bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of and Screening for Human Helicobacter cinaedi Infections and Carriers via Nested PCR
Authors: Oyama Kohta , Khan Shahzada , Okamoto Tatsuya , Fujii Shigemoto , Ono Katsuhiko , Matsunaga Tetsuro , Yoshitake Jun , Sawa Tomohiro , Tomida Junko , Kawamura Yoshiaki , Akaike Takaaki
Published: September 2012

Helicobacter cinaedi
is the most frequently reported enterohepatic
Helicobacter
species isolated from humans. Earlier research suggested that certain patients with
H. cinaedi
infection may remain undiagnosed or incorrectly diagnosed because of difficulties in detecting the bacteria by conventional culture methods. Here, we report a nested PCR assay that rapidly detects the cytolethal distending toxin gene (
cdt
) of
H. cinaedi
with high specificity and sensitivity. Specificity of the assay was validated by using different species of
Helicobacter
and
Campylobacter
, as well as known
H. cinaedi
-positive and -negative samples. The sensitivity of detection for the
cdt
gene in the assay was 10
2
CFU/ml urine or 10
2
CFU/10
5
infected RAW 264.7 cells. In an
H. cinaedi
-infected mouse model, the
cdt
gene of
H. cinaedi
was effectively detected via the assay with urine (6/7), stool (2/3), and blood (2/6) samples. Importantly, it detected
H. cinaedi
in blood, urine, and stool samples from one patient with a suspected
H. cinaedi
infection and three patients with known infections. The assay was further used clinically to follow up two
H. cinaedi
-infected patients after antibiotic treatment. Stool samples from these two patients evaluated by nested PCR after antibiotic therapy showed clearance of bacterial DNA. Finally, analysis of stool specimens from healthy volunteers showed occasional positive reactions (4/30) to
H. cinaedi
DNA, which suggests intestinal colonization by
H. cinaedi
in healthy subjects. In conclusion, this nested PCR assay may be useful for the rapid diagnosis, antimicrobial treatment evaluation, and epidemiological study of
H. cinaedi
infection.
Prospective Multicenter Study of Pneumocystis jirovecii Colonization among Cystic Fibrosis Patients in France
Authors: Hernández-Hernández Francisca , Fréalle Emilie , Caneiro Patrick , Salleron Julia , Durand-Joly Isabelle , Accoceberry Isabelle , Bouchara Jean-Philippe , Wallaert Benoit , Dei-Cas Eduardo , Delhaes Laurence
Published: September 2012

Pneumocystis
carriage was detected in 12.5% of 104 cystic fibrosis (CF) patients during a prospective multicenter French study, with a prevalence of genotype 85C/248C and geographic variations. It was significantly associated with the absence of
Pseudomonas aeruginosa
colonization and a greater forced expiratory volume in 1 s. Results are discussed considering the natural history of CF.
Activating KIR Copy Number Variation Is Associated with Granzyme B Release by NK Cells during Primary Simian Immunodeficiency Virus Infection in Rhesus Monkeys
Authors: Hellmann Ina , Schmitz Jörn E. , Letvin Norman L.
Published: September 2012

Here we show that the number of activating killer cell immunoglobulin-like receptor (KIR) copies in rhesus monkeys is associated with the extent of release of cytotoxic granules by cytolytic NK cells during primary simian immunodeficiency virus SIVmac251 infection. These findings suggest that NK cells expressing high levels of activating KIRs efficiently kill SIVmac251-infected cells, and this efficient killing contributes to the NK cell-mediated control of replication of this virus during early infection.
In Vitro
Assembly of an Empty Picornavirus Capsid follows a Dodecahedral Path
Authors: Li Chenglei , Wang Joseph Che-Yen , Taylor Milton W. , Zlotnick Adam
Published: September 2012

The
Picornaviridae
are a large family of small, spherical RNA viruses that includes numerous pathogens. The picornavirus structural proteins VP0, VP1, and VP3 are believed to first form protomers, which then form 14S particles and subsequently assemble to form empty and RNA-filled particles. 14S particles have long been presumed to be pentamers. However, the structure of the 14S particles, their mechanism of assembly, and the role of empty particles during infection are all unknown. We established an
in vitro
assembly system for bovine enterovirus (BEV) by using purified baculovirus-expressed proteins. By Rayleigh scattering, we determined that 14S particles are 488 kDa, confirming they are pentamers. Image reconstructions based on negative-stain electron microscopy showed that 14S particles have 5-fold symmetry, and their structures correlate extremely well with the corresponding pentamer from crystal structures of mature BEV. Purified 14S particles readily assemble in response to increasing ionic strength or temperature to form 5.8-MDa 12-pentamer particles, indistinguishable from native empty particles. Surprisingly, empty particles were sufficiently stable that, under physiological conditions, dissociation is unlikely to be a biologically relevant reaction. This suggests that empty particles are not a storage form of 14S particles, at least for bovine enterovirus, but are either a dead-end product or direct precursor into which viral RNA is packaged by as-yet-unidentified machinery.
Human Metapneumovirus M2-2 Protein Inhibits Innate Cellular Signaling by Targeting MAVS
Authors: Ren Junping , Wang Qingrong , Kolli Deepthi , Prusak Deborah J. , Tseng Chien-Te K. , Chen Zhijian J. , Li Kui , Wood Thomas G. , Bao Xiaoyong
Published: September 2012

