authors: Dominici Francesca , Greenstone Michael , Sunstein Cass R.
Published: April 2014

Quasi-experimental evidence is needed on the relations between human health and airborne particulate matter.
A Critical Period of Sleep for Development of Courtship Circuitry and Behavior in
Drosophila
Authors: Kayser Matthew S. , Yue Zhifeng , Sehgal Amita
Published: April 2014

Sleep Tight, Fly

Shortly after eclosion, young flies sleep a lot and are resistant to being woken. Several days later, the same flies sleep less and are more easily woken.

Kayser
et al.

(p.
269
) show that the different sleep pattern characteristic of youthful flies is critical to correct development of their brains. When sleep is disrupted in young flies, dopaminergic signaling is also disturbed and a glomerulus in the courtship behavior circuit does not develop properly, leading to inadequate courtship behavior and failure to reproduce.
The Structural Basis of Pathogenic Subgenomic Flavivirus RNA (sfRNA) Production
Authors: Chapman Erich G. , Costantino David A. , Rabe Jennifer L. , Moon Stephanie L. , Wilusz Jeffrey , Nix Jay C. , Kieft Jeffrey S.
Published: April 2014

Resisting the Chop

Dengue, West Nile, and Yellow Fever viruses are all flaviviruses that have single-stranded RNA genomes and form specific, short flaviviral RNAs (sfRNAs) during infection that cause viral pathogenicity. These sfRNAs are produced by the incomplete degradation of viral RNA by the host-cell exonuclease Xrn1. What stops the host enzyme from completely chopping up the viral RNA?

Chapman
et al.

(p.
307
) reveal a pseudoknot in the structure of the Xrn1-resistant segment of a sfRNA from Murray Valley Encephalitis Virus, which, perhaps, the host Xrn1 exonuclease cannot untangle.
On Scientists and Advocacy
Behind the Curve
Science and the Politics of Global Warming

by Joshua P. Howe

University of Washington Press, Seattle, 2014. 311 pp. $34.95. ISBN 9780295993683. Weyerhaeuser Environmental Books.
Authors: Schmidt Gavin A.
Published: April 2014

Howe considers how researchers, environmentalists, and the public have interpreted and responded to the problem of global warming.
Paired Plant Immune Receptors
Authors: Nishimura Marc T. , Dangl Jeffery L.
Published: April 2014

The ability of plant cell immune sensors to combine in different pairs could expand the host's defense against pathogens.

[Also see Report by

Williams
et al.

]
'Humanized' Mouse Detects Deadly Drug Side Effects
Authors: Cohen Jon
Published: April 2014

A chimeric mouse with a humanized liver offers a novel window into drug toxicity.
Ancient DNA Holds Clues to Gene Activity in Extinct Humans
Authors: Pennisi Elizabeth
Published: April 2014

Researchers have harnessed the chemical degradation of fossil DNA to determine methylation patterns that may reveal which genes were turned on, or off, in ancient human species.
More Sophisticated Forecasts Yield Glimmer of Hope in Climate Gloom
Authors: Kintisch Eli
Published: April 2014

The latest Intergovernmental Panel on Climate Change report, on mitigating emissions, finds a few glimmers of hope amid gloomy projections.
A Quality Control Program within a Clinical Trial Consortium for PCR Protocols To Detect Plasmodium Species
Authors: Taylor Steve M. , Mayor Alfredo , Mombo-Ngoma Ghyslain , Kenguele Hilaire M. , Ouédraogo Smaïla , Ndam Nicaise Tuikue , Mkali Happy , Mwangoka Grace , Valecha Neena , Singh Jai Prakash Narayan , Clark Martha A. , Verweij Jaco J. , Adegnika Ayola Akim , Severini Carlo , Menegon Michela , Macete Eusebio , Menendez Clara , Cisteró Pau , Njie Fanta , Affara Muna , Otieno Kephas , Kariuki Simon , ter Kuile Feiko O. , Meshnick Steven R.
Published: April 2014