Human metapneumovirus (hMPV) is a leading cause of respiratory infections in pediatric populations globally, with no prophylactic or therapeutic measures. Recently, a recombinant hMPV lacking the M2-2 protein (rhMPV-ΔM2-2) demonstrated reduced replication in the respiratory tract of animal models, making it a promising live vaccine candidate. However, the exact nature of the interaction between the M2-2 protein and host cells that regulates viral infection/propagation is largely unknown. By taking advantage of the available reverse genetics system and ectopic expression system for viral protein, we found that M2-2 not only promotes viral gene transcription and replication but subverts host innate immunity, therefore identifying M2-2 as a novel virulence factor, in addition to the previously described hMPV G protein. Since we have shown that the RIG-I/MAVS pathway plays an important role in hMPV-induced signaling in airway epithelial cells, we investigated whether M2-2 antagonizes the host cellular responses by targeting this pathway. Reporter gene assays and coimmunoprecipitation studies indicated that M2-2 targets MAVS, an inhibitory mechanism different from what we previously reported for hMPV G, which affects RIG-I- but not MAVS-dependent gene transcription. In addition, we found that the domains of M2-2 responsible for the regulation of viral gene transcription and antiviral signaling are different. Our findings collectively demonstrate that M2-2 contributes to hMPV immune evasion through the inhibition of MAVS-dependent cellular responses.
Heparan Sulfate Facilitates Rift Valley Fever Virus Entry into the Cell
Authors: de Boer S. M. , Kortekaas J. , de Haan C. A. M. , Rottier P. J. M. , Moormann R. J. M. , Bosch B. J.
Published: September 2012

Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.
Influenza Virus Neuraminidases with Reduced Enzymatic Activity That Avidly Bind Sialic Acid Receptors
Authors: Zhu Xueyong , McBride Ryan , Nycholat Corwin M. , Yu Wenli , Paulson James C. , Wilson Ian A.
Published: September 2012

Influenza virus neuraminidase (NA) cleaves off sialic acid from cellular receptors of hemagglutinin (HA) to enable progeny escape from infected cells. However, NA variants (D151G) of recent human H3N2 viruses have also been reported to bind receptors on red blood cells, but the nature of these receptors and the effect of the mutation on NA activity were not established. Here, we compare the functional and structural properties of a human H3N2 NA from A/Tanzania/205/2010 and its D151G mutant, which supports HA-independent receptor binding. While the wild-type NA efficiently cleaves sialic acid from both α2-6- and α2-3-linked glycans, the mutant exhibits much reduced enzymatic activity toward both types of sialosides. Conversely, while wild-type NA shows no detectable binding to sialosides, the D151G NA exhibits avid binding with broad specificity toward α2-3 sialosides. D151G NA binds the 3′ sialyllactosamine (3′-SLN) and 6′-SLN sialosides with equilibrium dissociation constant (

K
D

) values of 30.0 μM and 645 μM, respectively, which correspond to much higher affinities than the corresponding affinities (low mM) of HA to these glycans. Crystal structures of wild-type and mutant NAs reveal the structural basis for glycan binding in the active site by exclusively impairing the glycosidic bond hydrolysis step. The general significance of D151 among influenza virus NAs was further explored by introducing the D151G mutation into three N1 NAs and one N2 NA, which all exhibited reduced enzymatic activity and preferential binding to α2-3 sialosides. Since the enzymatic and binding activities of NAs are not routinely assessed, the potential for NA receptor binding to contribute to influenza virus biology may be underappreciated.
Properties of the Adeno-Associated Virus Assembly-Activating Protein
Authors: Naumer Matthias , Sonntag Florian , Schmidt Kristin , Nieto Karen , Panke Christian , Davey Norman E. , Popa-Wagner Ruth , Kleinschmidt Jürgen A.
Published: September 2012

Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the
cap
gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.
Interferon-Inducible Protein Mx1 Inhibits Influenza Virus by Interfering with Functional Viral Ribonucleoprotein Complex Assembly
Authors: Verhelst Judith , Parthoens Eef , Schepens Bert , Fiers Walter , Saelens Xavier
Published: September 2012

Mx1 is a GTPase that is part of the antiviral response induced by type I and type III interferons in the infected host. It inhibits influenza virus infection by blocking viral transcription and replication, but the molecular mechanism is not known. Polymerase basic protein 2 (PB2) and nucleoprotein (NP) were suggested to be the possible target of Mx1, but a direct interaction between Mx1 and any of the viral proteins has not been reported. We investigated the interplay between Mx1, NP, and PB2 to identify the mechanism of Mx1's antiviral activity. We found that Mx1 inhibits the PB2-NP interaction, and the strength of this inhibition correlated with a decrease in viral polymerase activity. Inhibition of the PB2-NP interaction is an active process requiring enzymatically active Mx1. We also demonstrate that Mx1 interacts with the viral proteins NP and PB2, which indicates that Mx1 protein has a direct effect on the viral ribonucleoprotein complex. In a minireplicon system, avian-like NP from swine virus isolates was more sensitive to inhibition by murine Mx1 than NP from human influenza A virus isolates. Likewise, murine Mx1 displaced avian NP from the viral ribonucleoprotein complex more easily than human NP. The stronger resistance of the A/H1N1 pandemic 2009 virus against Mx1 also correlated with reduced inhibition of the PB2-NP interaction. Our findings support a model in which Mx1 interacts with the influenza ribonucleoprotein complex and interferes with its assembly by disturbing the PB2-NP interaction.
Genome-Wide Analyses of Zta Binding to the Epstein-Barr Virus Genome Reveals Interactions in both Early and Late Lytic Cycles and an Epigenetic Switch Leading to an Altered Binding Profile
Authors: Ramasubramanyan Sharada , Kanhere Aditi , Osborn Kay , Flower Kirsty , Jenner Richard G. , Sinclair Alison J.
Published: September 2012

The Epstein-Barr virus (EBV) genome sustains substantial epigenetic modification involving chromatin remodelling and DNA methylation during lytic replication. Zta (ZEBRA, BZLF1), a key regulator of the EBV lytic cycle, is a transcription and replication factor, binding to Zta response elements (ZREs) in target promoters and EBV lytic origins of replication.
In vitro
, Zta binding is modulated by DNA methylation; a subset of CpG-containing Zta binding sites (CpG ZREs) is bound only in a DNA methylation-dependent manner. The question of how the dynamic epigenetic environment impacts Zta interaction during the EBV lytic cycle is unknown. To address this, we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) to identify Zta binding sites across the EBV genome before and after viral DNA replication. Replication did not alter the association of Zta across many regions of the EBV genome, but a striking reduction in Zta binding occurred at some loci that contain CpG ZREs. Separating Zta-bound DNA into methylated and nonmethylated fractions, we found that promoters that contain CpG ZREs were enriched in the methylated fraction but that Zta binding to promoters lacking CpG ZREs was not reduced. We hypothesize that the loss of DNA methylation on the EBV genome during the lytic cycle causes the reduced binding to CpG ZREs; this may act as a lytic cycle epigenetic switch. However, the epigenetic changes associated with the replicated EBV genome do not affect the interaction of Zta with many loci that are rich in non-CpG ZREs; this leads to sustained binding at these regions.
Genetic Characterization of Simian Foamy Viruses Infecting Humans
Authors: Rua Réjane , Betsem Edouard , Calattini Sara , Saib Ali , Gessain Antoine
Published: September 2012

Simian foamy viruses (SFVs) are retroviruses that are widespread among nonhuman primates (NHPs). SFVs actively replicate in their oral cavity and can be transmitted to humans after NHP bites, giving rise to a persistent infection even decades after primary infection. Very few data on the genetic structure of such SFVs found in humans are available. In the framework of ongoing studies searching for SFV-infected humans in south Cameroon rainforest villages, we studied 38 SFV-infected hunters whose times of infection had presumably been determined. By long-term cocultures of peripheral blood mononuclear cells with BHK-21 cells, we isolated five new SFV strains and obtained complete genomes of SFV strains from chimpanzee (Pan troglodytes troglodytes; strains BAD327 and AG15), monkey (Cercopithecus nictitans; strain AG16), and gorilla (Gorilla gorilla; strains BAK74 and BAD468). These zoonotic strains share a very high degree of similarity with their NHP counterparts and have a high degree of conservation of the genetic elements important for viral replication. Interestingly, analysis of FV DNA sequences obtained before cultivation revealed variants with deletions in both the U3 region andtasthat may correlate within vivochronicity in humans. Genomic changes inbet(a premature stop codon) andgagwere also observed. To determine if such changes were specific to zoonotic strains, we studied local SFV-infected chimpanzees and found the same genomic changes. Our study reveals that natural polymorphism of SFV strains does exist at both the intersubspecies level (gag,bet) and the intrasubspecies (U3,tas) levels but does not seem to reflect a viral adaptation specific to zoonotic SFV strains.
Cytosolic Phospholipase A2 Gamma Is Involved in Hepatitis C Virus Replication and Assembly
Authors: Xu Song , Pei Rongjuan , Guo Min , Han Qingxia , Lai Juan , Wang Yun , Wu Chunchen , Zhou Yuan , Lu Mengji , Chen Xinwen
Published: September 2012