Malaria parasite infections that are only detectable by molecular methods are highly prevalent and represent a potential transmission reservoir. The methods used to detect these infections are not standardized, and their operating characteristics are often unknown. We designed a proficiency panel of
Plasmodium
spp. in order to compare the accuracy of parasite detection of molecular protocols used by labs in a clinical trial consortium. Ten dried blood spots (DBSs) were assembled that contained
P. falciparum
,
P. vivax
,
P. malariae
, and
P. ovale
; DBSs contained either a single species or a species mixed with
P. falciparum
. DBS panels were tested in 9 participating laboratories in a masked fashion. Of 90 tests, 68 (75.6%) were correct; there were 20 false-negative results and 2 false positives. The detection rate was 77.8% (49/63) for
P. falciparum
, 91.7% (11/12) for
P. vivax
, 83.3% (10/12) for
P. malariae
, and 70% (7/10) for
P. ovale
. Most false-negative
P. falciparum
results were from samples with an estimated ≤5 parasites per μl of blood. Between labs, accuracy ranged from 100% to 50%. In one lab, the inability to detect species in mixed-species infections prompted a redesign and improvement of the assay. Most PCR-based protocols were able to detect
P. falciparum
and
P. vivax
at higher densities, but these assays may not reliably detect parasites in samples with low
P. falciparum
densities. Accordingly, formal quality assurance for PCR should be employed whenever this method is used for diagnosis or surveillance. Such efforts will be important if PCR is to be widely employed to assist malaria elimination efforts.
Profiling of
rpoB
Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis
Authors: Jamieson F. B. , Guthrie J. L. , Neemuchwala A. , Lastovetska O. , Melano R. G. , Mehaffy C.
Published: April 2014

Resistance to rifampin (RIF) and rifabutin (RFB) in
Mycobacterium tuberculosis
is associated with mutations within an 81-bp region of the
rpoB
gene (RIF resistance-determining region [RRDR]). Previous studies have shown that certain mutations in this region are more likely to confer high levels of RIF resistance, while others may be found in phenotypically susceptible isolates. In this study, we sought to determine the relationship between the MICs of RIF and RFB and
rpoB
RRDR mutations in 32 multidrug-resistant (MDR), 4 RIF-monoresistant, and 5 susceptible
M. tuberculosis
clinical isolates. The MICs were determined using the MGIT 960 system. Mutations in the
rpoB
RRDR were determined by Sanger sequencing. RpoB proteins with mutations S531L (a change of S to L at position 531), S531W, H526Y, and H526D and the double mutation D516A-R529Q were associated with high MICs for RIF and RFB. Five isolates carrying the mutations L511P, H526L, H526N, and D516G-S522L were found to be susceptible to RIF. Several mutations were associated with resistance to RIF and susceptibility to RFB (F514FF, D516V, and S522L). Whole-genome sequencing of two MDR isolates without
rpoB
RRDR mutations revealed a mutation outside the RRDR (V146F; RIF MIC of 50 μg/ml). The implications of the polymorphisms identified in the second of these isolates in RIF resistance need to be further explored. Our study further establishes a correlation between the mutations and the MICs of RIF and, also, RFB in
M. tuberculosis
. Several
rpoB
mutations were identified in RIF- and RFB-susceptible isolates. The clinical significance of these findings requires further exploration. Until then, a combination of phenotypic and molecular testing is advisable for drug susceptibility testing.
Within-Host Whole-Genome Deep Sequencing and Diversity Analysis of Human Respiratory Syncytial Virus Infection Reveals Dynamics of Genomic Diversity in the Absence and Presence of Immune Pressure
Authors: Grad Yonatan H. , Newman Ruchi , Zody Michael , Yang Xiao , Murphy Ryan , Qu James , Malboeuf Christine M. , Levin Joshua Z. , Lipsitch Marc , DeVincenzo John
Published: April 2014