Similar to other positive-sense, single-stranded RNA viruses, hepatitis C virus (HCV) replicates its genome in a remodeled intracellular membranous structure known as the membranous web (MW). To date, the process of MW formation remains unclear. It is generally acknowledged that HCV nonstructural protein 4B (NS4B) can induce MW formation through interaction with the cytosolic endoplasmic reticulum (ER) membrane. Many host proteins, such as phosphatidylinositol 4-kinase IIIα (PI4KIIIα), have been identified as critical factors required for this process. We now report a new factor, the cytosolic phospholipase A2 gamma (PLA2G4C), which contributes to MW formation, HCV replication, and assembly. The PLA2G4C gene was identified as a host gene with upregulated expression upon HCV infection. Knockdown of PLA2G4C in HCV-infected cells or HCV replicon-containing cells by small interfering RNA (siRNA) significantly suppressed HCV replication and assembly. In addition, the chemical inhibitor methyl arachidonyl fluorophosphonate (MAFP), which specifically inhibits PLA2, reduced HCV replication and assembly. Electron microscopy demonstrated that MW structure formation was defective after PLA2G4C knockdown in HCV replicon-containing cells. Further analysis by immunostaining and immunoprecipitation assays indicated that PLA2G4C colocalized with the HCV proteins NS4B and NS5A in cells infected with JFH-1 and interacted with NS4B. In addition, PLA2G4C was able to transport the HCV nonstructural proteins from replication sites to lipid droplets, the site for HCV assembly. These data suggest that PLA2G4C plays an important role in the HCV life cycle and might represent a potential target for anti-HCV therapy.
Cross-Allele Cytotoxic T Lymphocyte Responses against 2009 Pandemic H1N1 Influenza A Virus among HLA-A24 and HLA-A3 Supertype-Positive Individuals
Authors: Liu Jun , Zhang Shihong , Tan Shuguang , Yi Yong , Wu Bin , Cao Bin , Zhu Fengcai , Wang Chen , Wang Hua , Qi Jianxun , Gao George F.
Published: September 2012

Lack of a universal vaccine against all serotypes of influenza A viruses and recent progress on T cell-related vaccines against influenza A virus illuminate the important role of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTLs) in anti-influenza virus immunity. However, the diverse HLA alleles among humans complicate virus-specific cellular immunity research, and elucidation of cross-HLA allele T cell responses to influenza virus specificity requires further detailed work. An ideal CTL epitope-based vaccine would cover a broad spectrum of epitope antigens presented by most, if not all, of the HLAs. Here, we evaluated the 2009 pandemic influenza A (H1N1) virus-specific T cell responses among the HLA-A24+population using a rationally designed peptide pool during the 2009 pandemic. Unexpectedly, cross-HLA allele T cell responses against the influenza A virus peptides were detected among both HLA-A11+and HLA-A24+donors. Furthermore, we found cross-responses in the entire HLA-A3 supertype population (including HLA-A11, -A31, -A33, and -A30). The cross-allele antigenic peptides within the peptide pool were identified and characterized, and the crystal structures of the major histocompatibility complex (MHC)-peptide complexes were determined. The subsequent HLA-A24-defined cross-allele peptides recognized by the HLA-A11+population were shown to mildly bind to the HLA-A*1101 molecule. Together with the structural models, these results partially explain the cross-allele responses. Our findings elucidate the promiscuity of the cross-allele T cell responses against influenza A viruses and are beneficial for the development of a T cell epitope-based vaccine applied in a broader population.
Intrinsic Temperature Sensitivity of Influenza C Virus Hemagglutinin-Esterase-Fusion Protein
Authors: Takashita Emi , Muraki Yasushi , Sugawara Kanetsu , Asao Hironobu , Nishimura Hidekazu , Suzuki Koji , Tsuji Takashi , Hongo Seiji , Ohara Yoshihiro , Kawaoka Yoshihiro , Ozawa Makoto , Matsuzaki Yoko
Published: September 2012

Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.
In Vitro
and
In Vivo
Analysis of the Interaction between RNA Helicase A and HIV-1 RNA
Authors: Xing Li , Niu Meijuan , Kleiman Lawrence
Published: September 2012

RNA helicase A (RHA) promotes multiple steps of HIV-1 RNA metabolism during viral replication, including transcription, translation, and the annealing of primer tRNA
3
Lys
to the viral RNA. RHA is a member of the DExH subclass of RNA helicases that uniquely contains two double-stranded RNA binding domains (dsRBDs) at its N terminus. Here, we performed a genome-wide analysis of the interaction of RHA with HIV-1 RNA both
in vitro
, using fluorescence polarization, and during viral replication, using an RNA-protein coprecipitation assay.
In vitro
, RHA binds to all the isolated regions of the HIV-1 RNA genome tested, with

K
d

(equilibrium dissociation constant) values ranging from 44 to 178 nM. In contrast, during viral replication, RNA-protein coprecipitation assays detected only a major interaction of RHA with the 5′-untranslated region (5′-UTR) and a minor interaction with the Rev response element (RRE) of HIV-1 RNA. Since RHA does not associate well with all the highly structured regions of HIV-1 RNA tested
in vivo
, the results suggest that other viral or cellular factors not present
in vitro
may modulate the direct interaction of RHA with HIV-1 RNA during virus replication. Nevertheless, a role for duplex RNA as a target for RHA binding
in vivo
is suggested by the fact that the deletion of either one or both dsRBDs eliminates the
in vivo
interaction of RHA with HIV-1 RNA. Furthermore, these mutant RHAs do not promote the
in vivo
annealing of tRNA
3
Lys
to viral RNA, nor are they packaged into virions, demonstrating that the dsRBDs are essential for the role of RHA in HIV-1 replication.
Intersubtype Differences in the Effect of a Rare p24 Gag Mutation on HIV-1 Replicative Fitness
Authors: Chopera Denis R. , Cotton Laura A. , Zawaira Alexander , Mann Jaclyn K. , Ngandu Nobubelo K. , Ntale Roman , Carlson Jonathan M. , Mlisana Koleka , Woodman Zenda , de Assis Rosa Debra , Martin Eric , Miura Toshiyuki , Pereyra Florencia , Walker Bruce D. , Gray Clive M. , Martin Darren P. , Ndung'u Thumbi , Brockman Mark A. , Karim Salim Abdool , Brumme Zabrina L. , Williamson Carolyn ,   the CAPRISA 002 Study Team
Published: September 2012