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children and an important respiratory pathogen in the elderly and immunocompromised. While population-wide molecular epidemiology studies have shown multiple cocirculating RSV genotypes and revealed antigenic and genetic change over successive seasons, little is known about the extent of viral diversity over the course of an individual infection, the origins of novel variants, or the effect of immune pressure on viral diversity and potential immune-escape mutations. To investigate viral population diversity in the presence and absence of selective immune pressures, we studied whole-genome deep sequencing of RSV in upper airway samples from an infant with severe combined immune deficiency syndrome and persistent RSV infection. The infection continued over several months before and after bone marrow transplant (BMT) from his RSV-immune father. RSV diversity was characterized in 26 samples obtained over 78 days. Diversity increased after engraftment, as defined by T-cell presence, and populations reflected variation mostly within the G protein, the major surface antigen. Minority populations with known palivizumab resistance mutations emerged after its administration. The viral population appeared to diversify in response to selective pressures, showing a statistically significant growth in diversity in the presence of pressure from immunity. Defining escape mutations and their dynamics will be useful in the design and application of novel therapeutics and vaccines. These data can contribute to future studies of the relationship between within-host and population-wide RSV phylodynamics.

IMPORTANCE
Human RSV is an important cause of respiratory disease in infants, the elderly, and the immunocompromised. RSV circulating in a community appears to change season by season, but the amount of diversity generated during an individual infection and the impact of immunity on this viral diversity has been unclear. To address this question, we described within-host RSV diversity by whole-genome deep sequencing in a unique clinical case of an RSV-infected infant with severe combined immunodeficiency and effectively no adaptive immunity who then gained adaptive immunity after undergoing bone marrow transplantation. We found that viral diversity increased in the presence of adaptive immunity and was primarily within the G protein, the major surface antigen. These data will be useful in designing RSV treatments and vaccines and to help understand the relationship between the dynamics of viral diversification within individual hosts and the viral populations circulating in a community.
Lack of Significant Elevation of Myeloid-Derived Suppressor Cells in Peripheral Blood of Chronically Hepatitis C Virus-Infected Individuals
Authors: Nonnenmann Julia , Stirner Renate , Roider Julia , Jung Maria C. , Schrödl Kathrin , Bogner Johannes R. , Draenert Rika
Published: April 2014

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive function. Compared to the level in healthy controls (HC), no elevation of MDSC in chronic hepatitis C (cHEP-C) patients was found, and there was no difference in MDSC based on genotype or viral load (
P
> 0.25). Moreover, MDSC of cHEP-C patients inhibited CD8 T cell function as efficiently as MDSC of HC did. Since we detected neither quantitative nor qualitative differences in MDSC of cHEP-C patients relative to those of HC, we postulate that MDSC in peripheral blood are most likely not significant regarding immune dysfunction in cHEP-C.
Enhanced Viral Replication and Modulated Innate Immune Responses in Infant Airway Epithelium following H1N1 Infection
Authors: Clay Candice C. , Reader J. Rachel , Gerriets Joan E. , Wang Theodore T. , Harrod Kevin S. , Miller Lisa A.
Published: April 2014

Influenza is the cause of significant morbidity and mortality in pediatric populations. The contribution of pulmonary host defense mechanisms to viral respiratory infection susceptibility in very young children is poorly understood. As a surrogate to compare mucosal immune responses of infant and adult lungs, rhesus monkey primary airway epithelial cell cultures were infected with pandemic influenza A/H1N1 virusin vitro. Virus replication, cytokine secretion, cell viability, and type I interferon (IFN) pathway PCR array profiles were evaluated for both infant and adult cultures. In comparison with adult cultures, infant cultures showed significantly increased levels of H1N1 replication, reduced alpha interferon (IFN-α) protein synthesis, and no difference in cell death following infection. Age-dependent differences in expression levels of multiple genes associated with the type I IFN pathway were observed in H1N1-infected cultures. To investigate the pulmonary and systemic responses to H1N1 infection in early life, infant monkeys were inoculated with H1N1 by upper airway administration. Animals were monitored for virus and parameters of inflammation over a 14-day period. High H1N1 titers were recovered from airways at day 1, with viral RNA remaining detectable until day 9 postinfection. Despite viral clearance, bronchiolitis and alveolitis persisted at day 14 postinfection; histopathological analysis revealed alveolar septal thickening and intermittent type II pneumocyte hyperplasia. Our overall findings are consistent with the known susceptibility of pediatric populations to respiratory virus infection and suggest that intrinsic developmental differences in airway epithelial cell immune function may contribute to the limited efficacy of host defense during early childhood.

IMPORTANCETo the best of our knowledge, this study represents the first report of intrinsic developmental differences in infant airway epithelial cells that may contribute to the increased susceptibility of the host to respiratory virus infections. Despite the global burden of influenza, there are currently no vaccine formulations approved for children <6 months of age. Given the challenges of conducting experimental studies involving pediatric patients, rhesus monkeys are an ideal laboratory animal model to investigate the maturation of pulmonary mucosal immune mechanisms during early life because they are most similar to those of humans with regard to postnatal maturation of the lung structure and the immune system. Thus, our findings are highly relevant to translational medicine, and these data may ultimately lead to novel approaches that enhance airway immunity in very young children.
Characteristics of Nucleocytoplasmic Transport of H1N1 Influenza A Virus Nuclear Export Protein
Authors: Gao Shengyan , Wang Shanshan , Cao Shuai , Sun Lei , Li Jing , Bi Yuhai , Gao George F. , Liu Wenjun
Published: April 2014

The influenza A virus nuclear export protein (NEP) plays crucial roles in the nuclear export of the viral ribonucleoprotein complex through the chromosome region maintenance 1 (CRM1)-mediated cellular protein transport system. However, the detailed mechanism of NEP nucleocytoplasmic trafficking remains incompletely understood. Here, we investigated the subcellular localization of NEP from two strains of H1N1 influenza A virus and found that 2009 swine-origin H1N1 influenza A virus A/California/04/2009 (CA04) NEP displayed a distinct cellular distribution pattern, forming unique nuclear aggregates, compared to A/WSN/33 (H1N1) (WSN) NEP. Characterization of the nucleocytoplasmic transport pathways of these two NEPs showed that they both enter the nucleus by passive diffusion but are exported through the nuclear export receptor CRM1-mediated pathway with different efficiencies. The two identified nuclear export signals (NESs) on the two NEPs functioned similarly despite differences in their amino acid sequences. Using a two-hybrid assay, we confirmed that the CA04 NEP interacts less efficiently with CRM1 and that a threonine residue at position 48 is responsible for the nuclear aggregation. The present study revealed the dissimilarity in subcellular NEP transport processes between the 2009 pandemic (H1N1) influenza A virus CA04 and the laboratory-adapted H1N1 virus WSN and uncovered the mechanism responsible for this difference.

IMPORTANCE
Because the efficiency of the nucleocytoplasmic transport of viral components is often correlated with the viral RNA polymerase activity, propagation, and host range of influenza viruses, the present study investigated the subcellular localization of NEP from two strains of H1N1 influenza virus. We found that the NEPs of both A/California/04/2009 (H1N1) (CA04) and A/WSN/33 (H1N1) (WSN) enter the nucleus by passive diffusion but are exported with different efficiencies, which were caused by weaker binding activity between the CA04 NEP and CRM1. The results of the present study revealed characteristics of the nuclear import and export pathways of NEP and the mechanism responsible for the differences in the cellular distribution of NEP between two H1N1 strains.
Hepatitis Delta Antigen Requires a Flexible Quasi-Double-Stranded RNA Structure To Bind and Condense Hepatitis Delta Virus RNA in a Ribonucleoprotein Complex
Authors: Griffin Brittany L. , Chasovskikh Sergey , Dritschilo Anatoly , Casey John L.
Published: April 2014

The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2′OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the
in vitro
binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed
in vitro
revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg.