Certain immune-driven mutations in HIV-1, such as those arising in p24
Gag
, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24
Gag
M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (
P
= 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (
P
< 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces
in vitro
viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24
Gag
codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited
in vitro
replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.
Loss of the Protease Dimerization Inhibition Activity of Tipranavir (TPV) and Its Association with the Acquisition of Resistance to TPV by HIV-1
Authors: Aoki Manabu , Danish Matthew L. , Aoki-Ogata Hiromi , Amano Masayuki , Ide Kazuhiko , Das Debananda , Koh Yasuhiro , Mitsuya Hiroaki
Published: September 2012

Tipranavir (TPV), a protease inhibitor (PI) inhibiting the enzymatic activity and dimerization of HIV-1 protease, exerts potent activity against multi-PI-resistant HIV-1 isolates. When a mixture of 11 multi-PI-resistant (but TPV-sensitive) clinical isolates (HIV
11MIX
), which included HIV
B
and HIV
C
, was selected against TPV, HIV
11MIX
rapidly (by 10 passages [HIV
11MIX
P10
]) acquired high-level TPV resistance and replicated at high concentrations of TPV. HIV
11MIX
P10
contained various amino acid substitutions, including I54V and V82T. The intermolecular FRET-based HIV-1 expression assay revealed that TPV's dimerization inhibition activity against cloned HIV
B
(cHIV
B
) was substantially compromised. The introduction of I54V/V82T into wild-type cHIV
NL4-3
(cHIV

NL4-3
I54V/V82T

) did not block TPV's dimerization inhibition or confer TPV resistance. However, the introduction of I54V/V82T into cHIV
B
(cHIV
B
I54V/V82T
) compromised TPV's dimerization inhibition and cHIV
B
I54V/V82T
proved to be significantly TPV resistant. L24M was responsible for TPV resistance with the cHIV
C
genetic background. The introduction of L24M into cHIV
NL4-3
(cHIV

NL4-3
L24M

) interfered with TPV's dimerization inhibition, while L24M increased HIV-1's susceptibility to TPV with the HIV
NL4-3
genetic background. When selected with TPV, cHIV

NL4-3
I54V/V82T

most readily developed TPV resistance and acquired E34D, which compromised TPV's dimerization inhibition with the HIV
NL4-3
genetic background. The present data demonstrate that certain amino acid substitutions compromise TPV's dimerization inhibition and confer TPV resistance, although the loss of TPV's dimerization inhibition is not always associated with significantly increased TPV resistance. The findings that TPV's dimerization inhibition is compromised with one or two amino acid substitutions may explain at least in part why the genetic barrier of TPV against HIV-1's development of TPV resistance is relatively low compared to that of darunavir.
Review of quality assessment tools for the evaluation of pharmacoepidemiological safety studies
Authors: Neyarapally George A , Hammad Tarek A , Pinheiro Simone P , Iyasu Solomon
Published: September 2012

Objectives

Pharmacoepidemiological studies are an important hypothesis-testing tool in the evaluation of postmarketing drug safety. Despite the potential to produce robust value-added data, interpretation of findings can be hindered due to well-recognised methodological limitations of these studies. Therefore, assessment of their quality is essential to evaluating their credibility. The objective of this review was to evaluate the suitability and relevance of available tools for the assessment of pharmacoepidemiological safety studies.

Design

We created an a priori assessment framework consisting of reporting elements (REs) and quality assessment attributes (QAAs). A comprehensive literature search identified distinct assessment tools and the prespecified elements and attributes were evaluated.

Primary and secondary outcome measures

The primary outcome measure was the percentage representation of each domain, RE and QAA for the quality assessment tools.

Results

A total of 61 tools were reviewed. Most tools were not designed to evaluate pharmacoepidemiological safety studies. More than 50% of the reviewed tools considered REs under the research aims, analytical approach, outcome definition and ascertainment, study population and exposure definition and ascertainment domains. REs under the discussion and interpretation, results and study team domains were considered in less than 40% of the tools. Except for the data source domain, quality attributes were considered in less than 50% of the tools.

Conclusions

Many tools failed to include critical assessment elements relevant to observational pharmacoepidemiological safety studies and did not distinguish between REs and QAAs. Further, there is a lack of considerations on the relative weights of different domains and elements. The development of a quality assessment tool would facilitate consistent, objective and evidence-based assessments of pharmacoepidemiological safety studies.
A national cross-sectional study on effects of fluoride-safe water supply on the prevalence of fluorosis in China
Authors: Wang Cheng , Gao Yanhui , Wang Wei , Zhao Lijun , Zhang Wei , Han Hepeng , Shi Yuxia , Yu Guangqian , Sun Dianjun
Published: September 2012

Objective

To assess the effects of provided fluoride-safe drinking-water for the prevention and control of endemic fluorosis in China.