IMPORTANCE
RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex.
Cellular Transcriptome Analysis Reveals Differential Expression of Pro- and Antiapoptosis Genes by Varicella-Zoster Virus-Infected Neurons and Fibroblasts
Authors: Markus Amos , Waldman Ben-Asher Hiba , Kinchington Paul R. , Goldstein Ronald S.
Published: April 2014

Transcriptional changes following varicella-zoster virus (VZV) infection of cultured human neurons derived from embryonic stem cells were compared to those in VZV-infected human foreskin fibroblasts. Transcription of 340 neuronal genes significantly altered by VZV infection included 223 transcript changes unique to neurons. Strikingly, genes inhibiting apoptosis were upregulated in neurons, while proapoptotic gene transcription was increased in fibroblasts. These data are a basis for discovery of differences in virus-host interactions between these VZV targets.
NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies
Authors: Boukadida Célia , Marnata Caroline , Montserret Roland , Cohen Lisette , Blumen Brigitte , Gouttenoire Jérôme , Moradpour Darius , Penin François , Martin Annette
Published: April 2014

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the
Hepacivirus
genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using
trans
-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently
trans
-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis.

IMPORTANCE
Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.
Prolonged Activity of the Pestiviral RNase E
rns
as an Interferon Antagonist after Uptake by Clathrin-Mediated Endocytosis
Authors: Zürcher Christoph , Sauter Kay-Sara , Mathys Veronika , Wyss Fabienne , Schweizer Matthias
Published: April 2014

The RNase activity of the envelope glycoprotein E
rns
of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E
rns
is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E
rns
. Here, we show that the activity of soluble E
rns
as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E
rns
protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E
rns
mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E
rns
remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E
rns
acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance.

IMPORTANCE
The pestiviral RNase E
rns
was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of E
rns
at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E
rns
is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E
rns
remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.
Vaginal Myeloid Dendritic Cells Transmit Founder HIV-1
Authors: Shen Ruizhong , Kappes John C. , Smythies Lesley E. , Richter Holly E. , Novak Lea , Smith Phillip D.
Published: April 2014

We report that primary human vaginal dendritic cells (DCs) display a myeloid phenotype and express CD4, CCR5, and CXCR4. Vaginal CD13
+
CD11c
+
DCs rapidly and efficiently bound transmitted/founder (T/F) CCR5-tropic (R5) viruses, transported them through explanted vaginal mucosa, and transmitted them in
trans
to vaginal and blood lymphocytes. Vaginal myeloid DCs may play a key role in capturing and disseminating T/F R5 HIV-1
in vivo
and are candidate “gatekeeper” cells in HIV-1 transmission.
CTCF Binding to the First Intron of the Major Immediate Early (MIE) Gene of Human Cytomegalovirus (HCMV) Negatively Regulates MIE Gene Expression and HCMV Replication
Authors: Martínez Francisco Puerta , Cruz Ruth , Lu Fang , Plasschaert Robert , Deng Zhong , Rivera-Molina Yisel A. , Bartolomei Marisa S. , Lieberman Paul M. , Tang Qiyi
Published: April 2014