Design

A national cross-sectional study in China.

Setting

In 1985, randomly selected villages in 27 provinces (or cities and municipalities) in 5 geographic areas all over China.

Participants

Involved 81 786 children aged from 8 to 12 and 594 698 adults aged over 16.

Main outcome measure

The prevalence of dental fluorosis and clinical skeletal fluorosis, the fluoride concentrations in the drinking-water in study villages and in the urine of subjects.

Results

The study showed that in the villages where the drinking-water fluoride concentrations were higher than the government standard of 1.2 mg/l, but no fluoride-safe drinking-water supply scheme was provided (FNB areas), the prevalence rate and index of dental fluorosis in children, and prevalence rate of clinical skeletal fluorosis in adults were all significantly higher than those in the historical endemic fluorosis villages after the fluoride-safe drinking-water were provided (FSB areas). Additionally, the prevalence rate of dental fluorosis as well as clinical skeletal fluorosis, and the concentration of fluoride in urine were found increased with the increase of fluoride concentration in drinking-water, with significant positive correlations in the FNB areas. While, the prevalence rate of dental fluorosis and clinical skeletal fluorosis in different age groups and their degrees of prevalence were significantly lower in the FSB areas than those in the FNB areas.

Conclusions

The provision of fluoride-safe drinking-water supply schemes had significant effects on the prevention and control of dental fluorosis and skeletal fluorosis. The study also indicated that the dental and skeletal fluorosis is still prevailing in the high-fluoride drinking-water areas in China.
Effects of nicotine mouth spray on urges to smoke, a randomised clinical trial
Authors: Hansson Anna , Hajek Peter , Perfekt Roland , Kraiczi Holger
Published: September 2012

Objective

A new nicotine mouth spray was shown to be an effective stop-smoking treatment. This study was set up to examine the speed with which it relieves urges to smoke, and how it compares with nicotine lozenge in this respect.

Design

Randomised, cross-over trial that compared nicotine mouth spray 2 mg versus nicotine lozenge 2 or 4 mg.

Setting

Clinical pharmacology research unit.

Participants

200 Volunteer smokers who smoked their first cigarette of the day within 30 min of waking.

Interventions

Subjects abstained from smoking the night before the morning they attended the laboratory. Treatment was administered following 5 h of witnessed abstinence.

Primary and secondary outcome measures

Urge to smoke was rated before and at 1, 3, 5, 10, 15, 25, 30, 45 min and 1, 1.5, and 2 h after treatment administration. The primary outcome concerned change during the first 1, 3 and 5 min after treatment administration.

Results

Nicotine mouth spray achieved greater reductions in craving than either lozenge during the first 1, 3 and 5 min postadministration. After using mouth spray, half of the users experienced 50% reduction in craving within 3.40 min, while the same treatment effect was achieved within 9.92 and 9.20 min for the 2 and 4 mg lozenge, respectively. Adverse events with both mouth spray and lozenge were mostly mild. Hiccups, local irritation, nausea and dyspepsia were more frequent with spray than lozenge.

Conclusions

Nicotine mouth spray provides a faster relief of cravings than nicotine lozenge.
Managing the after effects of serious patient safety incidents in the NHS: an online survey study
Authors: Pinto Anna , Faiz Omar , Vincent Charles
Published: September 2012

Objective

To examine the current state of practice in English NHS Trusts in relation to the communication of serious patient safety incidents to patients and families and support for all parties involved.

Design

Cross-sectional design using online surveys.

Participants

209 patient safety managers, responsible for the ‘Being Open’ policy (54% response rate).

Setting

English NHS Trusts.

Results

98% of the participants reported that they are familiar with the National Patient Safety Agency Being Open guidance and 82% that they implement it more than half the time when incidents occur. However, provision of timely information was not reported as routine, with two-thirds of the discussions taking place 3–6 weeks after the investigation. The frequency of taking responsibility for harm was low for incidents of different severity levels but significantly lower for less serious ones (p<0.001). Follow-ups of patients in the long term and ex gratia payments were provided less than half the time. The most highly rated barriers to being open were clinical staff's fear of negative reactions from patients or their families and anxiety about litigation. Support practices for staff, such as debriefing and training on being open, were acknowledged as highly important but not always available.

Conclusions

Awareness of the importance of being open is high among patient safety managers in English NHS Trusts, but there is still considerable scope for improvement in the management of the after effects of patient safety incidents. More research is needed on patients' and healthcare professionals' preferences for support after patient safety incidents.
Applicability of the CATCH, CHALICE and PECARN paediatric head injury clinical decision rules: pilot data from a single Australian centre
Authors: Lyttle Mark D , Cheek John A , Blackburn Carol , Oakley Ed , Ward Brenton , Fry Amanda , Jachno Kim , Babl Franz E
Published: September 2012

Background

Clinical decision rules (CDRs) for paediatric head injury (HI) exist to identify children at risk of traumatic brain injury. Those of the highest quality are the Canadian assessment of tomography for childhood head injury (CATCH), Children's head injury algorithm for the prediction of important clinical events (CHALICE) and Pediatric Emergency Care Applied Research Network (PECARN) CDRs. They target different cohorts of children with HI and have not been compared in the same setting. We set out to quantify the proportion of children with HI to which each CDR was applicable.

Methods

Consecutive children presenting to an Australian paediatric Emergency Department with HIs were enrolled. Published inclusion/exclusion criteria and predictor variables from the CDRs were collected prospectively. Using these we determined the frequency with which each CDR was applicable.

Results

1012 patients (69.9%) were enrolled with 949 available for analysis. Mean age was 6.8 years (21% <2 years). 95% had initial Glasgow Coma Scale 15. CT rate was 12.8% and neurosurgery rate was 0.7%. No CDR was applicable to all patients. CHALICE was applicable to the most (97%, 95% CI 96% to 98%) and CATCH to the fewest (26%, 95% CI 24% to 29%). PECARN was applicable to 76% (95% CI 70% to 82%) aged <2 years, and 74% (95% CI 71% to 77%) aged 2–<18 years.

Conclusions

Each CDR is applicable to a different proportion of children with HI. This makes a direct comparison of the CDRs difficult. Prior to selection of any for implementation they should undergo validation outside the derivation setting coupled with an analysis of their performance accuracy, usability and cost effectiveness.
Utilisation of fluorescent multiplex PCR and laser-induced capillary electrophoresis for the diagnosis of Ewing family of tumours in formalin-fixed paraffin-embedded tissues
Authors: Patócs Barbara , Németh Krisztina , Garami Miklós , Arató Gabriella , Kovalszky Ilona , Szendrői Miklós , Fekete György
Published: September 2012

Aims

The localisation of the translocation breakpoint of the Ewing sarcoma family of tumours shows significant variability on relatively large regions of fusion partner genes. As a consequence, many alternative forms of EWSR1-ETS translocation exist which make the RNA-based molecular diagnostics of Ewing sarcoma family of tumours complicated. In addition to the heterogeneity of fusion transcripts, the degradation of RNA also presents a significant difficulty in the molecular analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Our aim was to establish a sensitive method which is able to identify all combinatorially possible EWSR1-FLI1 and EWSR1-ERG translocation transcripts in FFPE tissue samples despite significant RNA-degradation.

Methods

The combination of fluorescent multiplex PCR with laser-induced capillary electrophoresis was used to detect and identify EWSR1-FLI1 and EWSR1-ERG chimeric transcripts on the basis of amplicon size, and forward primers labelled by distinct fluorophores.

Results

Using this method, we processed 60 FFPE samples of Ewing sarcoma family of tumours, and identified six types EWSR1-FLI1 and one type EWSR1-ERG chimeric transcripts acceptable for RT-PCR analysis in 27 out of 45 samples. This result shows 60% sensitivity for detecting the most frequent Ewing family of tumour (EFT)-related fusion transcripts.

Conclusions

The utilisation of fluorescent multiplex PCR and laser-induced fluorescent capillary electrophoresis is effective for the diagnosis of EFT in FFPE tissue, and after the defined modifications it can offer a sensitive method to overcome the diagnostic difficulties connected with heterogeneity of the variant translocations in EFT.
Magnetic resonance spectroscopy of the brain
Authors: Currie Stuart , Hadjivassiliou Marios , Craven Ian J , Wilkinson Iain D , Griffiths Paul D , Hoggard Nigel
Published: September 2012

Proton magnetic resonance (MR) spectroscopy of the brain is a non-invasive, in vivo technique that allows investigation into regional chemical environments. Its complementary use with MR imaging sequences provides valuable insights into brain tumour characteristics, progression and response to treatment. Additionally, its sensitivity to brain dysfunction in the presence of apparently normal structural imaging has galvanised interest in its use as a biomarker of neurodegenerative disorders such as Alzheimer's disease. Accordingly, its integration into clinical imaging protocols within many neuroscience centres throughout the world is increasing. This growing attention is encouraging but if the potential of MR spectroscopy is to be realised, fundamental questions need to be addressed, such as reproducibility of the technique and the biochemistry that underpins the neurometabolites measured. Failure to resolve these issues will continue to hinder the extent and accuracy of conclusions that can be drawn from its data. In this review we discuss the issues regarding MR spectroscopy in the brain with particular attention paid to its technique. Key examples of current clinical applications are provided and future directions are discussed.
Care and Justice orientations to moral decision making in veterinary students
Authors: Quinn C. , Kinnison T. , May S. A.
Published: September 2012