Human cytomegalovirus (HCMV) gene expression during infection is highly regulated, with sequential expression of immediate-early (IE), early (E), and late (L) gene transcripts. To explore the potential role of chromatin regulatory factors that may regulate HCMV gene expression and DNA replication, we investigated the interaction of HCMV with the cellular chromatin-organizing factor CTCF. Here, we show that HCMV-infected cells produce higher levels of CTCF mRNA and protein at early stages of infection. We also show that CTCF depletion by short hairpin RNA results in an increase in major IE (MIE) and E gene expression and an about 50-fold increase in HCMV particle production. We identified a DNA sequence (TTAACGGTGGAGGGCAGTGT) in the first intron (intron A) of the MIE gene that interacts directly with CTCF. Deletion of this CTCF-binding site led to an increase in MIE gene expression in both transient-transfection and infection assays. Deletion of the CTCF-binding site in the HCMV bacterial artificial chromosome plasmid genome resulted in an about 10-fold increase in the rate of viral replication relative to either wild-type or revertant HCMV. The CTCF-binding site deletion had no detectable effect on MIE gene-splicing regulation, nor did CTCF knockdown or overexpression of CTCF alter the ratio of IE1 to IE2. Therefore, CTCF binds to DNA within the MIE gene at the position of the first intron to affect RNA polymerase II function during the early stages of viral transcription. Finally, the CTCF-binding sequence in CMV is evolutionarily conserved, as a similar sequence in murine CMV (MCMV) intron A was found to interact with CTCF and similarly function in the repression of MCMV MIE gene expression mediated by CTCF.

IMPORTANCE
Our findings that CTCF binds to intron A of the cytomegalovirus (CMV) major immediate-early (MIE) gene and functions to repress MIE gene expression and viral replication are highly significant. For the first time, a chromatin-organizing factor, CTCF, has been found to facilitate human CMV gene expression, which affects viral replication. We also identified a CTCF-binding motif in the first intron (also called intron A) that directly binds to CTCF and is required for CTCF to repress MIE gene expression. Finally, we show that the CTCF-binding motif is conserved in CMV because a similar DNA sequence was found in murine CMV (MCMV) that is required for CTCF to bind to MCMV MIE gene to repress MCMV MIE gene expression.
Determinants of Moloney Murine Leukemia Virus Gag-Pol and Genomic RNA Proportions
Authors: Johnson Silas F. , Collins John T. , D'Souza Victoria M. , Telesnitsky Alice
Published: April 2014

The Moloney murine leukemia virus (MoMLV) ribonucleoprotein complex is composed of an approximately 20:1 mixture of Gag and Gag-Pol polyproteins plus a single genomic RNA (gRNA) dimer. The mechanisms that regulate these proportions are unknown. Here, we examined whether virion proportions of Gag, Gag-Pol, and gRNA were determined by sampling (that is, if they reflected expression ratios or intracellular concentrations) or more specific recruitment. To this end, MoMLV Gag, Gag-Pol, and gRNA were expressed separately or together in various ratios. Varying the expression ratios of Gag and Gag-Pol revealed that Gag-Pol incorporation was stochastic and that the conserved 20:1 Gag/Gag-Pol ratio coincided with maximal particle production. When skewed expression ratios resulted in excess Gag-Pol, the released virions maintained the intracellular Gag/Gag-Pol ratios and the infectivity per virion was largely maintained, but virion production decreased sharply with high levels of Gag-Pol. The determinants of gRNA proportions were addressed by manipulating the amounts and contexts of functional nucleocapsid (NC) and the ratios of Gag to gRNA. The results showed that the NC domain of either Gag or Gag-Pol could provide gRNA packaging functions equally well. Unlike Gag-Pol, gRNA incorporation was saturable. An upper limit of gRNA incorporation was observed, and particle production was not disrupted by excess gRNA expression. These results indicate that the determinants of Gag/Gag-Pol proportions differ from those for Gag/gRNA. On the basis of the assumption that MoMLV evolved to produce virion components in optimal proportions, these data provide a means of estimating the proportion of unspliced MoMLV RNA that serves as genomic RNA.