An adapted version of the Moral Justification Scale was used to assess moral decision‐making orientation in veterinary students, comparing sex and year of study. The Scale consists of vignettes and related statements, each of which was classified as Justice, Care for People or Care for Animals. The importance of each statement in the decision‐making process was rated by 204 students on a 10‐point Likert Scale. An average score of importance for Justice, Care for People and Care for Animals related statements were calculated for each individual. General inclination scores were calculated by subtracting an individual's average Care score from their average Justice score. Inclination scores suggested that two‐thirds of students have a balanced approach, using Justice and Care almost equally in approaching ethical dilemmas. The majority of students, however, show an overall Justice orientation. The attitude towards the importance of Justice did not vary between students of different years or sexes. Students' attitudes towards the importance of Care for People in their decision making were, however, significantly lower for final‐year students. Reasons hypothesised include the start of placements. Care for Animals scores were affected by sex, whereby females give more importance to such statements than males.
IDEAL-MODIFIED BOSONIC GAS TRAPPED IN AN ARBITRARY THREE-DIMENSIONAL POWER-LAW POTENTIAL
Authors: CASTELLANOS E. , LÄMMERZAHL C.
Published: September 2012

We analyze the effects caused by an anomalous single-particle dispersion relation suggested in several quantum-gravity models, upon the thermodynamics of a Bose–Einstein condensate trapped in a generic three-dimensional power-law potential. We prove that the shift in the condensation temperature, caused by a deformed dispersion relation, described as a non-trivial function of the number of particles and the shape associated to the corresponding trap, could provide bounds for the parameters associated to such deformation. In addition, we calculate the fluctuations in the number of particles as a criterium of thermodynamic stability for these systems. We show that the apparent instability caused by the anomalous fluctuations in the thermodynamic limit can be suppressed considering the lowest energy associated to the system in question.
TRANSITION OF ORDERED WAVES IN NEURONAL NETWORK INDUCED BY DIFFUSIVE POISONING OF ION CHANNELS
Authors: HUANG LONG , MA JUN , TANG JUN , LI FAN
Published: September 2012

Normal physiological activities are often affected by some drugs, and some ion channels are blocked due to the katogene of drugs. This paper investigates the propagation of ordered waves in neuronal networks induced by diffusive poisoning, where the process is measured by increasing the number of neurons in the poisoned area of the networks. A coefficient of poisoning K is defined to measure the time units from one poisoned site to the adjacent site, a smaller K means that more neurons are poisoned in a certain period (a higher poisoning speed). A statistical factor of synchronization R in the two-dimensional array is defined to detect the transition of spiral waves induced by ion channel blocking. It is confirmed that the evolution of the spiral waves depends on the coefficient of poisoning K and number of poisoned neurons. Furthermore, breakup of the spirals is observed when weak channel noise is considered. Finally, the formation of the spiral wave induced by blocking the target wave with line defects is briefly discussed.
TOWARDS A CANONICAL GRAVITY IN TWO TIME AND TWO SPACE DIMENSIONS
Authors: NIETO J. A.
Published: September 2012

We describe a program for developing a canonical gravity in 2 + 2 dimensions (two time and two space dimensions). Our procedure is similar to the usual canonical gravity but with two times rather than just one time. Our work may be of particular interest as an alternative approach to loop quantum gravity in 2 + 2 dimensions.
Serum- and glucocorticoid-inducible kinase SGK1 regulates reorganization of actin cytoskeleton in mast cells upon degranulation
Authors: Schmid Evi , Gu Shuchen , Yang Wenting , Münzer Patrick , Schaller Martin , Lang Florian , Stournaras Christos , Shumilina Ekaterina
Published: September 2012

Aggregation of the high-affinity IgE receptor (FcεRI) on mast cells (MCs) causes MC degranulation, a process that involves cortical F-actin disassembly. Actin depolymerization may be triggered by increase of cytosolic Ca2+. Entry of Ca2+ through the Ca2+ release-activated Ca2+ (CRAC) channels is under powerful regulation by the serum- and glucocorticoid-inducible kinase SGK1. Moreover, FcεRI-dependent degranulation is decreased in SGK1-deficient ( sgk1 −/−) MCs. The present study addressed whether SGK1 is required for actin cytoskeleton rearrangement in MCs and whether modulation of actin architecture could underlie decreased degranulation of sgk1 −/− MCs. Confirming previous results, release of β-hexosaminidase reflecting FcεRI-dependent degranulation was impaired in sgk1 −/− MCs compared with sgk1 +/+ MCs. When CRAC channels were inhibited by 2-aminoethoxydiphenyl borate (2-APB; 50 μM), MC degranulation was strongly decreased in both sgk1 +/+ and sgk1 −/− MCs and the difference between genotypes was abolished. Moreover, degranulation was impaired by actin-stabilizing (phallacidin) and enhanced by actin-disrupting (cytochalasin B) agents to a similar extent in sgk1 +/+ MCs and sgk1 −/− MCs, implying a regulatory role of actin reorganization in this event. In line with this, measurements of monomeric (G) and filamentous (F) actin content by FACS analysis and Western blotting of detergent-soluble and -insoluble cell fractions indicated an increase of the G/F-actin ratio in sgk1 +/+ MCs but not in sgk1 −/− MCs upon FcεRI ligation, an observation reflecting actin depolymerization. In sgk1 +/+ MCs, FcεRI-induced actin depolymerization was abolished by 2-APB. The observed actin reorganization was confirmed by confocal laser microscopic analysis. Our observations uncover SGK1-dependent Ca2+ entry in mast cells as a novel mechanism regulating actin cytoskeleton.