IMPORTANCE
Viruses assemble their progeny from within the cells that they parasitize, where they must sort through a rich milieu of host proteins and nucleic acids to gather together their own building blocks, which are also proteins and nucleic acids. The research described here addresses whether or not the proportions of viral proteins and nucleic acids that are brought together to form a retroviral particle are determined by random sampling from the cell—and thus dictated by the components' availabilities within the cell—or if the amounts of each molecule are specified by the virus replication process. The results indicated that protein components of the murine retrovirus studied here are recruited by chance but that a specific counting mechanism defines the amount of nucleic acid incorporated into each progeny virion.
Mechanism and Significance of Cell Type-Dependent Neutralization of Flaviviruses
Authors: Mukherjee Swati , Dowd Kimberly A. , Manhart Carolyn J. , Ledgerwood Julie E. , Durbin Anna P. , Whitehead Stephen S. , Pierson Theodore C.
Published: April 2014

The production of neutralizing antibodies (NAbs) is a correlate of protection for many human vaccines, including currently licensed vaccines against flaviviruses. NAbs are typically measured using a plaque reduction neutralization test (PRNT). Despite its extensive use, parameters that impact the performance of the PRNT have not been investigated from a mechanistic perspective. The results of a recent phase IIb clinical trial of a tetravalent dengue virus (DENV) vaccine suggest that NAbs, as measured using a PRNT performed with Vero cells, do not correlate with protection. This surprising finding highlights the importance of understanding how well the PRNT captures the complexity of the NAb response to DENV. In this study, we demonstrated that the structural heterogeneity of flaviviruses arising from inefficient virion maturation impacts the results of neutralization assays in a cell type-dependent manner. Neutralization titers of several monoclonal antibodies were significantly reduced when assayed on Vero cells compared to Raji cells expressing DC-SIGNR. This pattern can be explained by differences in the efficiency with which partially mature flaviviruses attach to each cell type, rather than a differential capacity of antibody to block infection. Vero cells are poorly permissive to the fraction of virions that are most sensitive to neutralization. Analysis of sera from recipients of live-attenuated monovalent DENV vaccine candidates revealed a strong correlation between the sensitivity of serum antibodies to the maturation state of DENV and cell type-dependent patterns of neutralization. Cross-reactive patterns of neutralization may be underrepresented by the “gold-standard” PRNT that employs Vero cells.

IMPORTANCE
Cell type-dependent patterns of neutralization describe a differential capacity of antibodies to inhibit virus infection when assayed on multiple cellular substrates. In this study, we established a link between antibodies that neutralize infection in a cell type-dependent fashion and those sensitive to the maturation state of the flavivirus virion. We demonstrated that cell type-dependent neutralization reflects a differential capacity to measure neutralization of viruses that are incompletely mature. Partially mature virions that most efficiently bind maturation state-sensitive antibodies are poorly represented by assays typically used in support of flavivirus vaccine development. The selection of cellular substrate for neutralization assays may significantly impact evaluation of the neutralization potency of the polyclonal response. These data suggest that current assays do not adequately capture the full complexity of the neutralizing antibody response and may hinder the identification of correlates of protection following flavivirus vaccination.
Transcription Factor Binding Site Mapping Using ChIP-Seq
Authors: Jaini Suma , Lyubetskaya Anna , Gomes Antonio , Peterson Matthew , Tae Park Sang , Raman Sahadevan , Schoolnik Gary , Galagan James
Published: April 2014

Transcription factors (TFs) play a central role in regulating gene expression in all bacteria. Yet until recently, studies of TF binding were limited to a small number of factors at a few genomic locations. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) provides the ability to map binding sites globally for TFs, and the scalability of the technology enables the ability to map binding sites for every DNA binding protein in a prokaryotic organism. We have developed a protocol for ChIP-Seq tailored for use with mycobacteria and an analysis pipeline for processing the resulting data. The protocol and pipeline have been used to map over 100 TFs from
Mycobacterium tuberculosis
, as well as numerous TFs from related mycobacteria and other bacteria. The resulting data provide evidence that the long-accepted spatial relationship between TF binding site, promoter motif, and the corresponding regulated gene may be too simple a paradigm, failing to adequately capture the variety of TF binding sites found in prokaryotes. In this article we describe the protocol and analysis pipeline, the validation of these methods, and the results of applying these methods to
M. tuberculosis
.
Solid pseudopapillary tumour of pancreas
Authors: Singh Ashish , Agarwal Akshay Anand , Sonkar Abhinav Arun , Kushwaha Jitendra Kumar
Published: April 2014

Solid pseudopapillary tumour of the pancreas is a rare neoplasm (1%). This tumour primarily affects young women and is usually treated with surgical resection with a relatively favourable prognosis. We report an 18-year-old female patient presenting with moderate grade abdominal pain for 5 weeks. Abdominal examination revealed a lump palpable in the right upper abdomen. Contrast-enhanced CT abdomen reported soft tissue lesion arising from uncinate process of pancreas causing adjacent compression. Endoscopic ultrasound-guided fine-needle aspiration biopsy yielded a cellular sample comprising pseudopapillary arrangement with bland appearing tumour cells consistent with benign neoplasm. And because of unusual location, Whipple procedure was performed. The patient was discharged under satisfactory conditions. Final histology confirmed the diagnosis. Solid pseudopapillary tumours of the pancreas are a rare but treatable pancreatic tumour. Complete surgical excision is the treatment of choice and can be achieved through an open or minimal access technique.
Amyotrophic lateral sclerosis and parkinsonism in Papua, Indonesia: 2001–2012 survey results
Authors: Okumiya Kiyohito , Wada Taizo , Fujisawa Michiko , Ishine Masayuki , Garcia del Saz Eva , Hirata Yutaka , Kuzuhara Shigeki , Kokubo Yasumasa , Seguchi Harumichi , Sakamoto Ryota , Manuaba Indrajaya , Watofa Paulina , Rantetampang Andreas L , Matsubayashi Kozo
Published: April 2014

Objective

Only one previous follow-up study of amyotrophic lateral sclerosis (ALS) and parkinsonism in Papua, Indonesia has been carried out since a survey undertaken in 1962–1981 by Gajdusek and colleagues. Therefore, to clarify the clinical epidemiology of ALS and parkinsonism in the southern coastal region of Papua, the clinical characteristics and prevalence of the diseases in this region were examined and assessed.

Methods

Cases of ALS and parkinsonism were clinically examined during a 2001–2012 survey in Bade and other villages along the Ia, Edera, Dumut and Obaa rivers in Papua, Indonesia. Possible, probable and definite ALS was diagnosed clinically by certified neurologists based on El Escorial criteria. The criteria for a diagnosis of parkinsonism were the presence of at least two of the four following signs: tremor, rigidity, bradykinesia and postural impairment with a progressive course.

Results

During the survey, 46 cases of ALS and/or parkinsonism were diagnosed within a population range of 7000 (2001–2002) to 13 900 (2007–2012). The 46 cases consisted of 17 probable-definite cases of ALS, including three with cognitive impairment (CI), 13 cases of overlapping possible, probable or definite ALS and parkinsonism, including five with CI, and 16 cases of parkinsonism, including one with CI. The crude point prevalence rate of pure ALS was estimated to be at least 73 (95% CI 0 to 156) to 133 (27 to 240)/100 000 people and that of overlapping ALS and parkinsonism at least 53 (0 to 126) to 98 (2 to 193)/100 000 in 2007, or 2010 in some regions.

Conclusions

While the prevalence of ALS in Papua has decreased over the past ∼30–35 years, it remains higher than the global average. There was a high prevalence of overlapping ALS, parkinsonism and CI, which has also been previously reported in Guam and Kii.
Hypoxia, haemorrhage and hypotension: the interface between emergency medicine and intensive care medicine
Authors: Foëx Bernard A
Published: April 2014

This subjective review is based on a presentation made at the College of Emergency Medicine Scientific Conference in September 2013. My theme was that there are certain features of the critically ill which cause understandable anxiety, namely hypoxia, haemorrhage and hypotension. So, I have selected papers relevant to the management of these frightening situations.